Datasets:

LA1512

/

train_pubmed_ORC_4096_20k

like 0

in its role as the principal glucocorticoid in humans , cortisol performs vital anti - inflammatory and immunosuppressive functions,13 as well as a host of other effects that assist metabolism and homeostasis.4 although a background concentration is present at all times,4 cortisol is elevated during times of physical or mental stress.5,6 in addition , cortisol may exhibit a diurnal fluctuation in many persons,7 typically with a peak concentration early in the morning ( e.g. about 8.00 am ) and a minimal concentration later in the afternoon or early evening.8 however , this diurnal fluctuation is not present in everyone , with 17% of some samples showing a lack of variation over the day - night period,8 about 30% showing variation in their diurnal cycle from day to day,8 and up to 50% showing depression of that cycle due to a range of influences.9 the precise points of maximum and minimum cortisol concentrations have also been shown to vary somewhat across individuals8 ( although the former is usually early in the morning and the latter later in the afternoon ) , thus rendering the presence of a typical adaptive10 in the same way that other circadian - driven physiological variations influence survival behavior by maximizing the use of physiological resources,11 perhaps developed via evolutionary selection processes.12 produced from a cascade in the hypothalamicpituitary - adrenal ( hpa ) axis that commences with secretion of corticotropin releasing hormone ( crh ) in the hypothalamus and then adrenocorticotropic hormone ( acth ) in the pituitary , cortisol is synthesised from cholesterol in the adrenal cortex and enters the bloodstream about eight minutes after crh is secreted by the hypothalamus.4 as well as being measured in blood , cortisol can also be assayed from saliva13 and urine.4 cortisol produced by the adrenal glands influences the organism s preparedness for activity by gluconeogenesis , mobilization of fats and proteins from the liver and other cells , and reduction of glucose consumption by cells ( leading to elevated blood glucose and stimulation of insulin).4 cortisol also assists survival by stabilizing lysosomal membranes , decreasing capillary permeability , depressing the immune system and reducing fever , plus making a multiple assault on inflammation via at least 17 different pathways.14 it is therefore of particular interest when considering the latter set of capabilities to note that cortisol is also secreted in melanocytes15,16 and hair follicles1724 and may perform similar immunological functions at a localized level15 as it does when secreted from the adrenal glands to the general bloodstream . as well its end product , the entire crh - acth - cortisol sequence is replicated in melanocytes15 and hair follicles,25 pointing to the existence of a peripheral hpa axis in skin and hair15,16,26,27 that may be related to , or independent from , the the concentration of cortisol in hair has also been shown to vary in response to physical and psychological stressors in the same fashion as cortisol from the central hpa axis , although not in ways that would reflect a single connected hpa axis.2224 however , because peripherally - secreted cortisol has not yet been shown to enter the general bloodstream and contribute to those effects which central cortisol has upon metabolism and preparation of the organism for activity , any benefit it may convey upon the organism would most likely be in terms of its local anti - inflammatory properties , thus further demarking the peripheral hpa in terms of its effects upon behavior . the presence of a link between the central and peripheral hpa axes has been discussed by several sources,15,26 with one recent comment15 that one of the major , as - yet unmet challenges in cutaneous stress research ( is ) the study of the cross - talk between peripheral and systemic ( cortisol ) responses ( p. 1697 ) . that is , if there is a lack of consistency in cortisol concentrations from the peripheral and the central hpa axes , it could be concluded that they were independent sources of cortisol as suggested by ito and colleagues,25 a finding which would confirm hair cortisol as playing a different role to that exhibited by central cortisol in terms of behaviour and survival . some previous data support this hypothesis of the independence of the peripheral hpa axis by showing that cortisol responsivity to a local pain stressor applied to one hand was immediate and transient in the same arm hair , but restricted to the local area ( i.e. not in the hair from the opposite leg).24 central cortisol reactions were also found in saliva , but about 20 minutes afterward , as expected.6,13 that is , had central cortisol responses to the pain stressor been responsible for the cortisol responses found in hair , then the latter should have occurred at least eight minutes after the onset of the pain stressor , rather than immediately ( as was noted ) or about 20 minutes later ( as found in saliva ) . these findings suggest that the peripheral hpa axis may be uncoupled from the central hpa axis , and even be uncoupled from other hpa sites on the skin . however , those data were collected during administration of a pain stressor ( ice water immersion for 1 min ) which subjects described as very painful . while there may be evidence of a regional uncoupling of hair cortisol under stressor conditions , it remains to be seen if the same discrete responses are present under non - stressful conditions . one way to assess the degree of linkage between central and peripheral hpa axes while subjects engage in normal daily activities is to collect hair and saliva during a day and determine ( a ) the existence of the diurnal fluctuation in hair cortisol and ( b ) whether such a fluctuation coincides with the cycle in the central hpa axis . in order to ensure a more fine - grained data - collection procedure than possible from a single observation ( as reported in a previous study of 24-hour urine),20 and thus determine if peripheral cortisol concentrations were correlated with central cortisol diurnal fluctuations,4 both hair and saliva should be collected at regular intervals during a typical daily period . therefore , as a pilot investigation of the presence and nature of diurnal variation in cortisol within scalp hair , the present study measured cortisol from scalp hair samples collected simultaneously with saliva at set three - hour intervals during a 15-hour ( waking ) period to determine the relationship between central and peripheral hpa axes cortisol production while the subjects were not under pain stress . while previous studies of central cortisol have shown frequent cycles of cortisol elevation and decrease,7 reflecting the value of very frequent collections of serum ( every 20 min ) , that procedure was not considered appropriate here because of ( a ) the exploratory nature of this study and ( b ) the extreme unlikelihood of collecting saliva and hair samples from sleeping subjects without arousing them and thus confounding the findings due to fatigue and/or stress occasioned by frequent waking . it is worth commenting that very early explorations of central cortisol10 also collected data at similar frequencies as that employed in this study , and this methodology may suffice at this stage of investigation of this phenomenon . further , while it is the common procedure in much research , the use of large group research designs and group - based statistical analyses may be unjustified under two circumstances.28 first , when exploratory studies are being conducted , ethical constraints may preclude applying the experimental procedure to the large groups of subjects that are required for adequate statistical power . second , as mentioned above , the presence of diurnal variation in central hpa axis cortisol concentrations is not consistent across subjects , with about 17% of people not showing the early morning elevation and late afternoon depression in cortisol measured from blood , urine or saliva , and a further 50% being likely to experience fluctuations to that typical diurnal variation because of a range of factors.9 therefore , as an initial exploration of the presence of a diurnal variation in peripheral ( hair ) cortisol that might be parallel to that from central ( saliva ) cortisol , an n = 1 experimental design protocol was adopted.28 under such a design , group mean data ( and relevant statistical procedures ) are considered to be less important than individual subject responses as a means of forming an initial understanding of the phenomenon under scrutiny so that further ( later ) group studies may be conducted . two healthy young females aged 18 and 20 years , plus one healthy male of 21 years and the first author ( to provide a comparison with an older person , age = 61 yr ) volunteered to collect saliva and hair samples ( of about 50 strands ) at the hours of 6.00 am , 9.00 am 12.00 pm , 4.00 pm , 6.00 pm and 9.00 pm . saliva was collected via subjects ( ss ) using a separate and labeled ( subject , time ) salivette ( sarstedt ) for each time period and stored at 20 c until assayed . hair was collected by ss cutting with scissors from as close to the posterior vertex as possible and then placed in a labeled ( s , time ) paper envelope . after being emptied from the envelopes and placed in separate glass vials ( 20 ml ) , hair was weighed and then chopped with scissors ( washed with methanol between chopping samples ) before being extracted with 3 ml of methanol for 24 hours . the methanol was then decanted into polypropylene tubes ( 3.5 ml ) and evaporated under vacuum . gel buffer ( 100 l ) ( phosphate buffered saline , ph 7.5 containing 0.1% gelatin ) was added and the solution allowed to stand at room temperature for 60 minutes prior to assay . cortisol concentrations in both saliva and hair were determined by radioimmunoassay as previously described.29 ss were instructed in the sample - collection and storage procedures in a group . all samples were returned to the second author within a few days and then frozen until assay . previous examination of hair samples has shown that they may be stored at room temperature for at least a year with no effects upon cortisol concentration.20 similarly , saliva may be stored at room temperature for at least two weeks with no effects on stability.30 all procedures were approved by the university of new england human experimentation ethics committee . two healthy young females aged 18 and 20 years , plus one healthy male of 21 years and the first author ( to provide a comparison with an older person , age = 61 yr ) volunteered to collect saliva and hair samples ( of about 50 strands ) at the hours of 6.00 am , 9.00 am 12.00 pm , 4.00 pm , 6.00 pm and 9.00 pm . saliva was collected via subjects ( ss ) using a separate and labeled ( subject , time ) salivette ( sarstedt ) for each time period and stored at 20 c until assayed . hair was collected by ss cutting with scissors from as close to the posterior vertex as possible and then placed in a labeled ( s , time ) paper envelope . after being emptied from the envelopes and placed in separate glass vials ( 20 ml ) , hair was weighed and then chopped with scissors ( washed with methanol between chopping samples ) before being extracted with 3 ml of methanol for 24 hours . the methanol was then decanted into polypropylene tubes ( 3.5 ml ) and evaporated under vacuum . gel buffer ( 100 l ) ( phosphate buffered saline , ph 7.5 containing 0.1% gelatin ) was added and the solution allowed to stand at room temperature for 60 minutes prior to assay . all samples were returned to the second author within a few days and then frozen until assay . previous examination of hair samples has shown that they may be stored at room temperature for at least a year with no effects upon cortisol concentration.20 similarly , saliva may be stored at room temperature for at least two weeks with no effects on stability.30 all procedures were approved by the university of new england human experimentation ethics committee . in order to compare the cortisol concentrations from the six sample periods across each of the four ss , each s s first sample ( i.e. taken at 6.00 am ) was set as a standard of 100% , and the remaining concentrations were compared to that value for each s. as an overview , figure 1 shows the mean hair and saliva percent cortisol concentrations for the combined four ss across all sample times , and suggests that the previously - reported diurnal fluctuation in central cortisol concentrations ( i.e. saliva ) were replicated in these group mean data , with a peak in the morning ( 9.00 am : 154.9% ) , then a drop to 90.7% at 12.00 pm and remaining at about that level for the rest of the sample period ( to 9.00 pm ) . cortisol concentrations in scalp hair also showed the same morning peak ( 9.00 am : 207.5% of 6.00 am ) and a drop to 130.5% at 12.00 pm , a plateau until a further drop at 6.00 pm ( 88.1% ) and then a rise at 9.00 pm ( 143.7% ) . however , figure 1 presents only mean cortisol percentage data and , as mentioned in the introduction to this paper , there are marked variations in the presence , consistency and peak / low concentration times across persons . the presence of such variability suggested that the principal examination of the data obtained from this exploratory study should be performed on an individual subject basis ( i.e. from an n = 1 methodology perspective ) . therefore , each s s hair and saliva cortisol concentrations were plotted separately and are presented in figure 2 . it is apparent from figure 2 that the kind of individual differences that might be expected from the wider literature6,8,9 were also present across the four ss in the pattern of diurnal fluctuations shown for saliva and hair cortisol . s1 showed the typical peak cortisol concentrations for both saliva ( 316.7% ) and hair ( 293.4% ) at 9.00am , followed by a drop to 12.00 pm ( saliva = 146.5% , hair = 169.6% ) and then a gradual rise for saliva at 9.00 pm ( 193.6% ) . by contrast , hair cortisol dropped again from the 12.00 pm values to 136.2% at 6.00 pm and then rose at 9.00 pm ( 209.3% ) . s2 showed the expected pattern for saliva , with a minor peak at 9.00 am = 113.5% , followed by a gradual decrease to a nadir at 9.00 pm = 54.4% ) but not for hair , with a pattern for the latter that was almost the inverse of the former . the peak hair cortisol concentration for this s was 100% at 6.00 am and did not rise above this during the rest of the day , although the drop to 69.1% at 9.00 am and subsequent rise to 99.7% at 3.00 pm was suggestive of a bimodal pattern . overall , this s s cortisol concentrations did not show the same degree of variability across the 15 hours of the study as s1 . s3 showed a peak saliva cortisol concentration at 6.00am , and generally declined gradually after that to the minimum value at 9.00 pm ( 45.6% ) . hair cortisol concentration variations were also fairly flat for this s , with two very minor peaks , one at 6.00 am ( 100% ) and another at 9.00 pm ( 100.8% ) , following a nadir at 9.00 am ( 67.6% ) . s4 ( the first author ) showed a peak in salivary cortisol at 9.00 am ( 124.1% ) followed by a fairly flat set of cortisol concentration values to a minimum value of 52.5% at 9.00 pm . hair cortisol values were quite different for this s , with a dramatic increase from 100% at 6.00 am to a peak of 399.9% at 9.00 am , then a gradual decrease to 75.9% at 6.00 pm , followed by a rise to 143.0% at 9.00 pm . the group mean data on salivary cortisol approximated the expected single daily peak during the early morning for three of the four ss , followed by either gradual decreases in concentrations for the rest of the day ( in three ss ) or a drop to 12.00 pm and a slight decrease to the end of the day ( in one s ) . however , group mean hair cortisol concentrations for all four ss showed a bimodal pattern , with two peaks , one at either 6.00 am or 9.00 am and another at 9.00 pm ( in three ss ) or 3.00 pm ( in one s ) . although the mean pattern of salivary cortisol fluctuation found here was consistent with that reported in the literature,4,7,10 with a peak during the morning followed by a drop three hours later and then a fairly flat plateau during the rest of the day , mean hair cortisol data showed evidence of a bimodal peak in concentrations , the first at 9.00 am and the second at 9.00 pm . the central cortisol fluctuations in saliva did not show the expected steep decline during the afternoon that has been reported in some other studies,4,7,10 perhaps suggesting that this group of ss experienced some exogenous stressful stimuli during the afternoon . hair cortisol data did show that steep decline during the afternoon , with the lowest concentration being clearly at 6.00 pm . however , while group mean data are valuable in describing general trends within large samples , they do not amplify the range of responses present within a sample and which have been previously reported in the literature.8,9 figure 2 shows those inter - subject differences clearly , and supports the previous literature that found variability between persons in the diurnal fluctuation pattern that was mentioned in the introduction to this paper . up to 17% of some samples have shown no detectable diurnal cycle,9 30% have inconsistent diurnal variations from day to day8 and up to 50% have shown major variations to the standard fluctuation pattern.8 factors that have been shown to influence this departure from the typical diurnal cycle pattern include depression,31 relationship functioning , 32 prenatal anxiety,33 smoking,34 financial strain,35 stress and fear,36 relative dominance - submissive position with a social group,37 over - the - counter medications , 38 and even time of awakening.39 some of these factors may have been present in this sample . the current data are the first to report on the diurnal fluctuation in cortisol produced by the peripheral hpa axis in hair follicles , and the presence of inter - subject differences in this source of cortisol adds to an understanding of the peripheral secretion of this important hormone . that is , just as central cortisol diurnal fluctuation patterns vary ( and are even absent in about 17% of people ) across individuals , so do peripheral cortisol concentrations show some differences between persons also . the presence of inter - subject variability in both central and peripheral cortisol argues for the presence of exogenous factors in the determination of cortisol concentrations produced from the adrenal glands and also from hair follicles . while some of these that influence central cortisol secretion have been suggested above , many do not easily apply to the production of peripheral cortisol . that is , the central hpa axis is integrally involved with overall physiological regulation of many functions , and ( for example ) taking medications , time of awakening and psychological factors such as financial factors and depression may have direct impact upon the hypothalamus , pituitary and adrenal glands because these are key homeostatic agents for the body . however , a similar functional response in hair follicles to depression or financial stress is not so easily linked with overall functioning , and may be related to other , more localized , functions . for example , the diurnal fluctuation in central cortisol is a function of the influence of daylight upon specialized light sensitive retinal ganglia that express the photopigment melanopsin and project directly to the suprachiasmatic nucleus in the hypothalamus , creating a circadian clock which generates higher cortisol levels during the early part of the morning and lower concentrations during the later afternoon and evening.40 described by yates and urquhart10 as adaptive , the assumed selective benefit of increased cortisol secretion in the early hours of daylight may be in terms of the effects of ( central ) cortisol release upon the organism s ability to move from the relative inactivity of sleep to daily activity . that is , many aspects of the body s functioning are in train to the circadian rhythm or clock,11,12 and cortisol has been described in this fashion.40 as well as preparing the organism for increases in general activity levels following sleep , some of the particular physical demands faced by ancestors of h. sapiens may have included foraging and general food - gathering , plus hunting . that is , under this hypothesis , the gluconeogenesis , protein mobilization , fat mobilization and associated activation effects of central cortisol secretion may be congruent with increased physical work and demands undertaken by hungry primates during the early morning . by contrast , in terms of peripheral secretion of cortisol in hair , it is relevant to note that the central circadian clock mentioned above that controls the diurnal cycle of cortisol secretion from the central hpa is not the only such clock . in fact , it is replicated throughout the body by expression of clock genes,41 even in non - neural tissue.42 these peripheral clocks may function to modulate physiological dynamics in the particular tissue where they occur by secretion of selected hormones such as cortisol.40 in this way , the difference of the hair cortisol diurnal fluctuation pattern from that shown by salivary cortisol may reflect the latter being controlled by a diurnal cycling ( and all circadian rhythms ) , the survival benefit of peripheral cortisol at mid - morning ( 9.00 am ) and mid - evening ( 9.00 pm ) may be unrelated to those centrally - activated physiological responses ( such as gluconeogenesis and mobilization of fat and protein ) which prepare the organism for major physical activity and demand early , if only because the cortisol secreted in hair follicles has not yet been shown to reach the general bloodstream.25 instead , hair cortisol appears to be restricted to the particular site of its release , and does not generalize across body sites.43 therefore , any survival benefit from the release of hair cortisol is most probably restricted to those anti - inflammatory effects previously described.14 these might be of particular value in the skin if wound punctures were experienced , especially if they were from animals claws during hunting which might be expected to have significant pathological consequences.44 alternatively , the peak hair cortisol at mid - evening may coincide with the need for anti - inflammatory effects to skin during the maximum period of uv radiation received during the day and consequent skin damage and inflammation . 45,46 thus , the comparative differences in the diurnal variation patterns shown by central vs. peripheral cortisol might be linked to the effects of cortisol secretion in each of these venues at particular times , plus the reproductive advantages which its release then and there might confer upon species that showed such responsiveness . however , this study is exploratory and therefore this hypothetical explanation for the observed differences between the diurnal cycle patterns of central and peripheral cortisol remains untested at this stage . further , because all the hair collected in this study was from the posterior vertex of the skull , comparisons with the diurnal fluctuation pattern shown by hair from other body sites was not possible . recent data have shown that hair from different body sites ( arms , legs ) does show significantly different concentrations of cortisol , although those data were collected at a single point in time ( early afternoon),42 and replication of the present study of diurnal fluctuations in cortisol concentrations in hair from different body sites would help clarify these findings . as acknowledged in the introduction , this study was exploratory and used a very small sample , applying the relevant n = 1 methodology . on the basis of these data , further research with a larger sample is suggested to enable the application of group - based statistical procedures that might produce more informative data regarding the overall group mean trend for diurnal fluctuation in hair cortisol concentrations . however , such a study would have to accommodate the findings from the previous literature regarding quite large between - subject variations in the presence , consistency and timing of cortisol diurnal fluctuations . therefore , as well as increasing subject numbers , replications of the current study should also conduct analyses of individual subject variability as has been done previously with such revealing outcomes.8 finally , although several factors which impinge upon cortisol and diurnal fluctuations were mentioned above ( e.g. mood , medication , financial and social status , fear and stress , etc . ) , few data have been reported as yet regarding the influence that any of those factors might have upon hair cortisol production per se and none upon their effects on diurnal fluctuation of peripheral cortisol in particular , and this remains a fruitful avenue for future research .

cortisol concentrations in hair and saliva collected from male and female adults over a 15-hour period were compared for differences in overall level and cyclic pattern . typical diurnal fluctuations were noted for both salivary and hair cortisol , with some individual differences that are congruent with the previous literature . issues of the link between central and peripheral hpa axes are raised for discussion and further investigation , and hypothetical explanations for the diurnal variability shown in these two sets of cortisol secretion patterns are discussed from an evolutionary advantage perspective .

Introduction Methods Participants Sample collection and assay Procedure Results Discussion

about 8.00 am ) and a minimal concentration later in the afternoon or early evening.8 however , this diurnal fluctuation is not present in everyone , with 17% of some samples showing a lack of variation over the day - night period,8 about 30% showing variation in their diurnal cycle from day to day,8 and up to 50% showing depression of that cycle due to a range of influences.9 the precise points of maximum and minimum cortisol concentrations have also been shown to vary somewhat across individuals8 ( although the former is usually early in the morning and the latter later in the afternoon ) , thus rendering the presence of a typical adaptive10 in the same way that other circadian - driven physiological variations influence survival behavior by maximizing the use of physiological resources,11 perhaps developed via evolutionary selection processes.12 produced from a cascade in the hypothalamicpituitary - adrenal ( hpa ) axis that commences with secretion of corticotropin releasing hormone ( crh ) in the hypothalamus and then adrenocorticotropic hormone ( acth ) in the pituitary , cortisol is synthesised from cholesterol in the adrenal cortex and enters the bloodstream about eight minutes after crh is secreted by the hypothalamus.4 as well as being measured in blood , cortisol can also be assayed from saliva13 and urine.4 cortisol produced by the adrenal glands influences the organism s preparedness for activity by gluconeogenesis , mobilization of fats and proteins from the liver and other cells , and reduction of glucose consumption by cells ( leading to elevated blood glucose and stimulation of insulin).4 cortisol also assists survival by stabilizing lysosomal membranes , decreasing capillary permeability , depressing the immune system and reducing fever , plus making a multiple assault on inflammation via at least 17 different pathways.14 it is therefore of particular interest when considering the latter set of capabilities to note that cortisol is also secreted in melanocytes15,16 and hair follicles1724 and may perform similar immunological functions at a localized level15 as it does when secreted from the adrenal glands to the general bloodstream . as well its end product , the entire crh - acth - cortisol sequence is replicated in melanocytes15 and hair follicles,25 pointing to the existence of a peripheral hpa axis in skin and hair15,16,26,27 that may be related to , or independent from , the the concentration of cortisol in hair has also been shown to vary in response to physical and psychological stressors in the same fashion as cortisol from the central hpa axis , although not in ways that would reflect a single connected hpa axis.2224 however , because peripherally - secreted cortisol has not yet been shown to enter the general bloodstream and contribute to those effects which central cortisol has upon metabolism and preparation of the organism for activity , any benefit it may convey upon the organism would most likely be in terms of its local anti - inflammatory properties , thus further demarking the peripheral hpa in terms of its effects upon behavior . the presence of a link between the central and peripheral hpa axes has been discussed by several sources,15,26 with one recent comment15 that one of the major , as - yet unmet challenges in cutaneous stress research ( is ) the study of the cross - talk between peripheral and systemic ( cortisol ) responses ( p. 1697 ) . that is , if there is a lack of consistency in cortisol concentrations from the peripheral and the central hpa axes , it could be concluded that they were independent sources of cortisol as suggested by ito and colleagues,25 a finding which would confirm hair cortisol as playing a different role to that exhibited by central cortisol in terms of behaviour and survival . some previous data support this hypothesis of the independence of the peripheral hpa axis by showing that cortisol responsivity to a local pain stressor applied to one hand was immediate and transient in the same arm hair , but restricted to the local area ( i.e. not in the hair from the opposite leg).24 central cortisol reactions were also found in saliva , but about 20 minutes afterward , as expected.6,13 that is , had central cortisol responses to the pain stressor been responsible for the cortisol responses found in hair , then the latter should have occurred at least eight minutes after the onset of the pain stressor , rather than immediately ( as was noted ) or about 20 minutes later ( as found in saliva ) . these findings suggest that the peripheral hpa axis may be uncoupled from the central hpa axis , and even be uncoupled from other hpa sites on the skin . while there may be evidence of a regional uncoupling of hair cortisol under stressor conditions , it remains to be seen if the same discrete responses are present under non - stressful conditions . one way to assess the degree of linkage between central and peripheral hpa axes while subjects engage in normal daily activities is to collect hair and saliva during a day and determine ( a ) the existence of the diurnal fluctuation in hair cortisol and ( b ) whether such a fluctuation coincides with the cycle in the central hpa axis . in order to ensure a more fine - grained data - collection procedure than possible from a single observation ( as reported in a previous study of 24-hour urine),20 and thus determine if peripheral cortisol concentrations were correlated with central cortisol diurnal fluctuations,4 both hair and saliva should be collected at regular intervals during a typical daily period . therefore , as a pilot investigation of the presence and nature of diurnal variation in cortisol within scalp hair , the present study measured cortisol from scalp hair samples collected simultaneously with saliva at set three - hour intervals during a 15-hour ( waking ) period to determine the relationship between central and peripheral hpa axes cortisol production while the subjects were not under pain stress . while previous studies of central cortisol have shown frequent cycles of cortisol elevation and decrease,7 reflecting the value of very frequent collections of serum ( every 20 min ) , that procedure was not considered appropriate here because of ( a ) the exploratory nature of this study and ( b ) the extreme unlikelihood of collecting saliva and hair samples from sleeping subjects without arousing them and thus confounding the findings due to fatigue and/or stress occasioned by frequent waking . it is worth commenting that very early explorations of central cortisol10 also collected data at similar frequencies as that employed in this study , and this methodology may suffice at this stage of investigation of this phenomenon . further , while it is the common procedure in much research , the use of large group research designs and group - based statistical analyses may be unjustified under two circumstances.28 first , when exploratory studies are being conducted , ethical constraints may preclude applying the experimental procedure to the large groups of subjects that are required for adequate statistical power . second , as mentioned above , the presence of diurnal variation in central hpa axis cortisol concentrations is not consistent across subjects , with about 17% of people not showing the early morning elevation and late afternoon depression in cortisol measured from blood , urine or saliva , and a further 50% being likely to experience fluctuations to that typical diurnal variation because of a range of factors.9 therefore , as an initial exploration of the presence of a diurnal variation in peripheral ( hair ) cortisol that might be parallel to that from central ( saliva ) cortisol , an n = 1 experimental design protocol was adopted.28 under such a design , group mean data ( and relevant statistical procedures ) are considered to be less important than individual subject responses as a means of forming an initial understanding of the phenomenon under scrutiny so that further ( later ) group studies may be conducted . two healthy young females aged 18 and 20 years , plus one healthy male of 21 years and the first author ( to provide a comparison with an older person , age = 61 yr ) volunteered to collect saliva and hair samples ( of about 50 strands ) at the hours of 6.00 am , 9.00 am 12.00 pm , 4.00 pm , 6.00 pm and 9.00 pm . cortisol concentrations in both saliva and hair were determined by radioimmunoassay as previously described.29 ss were instructed in the sample - collection and storage procedures in a group . two healthy young females aged 18 and 20 years , plus one healthy male of 21 years and the first author ( to provide a comparison with an older person , age = 61 yr ) volunteered to collect saliva and hair samples ( of about 50 strands ) at the hours of 6.00 am , 9.00 am 12.00 pm , 4.00 pm , 6.00 pm and 9.00 pm . in order to compare the cortisol concentrations from the six sample periods across each of the four ss , each s s first sample ( i.e. taken at 6.00 am ) was set as a standard of 100% , and the remaining concentrations were compared to that value for each s. as an overview , figure 1 shows the mean hair and saliva percent cortisol concentrations for the combined four ss across all sample times , and suggests that the previously - reported diurnal fluctuation in central cortisol concentrations ( i.e. saliva ) were replicated in these group mean data , with a peak in the morning ( 9.00 am : 154.9% ) , then a drop to 90.7% at 12.00 pm and remaining at about that level for the rest of the sample period ( to 9.00 pm ) . cortisol concentrations in scalp hair also showed the same morning peak ( 9.00 am : 207.5% of 6.00 am ) and a drop to 130.5% at 12.00 pm , a plateau until a further drop at 6.00 pm ( 88.1% ) and then a rise at 9.00 pm ( 143.7% ) . from an n = 1 methodology perspective ) . therefore , each s s hair and saliva cortisol concentrations were plotted separately and are presented in figure 2 . it is apparent from figure 2 that the kind of individual differences that might be expected from the wider literature6,8,9 were also present across the four ss in the pattern of diurnal fluctuations shown for saliva and hair cortisol . s1 showed the typical peak cortisol concentrations for both saliva ( 316.7% ) and hair ( 293.4% ) at 9.00am , followed by a drop to 12.00 pm ( saliva = 146.5% , hair = 169.6% ) and then a gradual rise for saliva at 9.00 pm ( 193.6% ) . by contrast , hair cortisol dropped again from the 12.00 pm values to 136.2% at 6.00 pm and then rose at 9.00 pm ( 209.3% ) . s2 showed the expected pattern for saliva , with a minor peak at 9.00 am = 113.5% , followed by a gradual decrease to a nadir at 9.00 pm = 54.4% ) but not for hair , with a pattern for the latter that was almost the inverse of the former . the peak hair cortisol concentration for this s was 100% at 6.00 am and did not rise above this during the rest of the day , although the drop to 69.1% at 9.00 am and subsequent rise to 99.7% at 3.00 pm was suggestive of a bimodal pattern . overall , this s s cortisol concentrations did not show the same degree of variability across the 15 hours of the study as s1 . hair cortisol concentration variations were also fairly flat for this s , with two very minor peaks , one at 6.00 am ( 100% ) and another at 9.00 pm ( 100.8% ) , following a nadir at 9.00 am ( 67.6% ) . s4 ( the first author ) showed a peak in salivary cortisol at 9.00 am ( 124.1% ) followed by a fairly flat set of cortisol concentration values to a minimum value of 52.5% at 9.00 pm . hair cortisol values were quite different for this s , with a dramatic increase from 100% at 6.00 am to a peak of 399.9% at 9.00 am , then a gradual decrease to 75.9% at 6.00 pm , followed by a rise to 143.0% at 9.00 pm . the group mean data on salivary cortisol approximated the expected single daily peak during the early morning for three of the four ss , followed by either gradual decreases in concentrations for the rest of the day ( in three ss ) or a drop to 12.00 pm and a slight decrease to the end of the day ( in one s ) . however , group mean hair cortisol concentrations for all four ss showed a bimodal pattern , with two peaks , one at either 6.00 am or 9.00 am and another at 9.00 pm ( in three ss ) or 3.00 pm ( in one s ) . although the mean pattern of salivary cortisol fluctuation found here was consistent with that reported in the literature,4,7,10 with a peak during the morning followed by a drop three hours later and then a fairly flat plateau during the rest of the day , mean hair cortisol data showed evidence of a bimodal peak in concentrations , the first at 9.00 am and the second at 9.00 pm . hair cortisol data did show that steep decline during the afternoon , with the lowest concentration being clearly at 6.00 pm . however , while group mean data are valuable in describing general trends within large samples , they do not amplify the range of responses present within a sample and which have been previously reported in the literature.8,9 figure 2 shows those inter - subject differences clearly , and supports the previous literature that found variability between persons in the diurnal fluctuation pattern that was mentioned in the introduction to this paper . up to 17% of some samples have shown no detectable diurnal cycle,9 30% have inconsistent diurnal variations from day to day8 and up to 50% have shown major variations to the standard fluctuation pattern.8 factors that have been shown to influence this departure from the typical diurnal cycle pattern include depression,31 relationship functioning , 32 prenatal anxiety,33 smoking,34 financial strain,35 stress and fear,36 relative dominance - submissive position with a social group,37 over - the - counter medications , 38 and even time of awakening.39 some of these factors may have been present in this sample . the current data are the first to report on the diurnal fluctuation in cortisol produced by the peripheral hpa axis in hair follicles , and the presence of inter - subject differences in this source of cortisol adds to an understanding of the peripheral secretion of this important hormone . the presence of inter - subject variability in both central and peripheral cortisol argues for the presence of exogenous factors in the determination of cortisol concentrations produced from the adrenal glands and also from hair follicles . while some of these that influence central cortisol secretion have been suggested above , many do not easily apply to the production of peripheral cortisol . that is , the central hpa axis is integrally involved with overall physiological regulation of many functions , and ( for example ) taking medications , time of awakening and psychological factors such as financial factors and depression may have direct impact upon the hypothalamus , pituitary and adrenal glands because these are key homeostatic agents for the body . however , a similar functional response in hair follicles to depression or financial stress is not so easily linked with overall functioning , and may be related to other , more localized , functions . for example , the diurnal fluctuation in central cortisol is a function of the influence of daylight upon specialized light sensitive retinal ganglia that express the photopigment melanopsin and project directly to the suprachiasmatic nucleus in the hypothalamus , creating a circadian clock which generates higher cortisol levels during the early part of the morning and lower concentrations during the later afternoon and evening.40 described by yates and urquhart10 as adaptive , the assumed selective benefit of increased cortisol secretion in the early hours of daylight may be in terms of the effects of ( central ) cortisol release upon the organism s ability to move from the relative inactivity of sleep to daily activity . that is , under this hypothesis , the gluconeogenesis , protein mobilization , fat mobilization and associated activation effects of central cortisol secretion may be congruent with increased physical work and demands undertaken by hungry primates during the early morning . by contrast , in terms of peripheral secretion of cortisol in hair , it is relevant to note that the central circadian clock mentioned above that controls the diurnal cycle of cortisol secretion from the central hpa is not the only such clock . in fact , it is replicated throughout the body by expression of clock genes,41 even in non - neural tissue.42 these peripheral clocks may function to modulate physiological dynamics in the particular tissue where they occur by secretion of selected hormones such as cortisol.40 in this way , the difference of the hair cortisol diurnal fluctuation pattern from that shown by salivary cortisol may reflect the latter being controlled by a diurnal cycling ( and all circadian rhythms ) , the survival benefit of peripheral cortisol at mid - morning ( 9.00 am ) and mid - evening ( 9.00 pm ) may be unrelated to those centrally - activated physiological responses ( such as gluconeogenesis and mobilization of fat and protein ) which prepare the organism for major physical activity and demand early , if only because the cortisol secreted in hair follicles has not yet been shown to reach the general bloodstream.25 instead , hair cortisol appears to be restricted to the particular site of its release , and does not generalize across body sites.43 therefore , any survival benefit from the release of hair cortisol is most probably restricted to those anti - inflammatory effects previously described.14 these might be of particular value in the skin if wound punctures were experienced , especially if they were from animals claws during hunting which might be expected to have significant pathological consequences.44 alternatively , the peak hair cortisol at mid - evening may coincide with the need for anti - inflammatory effects to skin during the maximum period of uv radiation received during the day and consequent skin damage and inflammation . 45,46 thus , the comparative differences in the diurnal variation patterns shown by central vs. peripheral cortisol might be linked to the effects of cortisol secretion in each of these venues at particular times , plus the reproductive advantages which its release then and there might confer upon species that showed such responsiveness . however , this study is exploratory and therefore this hypothetical explanation for the observed differences between the diurnal cycle patterns of central and peripheral cortisol remains untested at this stage . further , because all the hair collected in this study was from the posterior vertex of the skull , comparisons with the diurnal fluctuation pattern shown by hair from other body sites was not possible . recent data have shown that hair from different body sites ( arms , legs ) does show significantly different concentrations of cortisol , although those data were collected at a single point in time ( early afternoon),42 and replication of the present study of diurnal fluctuations in cortisol concentrations in hair from different body sites would help clarify these findings . on the basis of these data , further research with a larger sample is suggested to enable the application of group - based statistical procedures that might produce more informative data regarding the overall group mean trend for diurnal fluctuation in hair cortisol concentrations . however , such a study would have to accommodate the findings from the previous literature regarding quite large between - subject variations in the presence , consistency and timing of cortisol diurnal fluctuations . therefore , as well as increasing subject numbers , replications of the current study should also conduct analyses of individual subject variability as has been done previously with such revealing outcomes.8 finally , although several factors which impinge upon cortisol and diurnal fluctuations were mentioned above ( e.g. , few data have been reported as yet regarding the influence that any of those factors might have upon hair cortisol production per se and none upon their effects on diurnal fluctuation of peripheral cortisol in particular , and this remains a fruitful avenue for future research .

ralstonia solanacearum is a vascular wilt pathogen that belongs to the subdivision of the proteobacteria and is one of the most destructive plant pathogens causing bacterial wilt ( bw ) in many crop plants . it has broad host range and infects around 54 plant families and 450 plant species . this pathogen also has a wide geographical distribution ranging from tropical , subtropical , and warm temperate regions of the world . cultivation of eggplant in the coastal state of goa , india , is severely affected by bw leading to 30100% crop loss . cell wall degrading exoenzymes disrupt the cell walls and facilitate its entry in the vascular system . inside the xylem vessels , the bacterial populations rapidly reach very high levels of 10 cells / cm of stem . high cell density and production of high molecular weight exopolysaccharides by r. solanacearum lead to clogging of xylem vessels , wilting , and eventually death of plant . xylem of healthy plants has been reported to be colonized by endophytic xylem residing bacteria ( xrb ) at low population levels and has been isolated from xylem of various crops , namely , citrus , sugar beets , maize , alfalfa , grape [ 11 , 12 ] , and bermuda grass . several endophytic bacteria have been reported to originate from the rhizosphere soil , initially entering the host plant during germination and radicle development , through wounds or by colonizing the cracks formed in lateral root junctions when the endodermis and casparian strips are disrupted thus gaining an easy access to the stele [ 14 , 15 ] . after their initial entry , depending on the endophytic colonization ability , bacteria may remain localized in the roots or colonize intercellular spaces and vascular system and move to the stems . few endophytes have been reported to be able to migrate to aerial plant parts through the vascular system passively with the transpirational flow or through additional assistance by production of cell wall degrading enzymes [ 18 , 19 ] . these systemically migrated endophytes have been isolated from leaves , inflorescence , fruits , and seeds . among the several methods of plant disease management biocontrol agents may be used as an alternative pathogen management strategy or can be combined with other management practices . biological control not only helps in suppressing the disease and increasing crop yield but also has importance in reducing the environmental pollution due to use of chemical pesticides . several studies have shown that endophytic bacteria can be used as biocontrol agents against plant pathogens . the capability of colonizing internal host tissues and ability to produce volatile and diffusible substances which inhibit pathogen , induction of systemic resistance in the plant , and directly or indirectly promoting plant growth have made endophytes a valuable tool in agriculture to improve crop performance . endophytic biocontrol agents isolated from potato , tomato , chilli and eggplant have been used for management of bw . however , the wilt prevention ability of xylem residing bacteria of solanaceous crops that share an ecological niche with the bw pathogen has remained unexplored . this study was undertaken to identify and screen bacteria isolated from the xylem of eggplant , chilli , and s. torvum for their biocontrol activities against r. solanacearum and growth promotion abilities in eggplant . apparently healthy plants were collected from the major vegetable growing locales in north goa and south goa districts of the coastal state of goa , india . eggplant samples were from two different varieties , namely , bw susceptible and bw resistant variety . chilli samples were from the locally grown cultivar , which is moderately susceptible to bw . wild eggplant s. torvum is known to be naturally resistant to bw and was sampled from a field in icar research complex for goa , india . stem pieces of 1315 cm length were surface sterilized by dipping in 0.1% mercuric chloride for 1 min and rinsed several times in sterile water . wash water used for rinsing each surface sterilized stem piece was plated onto tryptic soy agar ( tsa ) ( hi media laboratories , mumbai ) to confirm surface sterilization of each stem piece . xylem sap was extracted by vacuum infiltration as described earlier [ 7 , 28 ] . briefly , after the surface sterilization of stem , one cm piece from each end was discarded . epidermis and cortex from each end were removed and the vascular cylinder was fitted to sterile glass tubing attached in a rubber cork . to the other end of the stem piece a sterile plastic tubing was attached that could hold at least 500 l of 1 x phosphate buffered saline ( pbs ) ( nacl 8 g / l , kcl 0.2 was then fitted onto a buchner flask . for extraction of pbs through the xylem vessels a suction pressure 8 a total of four successive infiltrations using 500 l of pbs were performed for each sample . the sap was collected directly in a sterile test tube placed inside the buchner flask . alternatively , maceration / trituration was performed for isolation of the xrb from young eggplant and chilli samples which had thin and soft stems . the epidermis and cortex from the surface sterilized stem piece the decorticated pieces were macerated in a sterile mortar and pestle using 2 ml of sterile 1x pbs . one hundred l of the vacuum in - filtered sap or macerate was plated onto tsa or medium 523 . different colonies from isolation plates were selected based on differences in their shape , color , and texture and purified onto medium 523 . pure cultures of the xylem residing bacteria ( xrb ) thus obtained were maintained at 80c , as glycerol stocks for long term , and 4c for temporary storage . quality and quantity of the dna were measured using nanodrop 1000 ( thermo scientific , usa ) . 16s rrna gene was amplified using universal primers 27f ( 5-agagtttgatcctggctcag-3 ) and 1492r ( 5-ggttaccttgttacgactt-3 ) . twenty l reaction mix contained 1x pcr buffer , 0.75 units of taq dna polymerase ( sigma aldrich , usa ) , 200 m dntps ( sigma aldrich , usa ) , 0.5m each primer ( chromous biotech , bangalore , india ) , and 50 ng/l of genomic dna . amplifications were carried out on eppendorf mastercycler pro thermal cycler ( eppendorf , germany ) . amplification cycle included a denaturation step of 94c for 5 min followed by 32 cycles of denaturation at 94c for 30s , annealing at 55c for 40s , extension at 72c for 1 min , and a final extension at 72c for 10 min . the amplification of the 1500 bp pcr product was determined by electrophoresis on 0.8% agarose gel . fifteen microliters of the pcr product was digested with one unit of mspi ( thermo scientific , usa ) for 4 h at 37c . restriction fragments were separated on a 2% agarose gel in 1x tris - acetate edta buffer containing 0.5 g / ml ethidium bromide at 60 volts for 2 h. gel was documented using alpha imager ( alpha innotech inc . ardra restriction fingerprints were compared visually and scored manually as 1 for presence and 0 for absence of fragment , and the binary data was entered in the nt edit software version 1.1 b ( applied biostatistics inc . the similarity matrix derived using the binary data of ardra restriction fragment was subjected to cluster analysis using unweighted pair group method for arithmetic average ( upgma ) using dice coefficient in the ntsyspc 2.02i software ( applied biostatistics inc . haplotypes were delineated at 80% similarity values of dice coefficient and numbered as m80 - 1 to m80 - 38 . one hundred and sixty - seven xrb were screened for inhibition of the bw pathogen . r. solanacearum strain rs-09 - 100 was isolated from bw infected eggplant cultivated in goa , india , and was used for screening in vitro and in planta . rs-09 - 100 belongs to phylotype i , race 1 , and biovar 3 of the r. solanacearum species complex . agassaim and causes 100% wilt within 15 days after inoculation under greenhouse conditions ( data not shown ) . briefly , single colony of r. solanacearum and xrb was grown in 5 ml cpg broth ( casein hydrolysate 1.0 g / l , peptone , 10.0 g / l and glucose , 5.0 g / l ) and king 's b broth ( peptone , 20.0 g / l , k2hpo4 , 1.5 g / l and glycerol 10.0 ml / l ) , respectively , at 28 2c for 48 h with constant shaking at 140 rpm . one hundred and fifty microliters of r. solanacearum was seeded every 100 ml molten cooled king 's b agar , mixed well , and poured into plates . after the plates solidified , three wells were made in each plate by removing a circular agar piece with the help of cork borer ( 8 mm diameter ) . twenty - five l of culture broth of xrb containing 8.0 log cfu / ml was added into each of the three wells . all the plates were incubated at 28 2c for 48 h. plates were observed for inhibition of r. solanacearum . zones of inhibition were measured as radius in mm from the edge of the agar well . strains that were found antagonistic to r. solanacearum were screened for in vitro production of antagonistic compounds and plant growth promoting substances and identified by 16s rrna gene sequencing . antagonistic strains were tested for hcn production ability in presence of glycine as described by saraf et al . , a slight modification being the use of broth for the hcn test . immediately after inoculation of strains in king 's b broth containing 4.4 g / l of glycine , sterile filter paper strips dipped in picric acid solution were introduced taking care that the strips did not touch the medium and walls of the tube . the tubes were sealed with parafilm and incubated at 28 2c for 4 days with constant shaking at 140 rpm . the color change of the filter paper strips from yellow to brick red during incubation indicated the production of hcn . to detect ammonia production , g / l ) with constant shaking at 140 rpm for 48 h at 28 2c . ammonia production was determined using nessler 's reagent as described by marques et al . . acetoin production by antagonistic isolates was tested in voges proskauer broth ( peptone 7.0 g / l , k2hpo4 5.0 g / l , dextrose 5.0 g / l ph 7.0 ) . after incubation for 30 h at 28 2c at 140 rpm , one ml each of 5% napthol and 40% koh were added to the culture and mixed well . antagonistic xrb were tested for siderophore production on a medium containing chrome azurol s ( cas ) . isolates producing orange haloes on the blue green colored medium after incubation at 28 2c for 48 h were positive for siderophore production . antagonistic strains were tested for their ability to produce phytohormone iaa in presence of tryptophan as described by gordon and paleg . l of tryptophan for 30 h at 28 2c at 140 rpm . one ml of supernatant was mixed with one ml of salkowsky 's reagent ( 50 ml 35% perchloric acid , 1 ml 0.5 m fecl3 ) . the mixture was allowed to stand at room temperature for five minutes and the absorbance was read at 530 nm . a standard curve was prepared using analytical grade iaa and the concentrations of iaa in the culture supernatants of xrb were estimated based on the curve . antagonistic strains were tested for their ability to produce enzyme acc deaminase as per the method described by godinho et al . . strains were streaked on dworkin and foster 's df salts agar containing 3.0 mm acc and incubated at 28 2c for 7 days . ability of the strains to grow on the medium containing acc as a sole nitrogen source was indicative of acc deaminase production . antagonistic strains were tested for phosphate solubilization by a method described by godinho et al . . all strains were spot inoculated on pikovskaya 's agar plates ( hi media laboratories , mumbai ) . plates were incubated at 28 2c for 48 h. transparent zones around the growth of xrb on the opaque white medium were indicative of solubilisation phosphate . twenty - eight strains of xrb were selected based on in vitro inhibition of r. solanacearum in the agar well bioassay . thirty - day - old seedlings raised in nonsterile soil in greenhouse were transplanted in pots filled with standard nonsterile pot mixture ( soil : sand : farmyard manure at 2 : 1 : 1 ratio ) . ten ml suspension of antagonistic xrb ( 8.0 log cfu / ml ) in sterile 1 x pbs was applied per seedling by soil drenching . each treatment consisted of two replicates with two pots per replication and five seedlings per pot . twenty days after treatment with the antagonistic xrb the seedlings were challenged by inoculating 10 ml suspension of r. solanacearum strain rs-09 - 100 ( 7.0 log cfu / ml ) by soil drenching . plants not treated with xrb , but challenged with r. solanacearum , served as control . plants were maintained with suitable watering and percentage of plants infected by wilt was noted until 25 days after challenging with r. solanacearum . ability of the xrb to prevent wilt in eggplant was expressed as biocontrol efficacy ( bce ) and was determined using the formula bce = ( [ percent disease in control ] [ percent disease in treatment]/percent disease in control ) 100 . strains with bce greater than 25% were evaluated for their effect on growth in eggplant under greenhouse conditions . sixteen strains of xrb exhibiting biocontrol efficacies greater than 25% were studied for their effect on growth in eggplant . agassaim was used as a measure to evaluate their growth promotion efficacy under greenhouse conditions . thirty - day - old seedlings raised in nonsterile soil in greenhouse were transplanted in pots filled with standard nonsterile pot mixture ( soil : sand : farmyard manure at 2 : 1 : 1 ratio ) . ten ml suspension of xrb ( 8.0 log cfu / ml ) in sterile 1 x pbs was applied per seedling by soil drenching . each treatment consisted of two replicates with two pots per replication and five seedlings per pot . plants were maintained with suitable watering and plant height was measured from the soil level to the shoot tip 40 days postinoculation . ability of antagonistic xrb to increase shoot length in eggplant was expressed as growth promotion efficacy ( gpe ) using the formula ( [ shoot length increase in treatment ] [ shoot length increase in control]/shoot length increase in control ) 100 . representative strains from each of the 38 ardra haplotypes and xrb exhibiting antagonism to r. solanacearum were selected for identification . fragments of the 16s rrna gene of size 1500 bp were amplified as described above in the ardra section . amplicons were purified using genejet pcr purification kit ( thermo scientific , usa ) and sequenced using 27f and 1492r primers ( xcelris labs pvt . ltd . , partial 16s rrna gene sequences ( about 1200 nt ) obtained were matched against the sequences available in the nucleotide database from national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/blast ) using the blastn ( basic local alignment search tool ) program . in this study , bacteria could be constantly isolated from the xylem of eggplant , chilli , and s. torvum by vacuum infiltration and maceration techniques . bacterial counts from each isolation ranged from 10 to 10 cfu / ml of xylem sap or macerate . colonies appeared between 24 and 120 h of aerobic incubation at 28 2c . amongst 167 isolates obtained , 99 were gram - negative rods ( 59.28% ) and 68 were gram - positive bacteria ( 40.72% ) comprising of 42 rods ( 61.76% ) , 25 cocci ( 36.76% ) , and one filamentous actinomycete ( table 1 ) . ardra generated three to six restriction fragments of the 16s rrna gene amplified from the xrb . analysis of ardra profiles by upgma using dice 's coefficient divided xrb with identical restriction profiles into several groups . at 80% similarity values of dice coefficient host based analysis of ardra revealed that 89 strains isolated from bw susceptible eggplant were grouped in 31 haplotypes , 36 strains from bw resistant eggplant into 17 haplotypes , 33 strains from chilli into 19 haplotypes , and 9 strains from s. torvum into 7 haplotypes , respectively ( table 1 ) . a detailed representation of the ardra based analysis of 167 strains is presented in table 2 . based on the ardra analysis of the collection of xrb , 153 strains were distributed over 24 different haplotypes and 14 xrb strains had unique profiles which formed 14 independent haplotypes with one strain in each . antagonistic strains ( n = 28 ) were distributed over 14 different ardra haplotypes wherein 6 antagonists formed independent haplotypes . haplotypes m80 - 9 and m80 - 15 were shared amongst bw susceptible and resistant eggplant , chilli , and s. torvum . haplotypes m80 - 1 , m8 - 12 , and m80 - 38 were unique to bw resistant eggplant whereas haplotypes m80 - 13 , m80 - 19 , and m80 - 26 comprised of strains isolated from chilli . these results indicate that bacterial communities from the xylem of mainly eggplant and chilli cultivated in different locations in goa comprise of diverse bacteria . however it is observed that each ardra group consists of bacteria from different plant species and plants collected from different locales . in addition there are certain xrb unshared between each of the plant species that form unique haplotypes . moreover , strains with biocontrol ability ( bce > 25% ) were restricted to only 8 haplotypes , namely , m80 - 6 , m80 - 7 , m80 - 10 , m80 - 15 , m80 - 20 , m80 - 29 , m80 - 31 , and m80 - 36 ( table 2 ) . haplotypes m80 - 6 , m80 - 7 , m80 - 29 , m80 - 31 , m80 - 36 , and m80 - 38 comprised of xrb with gpe > 10% . plate based bioassay was used for rapid screening of antagonism of xrb towards r. solanacearum strain rs-09 - 100 . results of the in vitro screening against r. solanacearum revealed that 28 amongst 167 xrb exhibited antagonism towards the pathogen ( table 3 ) . amongst the antagonists , 16 were strains from bw susceptible eggplant , 6 from bw resistant eggplant , and 3 each from chilli and s. torvum ( table 1 ) . amongst the 28 antagonists , 7 strains , namely , xb62 , xb99 , xb100 , xb114 , xb122 , xb196 , xb197 , and xb202 formed larger inhibition zones against r. solanacearum ranging from 4.0 mm to 8.17 mm ( table 3 ) . the majority of the antagonistic strains ( n = 16 ) produced inhibition zones ranging from 2.0 mm to 3.83 mm . xb27 , xb134 , xb165 , and xb169 formed smaller inhibition zones ranging from 1.5 mm to 2.0 mm . however , 139 strains of xrb did not inhibit r. solanacearum in the bioassay test . twenty - eight antagonistic xrb were screened for production of volatile inhibitory compounds , namely , acetoin , hcn , ammonia , and diffusible siderophore molecules in vitro ( table 3 ) . bacterial isolates xb7 and xb122 were found to produce both hcn as well as siderophores . xb62 , xb93 , and xb170 produced hcn whereas xb114 , xb140 , and xb203 produced siderophores only . results of the screening of antagonistic xrb for in vitro production of several plant growth promoting compounds is presented in table 3 . majority of the antagonistic strains produced the phytohormone iaa with concentrations ranging from 15.91 g / ml to 645.91 g / ml . xb202 was found to be the best acc deaminase producing strain based on its luxuriant growth on df salts medium supplemented with 3.0 mm acc as sole nitrogen source . 64.28% of the strains produced phosphate solubilizing organic acids as indicated by clear haloes on pikovskaya 's agar plate . ability to suppress bw was assessed as the difference in the percentage of wilt in xrb treated plants with respect to wilt in untreated control and was expressed as the biocontrol efficacy ( bce ) of the antagonists . bce of 16 strains with values ranging from 28.6 to 100% is presented in figure 1 . plants treated with strains xb86 , xb169 , and xb177 were free from bw and hence recorded 100% biocontrol efficacy . treatments with xb170 , xb197 , xb200 , xb202 , and xb203 recorded 30 percent or less wilt incidence ( 70% to 85% bce ) . xb1 , xb27 , xb70 , xb93 , and xb123 treatments recorded bce between 42.9 and 57.1% . bce of 28.6% was recorded in xb20 and xb165 treatments . however , 12 antagonistic xrb recorded bce of 25% and were least effective in wilt protection . further it is observed that all the antagonistic strains originating from bw resistant eggplant and s. torvum exhibited bce greater than 25% . five antagonistic strains from bw susceptible eggplant and three from chilli were effective in preventing wilt in eggplant ( table 1 ) . increase in shoot length of eggplant ( 40 days after treatment ) observed in xrb treated plants in relation to untreated control was expressed as growth promotion efficacy ( gpe ) and is shown in figure 1 . amongst the 16 strains which were effective in preventing wilt , six strains exhibited the highest increase in shoot length as indicated by their gpe values in the range of 13.922.3% . however , strains xb27 , xb196 , and xb203 stunted shoot growth in eggplant , in comparison to untreated control . when the source of xrb is considered , 55.55% strains that exhibited gpe greater than 10% were isolated from bw resistant eggplant . whereas , only two antagonists from bw susceptible plant and one each from chilli and s. torvum were able to promote growth in eggplant ( table 1 ) . strains with gpe > 10% belonged to six different ardra haplotypes ( table 2 ) . 16s rrna gene sequences of xrb were used to identify the diverse xylem inhabitants and antagonistic strains . identity of 55 xrb based on 16s rrna gene sequencing and their genbank accessions are presented in table 4 . based on the identification , phylum proteobacteria consisting of gram - negative bacteria of subdivisions alpha proteobacteria ( 12.73% ) , beta proteobacteria ( 3.64% ) , and gamma proteobacteria ( 25.45% ) were predominant ( 41.81% strains identified ) , followed by phyla firmicutes ( 29.09% ) , actinobacteria ( 25.45% ) , and bacteroidetes ( 3.64% ) ( figure 2(a ) ) . eleven antagonistic strains identified belonged to gamma subdivision of proteobacteria consisting of five strainseach of enterobacter sp . and fluorescent and nonfluorescent pseudomonas sp . and one strain of pantoea eucrina ( xb126 ) ( figure 2(b ) ) . ( xb200 ) , and janibacter melonis ( xb70 ) of phyla actinobacteria were found to be antagonistic . the statistical analysis of percentage wilts and shoot length of eggplant was performed using web agri statistical package ( wasp ) version 2.0 ( http://www.icargoa.res.in/wasp2.0/index/php ) . eggplant and chilli not only are of economic and cultural importance but also are common ingredients in the cuisine throughout india . in the coastal state of goa , r. solanacearum has been reported to be a destructive pathogen in cultivation of eggplant and chilli . isolation of biocontrol agents against the bw pathogen has been commonly restricted to endophytic tissue and plant rhizosphere [ 26 , 27 ] . studies on xylem colonizing endophytes were undertaken because we speculated existence of interactions between the xrb and vascular wilt pathogen r. solanacearum during xylem colonization . our study reveals the diversity , biocontrol potential , and identity of endophytic xylem colonizers from solanaceous crops cultivated in goa , india . a total of 167 bacteria were isolated from the xylem of eggplant , chilli , and s. torvum with gram - negative bacteria ( 59.28% ) predominating in the collection . congruent to our observation , gardner et al . and bell et al . have earlier reported isolation of more number of gram - negative rod shaped bacteria from xylem of citrus and grapevine using vacuum infiltration . scholander pressure bomb was found to be useful in extraction of diverse bacterial genera from xylem tissues in contrast to trituration methods that yielded higher number of gram - positive rod shaped bacteria . though , scholander pressure bomb was not used in this study , a combination of vacuum infiltration [ 7 , 11 ] and trituration of decorticated stems [ 10 , 12 ] was employed with an aim to isolate diverse xrb from xylem tissues . this is the first study reporting the use of vacuum infiltration and trituration methods for isolating xylem residing bacteria from eggplant , chilli , and s. torvum . traditionally bacteria have been characterized and grouped based on colony morphology and biochemical tests . however , whole genome fingerprinting or pcr - rflp based methods are rapid tools for determining genetic diversity of bacteria in a given collection . ardra which is a type of pcr based rflp method has been used widely in estimating genetic diversity endophytic bacterial populations and clustering genetically similar strains [ 3941 ] . in addition to earlier reports , our study demonstrates the usefulness of ardra as a tool to cluster genetically identical strains of endophytic xrb isolated from eggplant , chilli , and s. torvum . in our study 91.61% strains ( n = 153 ) were grouped in 24 haplotypes by using ardra . the majority of these haplotypes represent a combination of xrb isolated from different solanaceous plants from diverse locales . these results indicate that xylem of eggplant , chilli , and s. torvum largely bears similar population of xrb which can efficiently cross colonize eggplant , chilli , or s. torvum . this observation leads to a conclusion that a minor population of xylem inhabitants are restricted to a specific plant species and can not easily cross colonize xylem of other solanaceous plants . however , factors that determine the selection of xylem colonists or the ability of xrb to colonize eggplant , chilli , and s. torvum in this study remain unknown . interestingly , the structure of endophytic community in nicotiana attenuata a member of solanaceae family is shown to be influenced by soil composition and ethylene homeostasis . earlier evidence has shown that colonization by endophytic bacteria is also governed by plant genotype as well as root exudates . only 16.77% xrb out of 167 were antagonistic to r. solanacearum based on in vitro assays . antagonistic xrb produced volatile and diffusible inhibitory compounds , namely , hcn , ammonia , and acetoin and siderophores . these substances have been long known to be involved in disease suppression and indirect growth promotion in plants [ 4346 ] . these mechanisms possibly played a role in the evident biocontrol effect against bw exhibited by the xrb in the greenhouse screening . endophytic bacteria have been known to have plant growth promoting traits , namely , production of iaa , acc deaminase , and phosphate solubilization [ 25 , 33 , 36 ] . these traits were detected in the majority of antagonistic xrb tested in this study and may have resulted in the observed increase in shoot length of eggplant in our greenhouse experiments . in contrast , strains xb20 , xb196 , and xb203 suppressed growth in eggplant under greenhouse conditions ; however no visible symptoms of disease were observed . vascular plugging and production of certain metabolites toxic to plant cells , but not cell viability , may have resulted in stunted shoot in eggplant [ 11 , 47 ] . evaluation of efficacy of antagonistic organisms to suppress the plant diseases under greenhouse conditions is one of the key steps for selecting a potential biocontrol agent for disease management . endophytic strains from bw susceptible varieties of eggplant have been shown to prevent wilt and promote growth in eggplant earlier . our greenhouse screening shows that 38.46% of antagonistic xrb with biocontrol efficacies greater than 40% were isolates from bw resistant varieties of eggplant . this raises a question whether the bacteria from resistant varieties are involved in bw resistance and whether bw resistant varieties are able to selectively influence xylem colonization by antagonistic bacteria ? presence of higher number of endophytes with antagonistic abilities was reported in bw resistant varieties of tomato as compared to susceptible varieties , and their role in resistance to bw was proposed . similar observations on correlation of resistance of potato to soft rot and endophytic bacteria have been reported . thus bw resistant varieties can be considered a better host for isolating potential biocontrol strains for management of bacterial wilt . identification of 55 xrb strains by 16s rrna gene sequencing revealed the presence of 23 diverse genera of bacteria belonging to 4 phyla of eubacteria . strains belonging to phyla firmicutes , actinobacteria , and subdivision of proteobacteria were the major xylem colonists identified in this study . several genera of bacteria belonging to these phyla have also been reported to be present in endophytic tissues and xylem of a variety of other agricultural and horticultural plant species [ 14 , 51 ] . in addition , the majority of the antagonists identified belonged to enterobacter sp . , pseudomonas sp . congruent to our results , several researchers have reported bacteria isolated from solanaceous crops and belonging to similar genera to be antagonistic to r. solanacearum [ 26 , 5153 ] . however , flavobacterium sp.and janibacter melonis identified in this study have never been previously reported to be inhibitory to r. solanacearum . large population of the xylem inhabiting bacterial flora accounting for 83.23% exhibited no antagonism towards r. solanacearum . nonantagonistic xrb were identified predominantly as microbacterium sp . endophytic persistence and nematicidal activities of microbacterium sp . therefore the collection of xrb isolated in this study can be screened for inhibitory activities against other important agricultural pests . though few strains , namely , xb86 , xb169 , and xb177 exhibited plant beneficial properties in this study their usefulness in plant disease control remains to be seen . xb86 has been identified as agrobacterium tumefaciens , the crown gall disease pathogen , and its deployment as biocontrol agent is uncertain . xb169 ( staphylococcus gallinarum ) and xb177 ( bacillus cereus ) are reported as opportunistic animal pathogens and thus unsuitable for field applications . has earlier been reported as antagonistic to r. solanacearum and tested for management of wilt in potato and tomato [ 56 , 57 ] . is one of the xrb high bce and gpe ; it could be explored further for biocontrol of bacterial wilt after additional characterization and field evaluation . this study is the first report on the identity of novel and diverse xrb colonizing the xylem of eggplant , chilli , and s. torvum . xrb particularly from bw resistant varieties were found to protect eggplant from bacterial wilt and enhanced growth in eggplant in the greenhouse screening . therefore the repertoire of xrb reported in this study may be useful for cultivation of eggplant in bw affected areas .

eggplant ( solanum melongena l. ) is one of the solanaceous crops of economic and cultural importance and is widely cultivated in the state of goa , india . eggplant cultivation is severely affected by bacterial wilt caused by ralstonia solanacearum that colonizes the xylem tissue . in this study , 167 bacteria were isolated from the xylem of healthy eggplant , chilli , and solanum torvum sw . by vacuum infiltration and maceration . amplified rdna restriction analysis ( ardra ) grouped these xylem residing bacteria ( xrb ) into 38 haplotypes . twenty - eight strains inhibited growth of r. solanacearum and produced volatile and diffusible antagonistic compounds and plant growth promoting substances in vitro . antagonistic strains xb86 , xb169 , xb177 , and xb200 recorded a biocontrol efficacy greater than 85% against bw and exhibited 12%22 % increase in shoot length in eggplant in the greenhouse screening . 16s rrna based identification revealed the presence of 23 different bacterial genera . xrb with high biocontrol and plant growth promoting activities were identified as strains of staphylococcus sp . , bacillus sp . , streptomyces sp . , enterobacter sp . , and agrobacterium sp . this study is the first report on identity of bacteria from the xylem of solanaceous crops having traits useful in cultivation of eggplant .

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

ralstonia solanacearum is a vascular wilt pathogen that belongs to the subdivision of the proteobacteria and is one of the most destructive plant pathogens causing bacterial wilt ( bw ) in many crop plants . this pathogen also has a wide geographical distribution ranging from tropical , subtropical , and warm temperate regions of the world . cultivation of eggplant in the coastal state of goa , india , is severely affected by bw leading to 30100% crop loss . high cell density and production of high molecular weight exopolysaccharides by r. solanacearum lead to clogging of xylem vessels , wilting , and eventually death of plant . xylem of healthy plants has been reported to be colonized by endophytic xylem residing bacteria ( xrb ) at low population levels and has been isolated from xylem of various crops , namely , citrus , sugar beets , maize , alfalfa , grape [ 11 , 12 ] , and bermuda grass . these systemically migrated endophytes have been isolated from leaves , inflorescence , fruits , and seeds . the capability of colonizing internal host tissues and ability to produce volatile and diffusible substances which inhibit pathogen , induction of systemic resistance in the plant , and directly or indirectly promoting plant growth have made endophytes a valuable tool in agriculture to improve crop performance . endophytic biocontrol agents isolated from potato , tomato , chilli and eggplant have been used for management of bw . however , the wilt prevention ability of xylem residing bacteria of solanaceous crops that share an ecological niche with the bw pathogen has remained unexplored . this study was undertaken to identify and screen bacteria isolated from the xylem of eggplant , chilli , and s. torvum for their biocontrol activities against r. solanacearum and growth promotion abilities in eggplant . apparently healthy plants were collected from the major vegetable growing locales in north goa and south goa districts of the coastal state of goa , india . wild eggplant s. torvum is known to be naturally resistant to bw and was sampled from a field in icar research complex for goa , india . xylem sap was extracted by vacuum infiltration as described earlier [ 7 , 28 ] . different colonies from isolation plates were selected based on differences in their shape , color , and texture and purified onto medium 523 . pure cultures of the xylem residing bacteria ( xrb ) thus obtained were maintained at 80c , as glycerol stocks for long term , and 4c for temporary storage . twenty l reaction mix contained 1x pcr buffer , 0.75 units of taq dna polymerase ( sigma aldrich , usa ) , 200 m dntps ( sigma aldrich , usa ) , 0.5m each primer ( chromous biotech , bangalore , india ) , and 50 ng/l of genomic dna . amplification cycle included a denaturation step of 94c for 5 min followed by 32 cycles of denaturation at 94c for 30s , annealing at 55c for 40s , extension at 72c for 1 min , and a final extension at 72c for 10 min . ardra restriction fingerprints were compared visually and scored manually as 1 for presence and 0 for absence of fragment , and the binary data was entered in the nt edit software version 1.1 b ( applied biostatistics inc . r. solanacearum strain rs-09 - 100 was isolated from bw infected eggplant cultivated in goa , india , and was used for screening in vitro and in planta . rs-09 - 100 belongs to phylotype i , race 1 , and biovar 3 of the r. solanacearum species complex . briefly , single colony of r. solanacearum and xrb was grown in 5 ml cpg broth ( casein hydrolysate 1.0 g / l , peptone , 10.0 g / l and glucose , 5.0 g / l ) and king 's b broth ( peptone , 20.0 g / l , k2hpo4 , 1.5 g / l and glycerol 10.0 ml / l ) , respectively , at 28 2c for 48 h with constant shaking at 140 rpm . one hundred and fifty microliters of r. solanacearum was seeded every 100 ml molten cooled king 's b agar , mixed well , and poured into plates . twenty - five l of culture broth of xrb containing 8.0 log cfu / ml was added into each of the three wells . all the plates were incubated at 28 2c for 48 h. plates were observed for inhibition of r. solanacearum . zones of inhibition were measured as radius in mm from the edge of the agar well . strains that were found antagonistic to r. solanacearum were screened for in vitro production of antagonistic compounds and plant growth promoting substances and identified by 16s rrna gene sequencing . antagonistic strains were tested for hcn production ability in presence of glycine as described by saraf et al . to detect ammonia production , g / l ) with constant shaking at 140 rpm for 48 h at 28 2c . antagonistic strains were tested for their ability to produce phytohormone iaa in presence of tryptophan as described by gordon and paleg . a standard curve was prepared using analytical grade iaa and the concentrations of iaa in the culture supernatants of xrb were estimated based on the curve . antagonistic strains were tested for their ability to produce enzyme acc deaminase as per the method described by godinho et al . twenty - eight strains of xrb were selected based on in vitro inhibition of r. solanacearum in the agar well bioassay . twenty days after treatment with the antagonistic xrb the seedlings were challenged by inoculating 10 ml suspension of r. solanacearum strain rs-09 - 100 ( 7.0 log cfu / ml ) by soil drenching . ability of the xrb to prevent wilt in eggplant was expressed as biocontrol efficacy ( bce ) and was determined using the formula bce = ( [ percent disease in control ] [ percent disease in treatment]/percent disease in control ) 100 . strains with bce greater than 25% were evaluated for their effect on growth in eggplant under greenhouse conditions . sixteen strains of xrb exhibiting biocontrol efficacies greater than 25% were studied for their effect on growth in eggplant . plants were maintained with suitable watering and plant height was measured from the soil level to the shoot tip 40 days postinoculation . ability of antagonistic xrb to increase shoot length in eggplant was expressed as growth promotion efficacy ( gpe ) using the formula ( [ shoot length increase in treatment ] [ shoot length increase in control]/shoot length increase in control ) 100 . representative strains from each of the 38 ardra haplotypes and xrb exhibiting antagonism to r. solanacearum were selected for identification . fragments of the 16s rrna gene of size 1500 bp were amplified as described above in the ardra section . , partial 16s rrna gene sequences ( about 1200 nt ) obtained were matched against the sequences available in the nucleotide database from national center for biotechnology information ( http://www.ncbi.nlm.nih.gov/blast ) using the blastn ( basic local alignment search tool ) program . in this study , bacteria could be constantly isolated from the xylem of eggplant , chilli , and s. torvum by vacuum infiltration and maceration techniques . amongst 167 isolates obtained , 99 were gram - negative rods ( 59.28% ) and 68 were gram - positive bacteria ( 40.72% ) comprising of 42 rods ( 61.76% ) , 25 cocci ( 36.76% ) , and one filamentous actinomycete ( table 1 ) . ardra generated three to six restriction fragments of the 16s rrna gene amplified from the xrb . analysis of ardra profiles by upgma using dice 's coefficient divided xrb with identical restriction profiles into several groups . at 80% similarity values of dice coefficient host based analysis of ardra revealed that 89 strains isolated from bw susceptible eggplant were grouped in 31 haplotypes , 36 strains from bw resistant eggplant into 17 haplotypes , 33 strains from chilli into 19 haplotypes , and 9 strains from s. torvum into 7 haplotypes , respectively ( table 1 ) . haplotypes m80 - 9 and m80 - 15 were shared amongst bw susceptible and resistant eggplant , chilli , and s. torvum . haplotypes m80 - 1 , m8 - 12 , and m80 - 38 were unique to bw resistant eggplant whereas haplotypes m80 - 13 , m80 - 19 , and m80 - 26 comprised of strains isolated from chilli . these results indicate that bacterial communities from the xylem of mainly eggplant and chilli cultivated in different locations in goa comprise of diverse bacteria . however it is observed that each ardra group consists of bacteria from different plant species and plants collected from different locales . haplotypes m80 - 6 , m80 - 7 , m80 - 29 , m80 - 31 , m80 - 36 , and m80 - 38 comprised of xrb with gpe > 10% . results of the in vitro screening against r. solanacearum revealed that 28 amongst 167 xrb exhibited antagonism towards the pathogen ( table 3 ) . amongst the antagonists , 16 were strains from bw susceptible eggplant , 6 from bw resistant eggplant , and 3 each from chilli and s. torvum ( table 1 ) . amongst the 28 antagonists , 7 strains , namely , xb62 , xb99 , xb100 , xb114 , xb122 , xb196 , xb197 , and xb202 formed larger inhibition zones against r. solanacearum ranging from 4.0 mm to 8.17 mm ( table 3 ) . the majority of the antagonistic strains ( n = 16 ) produced inhibition zones ranging from 2.0 mm to 3.83 mm . xb27 , xb134 , xb165 , and xb169 formed smaller inhibition zones ranging from 1.5 mm to 2.0 mm . however , 139 strains of xrb did not inhibit r. solanacearum in the bioassay test . twenty - eight antagonistic xrb were screened for production of volatile inhibitory compounds , namely , acetoin , hcn , ammonia , and diffusible siderophore molecules in vitro ( table 3 ) . results of the screening of antagonistic xrb for in vitro production of several plant growth promoting compounds is presented in table 3 . majority of the antagonistic strains produced the phytohormone iaa with concentrations ranging from 15.91 g / ml to 645.91 g / ml . ability to suppress bw was assessed as the difference in the percentage of wilt in xrb treated plants with respect to wilt in untreated control and was expressed as the biocontrol efficacy ( bce ) of the antagonists . plants treated with strains xb86 , xb169 , and xb177 were free from bw and hence recorded 100% biocontrol efficacy . further it is observed that all the antagonistic strains originating from bw resistant eggplant and s. torvum exhibited bce greater than 25% . five antagonistic strains from bw susceptible eggplant and three from chilli were effective in preventing wilt in eggplant ( table 1 ) . increase in shoot length of eggplant ( 40 days after treatment ) observed in xrb treated plants in relation to untreated control was expressed as growth promotion efficacy ( gpe ) and is shown in figure 1 . amongst the 16 strains which were effective in preventing wilt , six strains exhibited the highest increase in shoot length as indicated by their gpe values in the range of 13.922.3% . however , strains xb27 , xb196 , and xb203 stunted shoot growth in eggplant , in comparison to untreated control . when the source of xrb is considered , 55.55% strains that exhibited gpe greater than 10% were isolated from bw resistant eggplant . whereas , only two antagonists from bw susceptible plant and one each from chilli and s. torvum were able to promote growth in eggplant ( table 1 ) . 16s rrna gene sequences of xrb were used to identify the diverse xylem inhabitants and antagonistic strains . identity of 55 xrb based on 16s rrna gene sequencing and their genbank accessions are presented in table 4 . eleven antagonistic strains identified belonged to gamma subdivision of proteobacteria consisting of five strainseach of enterobacter sp . the statistical analysis of percentage wilts and shoot length of eggplant was performed using web agri statistical package ( wasp ) version 2.0 ( http://www.icargoa.res.in/wasp2.0/index/php ) . eggplant and chilli not only are of economic and cultural importance but also are common ingredients in the cuisine throughout india . in the coastal state of goa , r. solanacearum has been reported to be a destructive pathogen in cultivation of eggplant and chilli . isolation of biocontrol agents against the bw pathogen has been commonly restricted to endophytic tissue and plant rhizosphere [ 26 , 27 ] . our study reveals the diversity , biocontrol potential , and identity of endophytic xylem colonizers from solanaceous crops cultivated in goa , india . a total of 167 bacteria were isolated from the xylem of eggplant , chilli , and s. torvum with gram - negative bacteria ( 59.28% ) predominating in the collection . have earlier reported isolation of more number of gram - negative rod shaped bacteria from xylem of citrus and grapevine using vacuum infiltration . scholander pressure bomb was found to be useful in extraction of diverse bacterial genera from xylem tissues in contrast to trituration methods that yielded higher number of gram - positive rod shaped bacteria . though , scholander pressure bomb was not used in this study , a combination of vacuum infiltration [ 7 , 11 ] and trituration of decorticated stems [ 10 , 12 ] was employed with an aim to isolate diverse xrb from xylem tissues . this is the first study reporting the use of vacuum infiltration and trituration methods for isolating xylem residing bacteria from eggplant , chilli , and s. torvum . in addition to earlier reports , our study demonstrates the usefulness of ardra as a tool to cluster genetically identical strains of endophytic xrb isolated from eggplant , chilli , and s. torvum . these results indicate that xylem of eggplant , chilli , and s. torvum largely bears similar population of xrb which can efficiently cross colonize eggplant , chilli , or s. torvum . however , factors that determine the selection of xylem colonists or the ability of xrb to colonize eggplant , chilli , and s. torvum in this study remain unknown . only 16.77% xrb out of 167 were antagonistic to r. solanacearum based on in vitro assays . antagonistic xrb produced volatile and diffusible inhibitory compounds , namely , hcn , ammonia , and acetoin and siderophores . these mechanisms possibly played a role in the evident biocontrol effect against bw exhibited by the xrb in the greenhouse screening . endophytic bacteria have been known to have plant growth promoting traits , namely , production of iaa , acc deaminase , and phosphate solubilization [ 25 , 33 , 36 ] . these traits were detected in the majority of antagonistic xrb tested in this study and may have resulted in the observed increase in shoot length of eggplant in our greenhouse experiments . in contrast , strains xb20 , xb196 , and xb203 suppressed growth in eggplant under greenhouse conditions ; however no visible symptoms of disease were observed . evaluation of efficacy of antagonistic organisms to suppress the plant diseases under greenhouse conditions is one of the key steps for selecting a potential biocontrol agent for disease management . endophytic strains from bw susceptible varieties of eggplant have been shown to prevent wilt and promote growth in eggplant earlier . our greenhouse screening shows that 38.46% of antagonistic xrb with biocontrol efficacies greater than 40% were isolates from bw resistant varieties of eggplant . presence of higher number of endophytes with antagonistic abilities was reported in bw resistant varieties of tomato as compared to susceptible varieties , and their role in resistance to bw was proposed . identification of 55 xrb strains by 16s rrna gene sequencing revealed the presence of 23 diverse genera of bacteria belonging to 4 phyla of eubacteria . strains belonging to phyla firmicutes , actinobacteria , and subdivision of proteobacteria were the major xylem colonists identified in this study . several genera of bacteria belonging to these phyla have also been reported to be present in endophytic tissues and xylem of a variety of other agricultural and horticultural plant species [ 14 , 51 ] . in addition , the majority of the antagonists identified belonged to enterobacter sp . congruent to our results , several researchers have reported bacteria isolated from solanaceous crops and belonging to similar genera to be antagonistic to r. solanacearum [ 26 , 5153 ] . however , flavobacterium sp.and janibacter melonis identified in this study have never been previously reported to be inhibitory to r. solanacearum . large population of the xylem inhabiting bacterial flora accounting for 83.23% exhibited no antagonism towards r. solanacearum . therefore the collection of xrb isolated in this study can be screened for inhibitory activities against other important agricultural pests . though few strains , namely , xb86 , xb169 , and xb177 exhibited plant beneficial properties in this study their usefulness in plant disease control remains to be seen . xb86 has been identified as agrobacterium tumefaciens , the crown gall disease pathogen , and its deployment as biocontrol agent is uncertain . has earlier been reported as antagonistic to r. solanacearum and tested for management of wilt in potato and tomato [ 56 , 57 ] . is one of the xrb high bce and gpe ; it could be explored further for biocontrol of bacterial wilt after additional characterization and field evaluation . this study is the first report on the identity of novel and diverse xrb colonizing the xylem of eggplant , chilli , and s. torvum . xrb particularly from bw resistant varieties were found to protect eggplant from bacterial wilt and enhanced growth in eggplant in the greenhouse screening . therefore the repertoire of xrb reported in this study may be useful for cultivation of eggplant in bw affected areas .

, blood pressure is measured using a sphygmomanometer ( manual or automatic ) over the brachial artery . the vast majority of the evidence supporting the benefits of blood pressure reduction is based on brachial blood pressure measurements . over the last 10 years , several antihypertensive drug treatment trials have shown an unexpected discrepancy between reduction in brachial blood pressure and observed clinical outcomes . in the heart outcomes prevention evaluation ( hope ) , the losartan intervention for endpoint reduction ( life ) in hypertension , and the australian national blood pressure 2 ( anbp2 ) trials , the observed clinical benefits tended to be greater than those expected from the decrease in brachial blood pressure . this may be explained by the pleiotropic effects of the antihypertensive drugs used in these trials . in particular , the beneficial effects of these agents on endothelial function and its vascular manifestations , such as changes in central aortic pressure and atheroma development . it has been proposed that central aortic pressure may have more pathophysiological relevance than peripheral blood pressure as a marker for the development of cardiovascular disease [ 6 , 7 ] . central aortic pressure is determined by the combination of cardiac output and peripheral vascular resistance that is modulated by arterial stiffness along with the timing and magnitude of pressure wave reflections in the arterial tree . the pressure wave generated by the left ventricle during cardiac systole travels through the vessels until it reaches the small muscular arteries and arterioles where it is reflected . the pressure waveform at any point in the arterial tree is therefore the sum of both forward and backward waveforms . when the large arteries are healthy and compliant , the reflected wave merges with the forward wave in the proximal aorta during diastole , augmenting diastolic blood pressure and aiding coronary perfusion . however , when the large arteries are stiff , pulse wave velocity increases , accelerating both incident and reflected waves . this results in the reflected wave merging with the incident wave in systole , thus augmenting central aortic systolic rather than diastolic pressure . thus , central aortic stiffness contributes directly to a wide pulse pressure with higher systolic and lower diastolic blood pressure . furthermore , the state of the vasculature in the peripheral circulation also affects the proportion of the incident wave that is reflected , thus affecting central pressure . observational studies have shown that the difference between brachial and central arterial pressures can vary by between 2 and 33 mm hg [ 9 , 10 ] . furthermore , different antihypertensive drugs have been shown to have similar effects on brachial blood pressure , but different effects on central aortic pressure [ 6 , 1113 ] . this may , in part , explain why central aortic pressure has been shown to have superior prognostic value with respect to cardiovascular events than brachial blood pressure in clinical studies [ 6 , 1113 ] . the conduit artery function evaluation ( cafe ) study [ 6 ] , was a substudy of the anglo - scandinavian cardiac outcomes trial ( ascot ) . in ascot , patients with hypertension and at least three additional cardiovascular risk factors were randomized to an atenolol plus bendroflumethiazide - based treatment regimen or an amlodipine plus perindopril - based regimen . the cafe study recruited 2,199 ascot patients after the first year of ascot follow - up ( mean age of the cohort at baseline , 63 years ) and followed them for up to 4 years . radial artery applanation tonometry and pulse wave analysis were used to derive central aortic pressures and hemodynamic indices . throughout follow up , derived central aortic systolic pressure was substantially lower in the amlodipine - based treatment group compared with the atenolol - based treatment group ( area under the curve [ auc ] difference , 4.3 mm hg ; 95 % confidence interval [ ci ] , 3.3 to 5.4 ; p < 0.0001 ) , whereas brachial systolic blood pressure was similar between the two treatment arms ( auc difference , 0.7 mm hg ; 95 % ci , 0.4 to 1.7 ; p = 0.2 ) . furthermore , central pulse pressure ( pp ) was associated with total cardiovascular events and procedures and the development of renal impairment ( unadjusted p < 0.0001 ; adjusted for baseline values p < 0.05 ) . the cafe study demonstrated that antihypertensive drugs can have different effects on central aortic pressure despite similar brachial blood pressure measurements . the authors of the cafe study proposed that their study elucidated a plausible mechanism to explain the superior clinical outcomes observed in the amlodipine - based treatment arm of ascot . they also speculated that central aortic pressure measurements may provide an explanation for the differences observed in other major outcome trials including life and hope . the findings of the cafe study [ 6 ] supported those of the preterax in regression of arterial stiffness in a controlled double - blind study ( reason ) , which reported that peripheral blood pressure measurements did not accurately reflect changes in central aortic pressure following treatment with different antihypertensive drugs . the finding that central pressure and wave reflection indices are strong independent predictors of all - cause and cardiovascular mortality has also been demonstrated in high - risk patient groups , including those with end - stage renal failure ( table 1 ) [ 11 , 12].table 1studies reporting differences in measured brachial and central blood pressure that may explain cardiovascular outcomesstudymeasurementspatient populationkey resultstrong heart study ( 2007 , 2009 ) [ 7 , 14]sphygmomanometer to measure brachial bp . radial applanation tonometry to determine central bppopulation - based , longitudinal study among 3,520 american indians followed for a mean of 4.8 yearscentral pp was strongly associated with carotid intima - media thickness , plaque score , and vascular mass . central pp was an independent predictor of cv outcomescafe ( 2006 ) [ 6]semiautomated oscillometric device to measure brachial bp . radial artery applanation tonometry and pulse wave velocity analysis to derive central aortic pressure and hemodynamic indexessubstudy of the ascot study . 2,199 patients with hypertension and 3 additional cv risk factors previously randomized to an amlodipine or atenolol - based regimen followed for up to 4 yearscentral aortic systolic bp was lower in the amlodipine versus atenolol arm throughout follow - up ( auc difference 4.3 mm hg , 3.3 to 5.4 , p < 0.0001 ) . brachial systolic bp similar ( auc difference 0.7 mm hg , 0.4 , 1.7 , p = 0.2 ) . may explain lower cv events in amlodipine armreason ( 2004 ) sphygmomanometer to measure brachial bp . pulse wave velocity analysis and pattern of wave reflections to derive central aortic pressure375 patients with hypertension randomized to atenolol or perindopril + indapamide , followed for 1 yeartreatment with perindopril + indapamide decreased brachial and central systolic bp significantly more than atenolol . in the perindopril + indapamide group the difference between brachial and central systolic bp was 8.28 1.53 mm hg versus 0.29 1.61 mm hg in the atenolol groupsafar ( 2002 ) , london ( 2001 ) aortic pulse wave velocity measurement and determination of arterial wave reflection by applanation tonometry on the common carotid artery180 patients with end - stage renal failure followed for a mean of 4.3 years . 40 cv and 30 non - cv events occurredaortic pulse wave velocity , increased augmentation index and carotid pp , were independent predictors of all - cause and cv mortalityascot anglo - scandinavian cardiac outcomes trial ; auc area under the curve ; bp blood pressure ; cafe conduit artery function evaluation ; cv cardiovascular ; pp pulse pressure ; reason preterax in regression of arterial stiffness in a controlled double - blind study studies reporting differences in measured brachial and central blood pressure that may explain cardiovascular outcomes ascot anglo - scandinavian cardiac outcomes trial ; auc area under the curve ; bp blood pressure ; cafe conduit artery function evaluation ; cv cardiovascular ; pp pulse pressure ; reason preterax in regression of arterial stiffness in a controlled double - blind study these findings are complemented by the results of the strong heart study [ 7 , 14 ] , a population - based , longitudinal study among 3,520 american indians ( mean age 58 years ) followed for a mean of 4.8 years . this study reported that a central pp greater than 50 mm hg and not brachial pp was an independent predictor of cardiovascular outcomes , regardless of age , sex , or diabetes . furthermore , central pp was strongly associated with carotid intima - media thickness , plaque score , and vascular mass , and was a stronger predictor of cardiovascular events than brachial blood pressure . together , these studies provide evidence to support the hypothesis that central aortic pressure may more accurately reflect the load on the central vasculature than brachial blood pressure ( table 1 ) [ 1113 , 6 , 7 ] . it is therefore a reasonable proposition that central pressure relates more directly to target organ damage and clinical cardiovascular disease . this has led to the suggestion that central and not brachial blood pressure should be a treatment target for cardiovascular disease risk reduction strategies . the mechanisms by which some antihypertensive drugs , including calcium channel blockers , affect the vasculature and lead to differential lowering of central and brachial blood pressure may be because of their effects on endothelial function , as will now be discussed in more detail . endothelial dysfunction , a key feature of hypertension , is primarily caused by enhanced oxidative stress , but other important contributors include age , vascular injury , metabolic disorders , deficiencies in essential substrates ( e.g. , l - arginine ) , and enzyme cofactors ( e.g. , tetrahydrobiopterin [ bh4 ] ) . endothelial dysfunction is characterized by reduced nitric oxide ( no ) bioavailability resulting in increased vascular resistance and reduced sensitivity to normal stimuli of vasodilation , such as shear stress and acetylcholine . the signaling molecule no is produced by the endothelium and has a key role in regulating vasomotor tone . in addition , no has atheroprotective effects by reducing smooth muscle cell proliferation and migration , adhesion of leukocytes to the endothelium , and platelet aggregation . no is derived from the conversion of l - arginine to l - citrulline by the enzymatic activity of endothelial no synthase ( enos ) . the activity of this electron transport enzyme requires calcium / calmodulin , flavin adenine dinucleotide , flavin mononucleotide , and bh4 as cofactors . normal physiologic levels of no increase vasodilation and interfere with the atherothrombotic process , thereby helping to maintain a healthy circulatory system . the net concentration of no in the circulation is dependent on a balance between the enzymatic production of no through the activity of enos and the production of superoxide ( o2 ) . many factors can influence enos activity , but studies have shown that its enzymatic cofactor bh4 has a particularly important role [ 1820 ] . when levels of bh4 are insufficient enos can not couple the reduction of molecular oxygen with the oxidation of l - arginine . oxidative modification of bh4 by various oxidases is a leading reason for abnormal low levels in the cell.fig . enos , endothelial nitric oxide synthase ; bh4 , tetrahydrobiopterin ; fad , flavin adenine dinucleotide ; fmn , flavin mononucleotide ; ca , calcium ; o2 , oxygen ; o2 , superoxide , no , nitric oxide ; onoo , peroxynitrite targeting mechanisms a global approach to cardiovascular risk management . enos , endothelial nitric oxide synthase ; bh4 , tetrahydrobiopterin ; fad , flavin adenine dinucleotide ; fmn , flavin mononucleotide ; ca , calcium ; o2 , oxygen ; o2 , superoxide , no , nitric oxide ; onoo , peroxynitrite reactive oxygen species ( ros ) are also generated in the vasculature by oxidases such as nad(p)h oxidase that contribute to oxidative stress . in the presence of excessive levels of o2 , no is rapidly converted to peroxynitrite ( onoo ) resulting in decreased no bioavailability and further impairment of endothelium - mediated vasodilation . furthermore , onoo molecules themselves are highly reactive and oxidize lipids , cause cellular injury , and enhanced arterial contraction ( fig . 1 ) [ 21 ] . animal models have shown that mice deficient in ros - generating enzymes have lower blood pressure levels compared with wild - type animals . further studies found that , compared with normotensive rats , spontaneously hypertensive rats had lower no bioavailability despite increased levels of enos . the effects were even more pronounced after induction of diabetes among the hypertensive animals [ 21 ] . the findings of reduced no bioavailability , despite increased levels of enos observed in these studies , may be explained by the increased production of o2 by uncoupled enos . glucose intolerance is also believed to impair enos activity directly through enhanced oxidative stress . in a human tissue study comparing endothelial cells from healthy african american and caucasian donors , those of african american origin this paradox was attributed to excessive o2 generation by nad(p)h oxidase , which contributes to increased onoo formation and uncoupled enos activity . these results are consistent with studies reporting differences in endothelial - dependent vasodilation in african american subjects compared with age- and gender - matched caucasians . reduced no bioavailability may partly explain the higher rates of hypertension observed among african americans compared with caucasians in various surveys . this has clinical importance as drug treatments that enhance endothelial no production may be of particular benefit in these populations . the finding of a relationship between reduced endothelial - derived no and increased oxidative stress led to the design of the a - heft study ( african american heart failure trial ) . that trial showed that the addition of isosorbide dinitrate and hydralazine to conventional therapy reduced relative 1-year mortality by 43 % among african american subjects with advanced heart failure . isosorbide dinitrate is an organic nitrate that directly increases vascular no levels , whereas hydralazine is a vasodilator with antioxidant activity that may scavenge oxyradical species , including o2. this adds further support to the theory that agents that enhance no bioavailability while reducing nitroxidative stress may have important benefits in this population . in summary , the relationship between no and ros is strongly implicated in the etiology of hypertension , and drugs that have both antihypertensive and antioxidant properties may be more effective at reducing blood pressure and subsequent pathology . indeed , improving no bioavailability may be an important treatment goal in the management of hypertension ( fig . 1 ) . dihydropyridine ( dhp)-type calcium channel blockers ( ccbs ) reversibly inhibit calcium entry into cardiac and vascular smooth muscle cells by binding to l - type voltage - sensitive calcium channels . of particular importance , certain dhp - type ccbs have been shown to modify endothelial function by enhancing enos activity , resulting in increased no production [ 29 , 30 ] . studies have also suggested that some of these drugs increase the antioxidant capacity of the endothelium by scavenging o2 [ 29 , 30 ] . this further protects the endothelium by reducing the availability of free radicals to react with no . the antioxidant activity is attributed to ccbs high lipophilicity , and a chemical structure that facilitates proton - donation and resonance - stabilization mechanisms that inhibit the free - radical reaction [ 30 , 31 ] . in animal models , the ccb amlodipine has been shown to increase enos activity and its mrna level in hypertensive rats . in coronary microvessels isolated from canine cardiac tissue , amlodipine caused a dose - dependent release of nitrite , the hydration product of no [ 33 ] . the effects of amlodipine on both nitrite release and the no - dependent regulation of cardiac oxygen consumption were inhibited with specific antagonists of enos such as l - n - monomethylarginine ( l - nmma ) . under identical conditions , other dhp and non - dhp - type ccbs , including nifedipine and diltiazem , failed to reproduce these effects . in clinical studies among patients with essential hypertension , nifedipine has been shown to attenuate circulating plasma levels of lipoperoxides and isoprostanes , increase plasma antioxidant capacity , and restore no bioavailability . in the elevation of nifedipine and cerivastatin on recovery of endothelial function ( encore ) i and encore ii studies , nifedipine significantly improved no - mediated coronary endothelial function in patients with coronary artery disease [ 35 , 36 ] . the safety and efficacy of ccb therapy and its role in reducing cardiovascular events and procedures has been demonstrated in several large clinical studies among patients with and without established cardiovascular disease [ 3746 , 47 , 48 ] . key findings from these studies are summarized in table 2 for both dhp - type ccbs and other members of this drug class.table 2outcome trials using calcium channel blockersstudydesign / drugpatient populationkey resultaccomplish ( 2008 ) [ 48]double - blind , randomized trial . mean follow - up 36 months.11,506 patients with hypertension at high risk of cv events.compared with benazepril + hydrochlorothiazide , fewer individuals on benazepril + amlodipine had a primary endpoint ( death from cv causes , nonfatal mi , nonfatal stroke , hospitalization for angina , resuscitation after sudden cardiac arrest , and coronary revascularization ) hr , 0.80 ; 95 % ci , 0.72 to 0.90 ; p < 0.001 . for the secondary endpoint of death from cv causes , nonfatal mi , and nonfatal stroke , hr 0.79 ; 95 % ci , 0.67 to 0.92 ; p = 0.002.ascot-bpla ( 2005 ) [ 47]open - label , randomized trial . mean 5.5 year follow - up.19,257 patients with hypertension and 3 additional cv risk factors.compared with the atenolol - based regimen , fewer individuals on the amlodipine - based regimen had a primary endpoint ( nonfatal mi and fatal chd ) hr , 0.90 ; 95 % ci , 0.79 to 1.02 ; p = 0.1052 ; fatal and nonfatal stroke , hr , 0.77 ; 95 % ci , 0.66 to 0.89 ; p = 0.0003 ; total cv events and procedures , hr , 0.84 ; 95 % ci , 0.78 to 0.90 ; p < 0.0001 ; all - cause mortality , hr , 0.89 ; 95 % ci , 0.81 to 0.99 ; p = 0.025.camelot ( 2004 ) double - blind , placebo - controlled , randomized trial . 24 month follow - up.1,991 patients with cad and dbp < 100 mm hg.compared with placebo , there was a 31 % reduction in cv events in the amlodipine group ( p = 0.003 ) and a 15 % reduction in the enalapril group ( p = 0.16 ) . in the amlodipine group , ivus showed evidence of slowing atherosclerosis progression.value ( 2004)double - blind , parallel - group , randomized trial . mean follow - up 4.2 years.15,245 patients with hypertension at high risk of cardiac events.no difference in the primary outcome ( cardiac mortality and morbidity ) between treatment groups , hr 1.04 ; 95 % ci , 0.94 to 1.15 ; p = 0.49 . bp reduced by both treatments , but amlodipine had greater effect especially in the early period . amlodipine was superior to valsartan at preventing mi and angina.invest ( 2003)open - label , blinded endpoint , randomized trial . . mean follow - up 2.7 years.22,576 patients with hypertension and cad.no difference in the primary outcome ( first occurrence of all - cause mortality , nonfatal mi or nonfatal stroke ) between treatment groups , rr 0.98 ; 95 % ci 0.90-1.06.convince ( 2003)double - blind , randomized trial . . mean follow - up 3 years.16,602 patients with hypertension and 1 additional cv risk factor.no difference in the primary outcome ( first occurrence of stroke , mi , or cv - related death ) between treatment groups , hr 1.02 ; 95 % ci , 0.88 to 1.18 ; p = 0.77.allhat ( 2002)double - blind , randomized trial . 3 treatment groups : chlorthalidone ; amlodipine ; lisinopril . mean follow - up 4.9 years.33,357 patients with hypertension and 1 additional chd risk factor.no difference in the primary outcome ( fatal chd or nonfatal mi ) between treatment groups . compared with chlorthalidone : rr for amlodipine 0.98 ; 95 % ci , 0.90 to 1.07 ; lisinopril 0.99 ; 95 % ci , 0.91 to 1.08.nordil ( 2000)open - label , blinded endpoint , randomized trial . mean follow - up 4.5 years.10,881 patients with dbp 100 mm hg.no difference in the primary outcome ( fatal and non - fatal stroke , mi , cv death ) between the 2 groups , rr 1.00 ; 95 % ci , 0.87 to 1.15 ; p = 0.97.insight ( 2000)double - blind , randomized trial . follow - up 3 years after recruitment of the last patient.6,321 patients with hypertension and 1 additional cv risk factor.no difference in the primary outcome ( cv death , mi , heart failure , or stroke ) between the 2 groups , rr , 1.10 ; 95 % ci , 0.91 to 1.34 ; p = 0.35.prevent ( 2000 ) double - blind , placebo - controlled , randomized trial . 36-month follow - up.825 patients with cad.no difference in coronary stenosis between the amlodipine and placebo group . amlodipine slowed the progression of carotid artery atherosclerosis ( imt : amlodipine 0.0126 versus placebo + 0.033 ; p = 0.007 ) and was associated with fewer hospitalizations for unstable angina and coronary revascularization.syst-eur ( 1997 ) double - blind , placebo - controlled , randomized trial . median follow - up 2 years.4695 patients with isolated systolic hypertension ( sbp 160 mmhg and dbp < 95 mmhg).compared with placebo , nitrendipine reduced the total rate of stroke by 42 % ( p = 0.003 ) ; nonfatal stroke by 44 % ( p = 0.007 ) and all fatal and nonfatal cardiac events by 26 % ( p = 0.03).midas ( 1996 ) double - blind , randomized trial . isradipine or hydrochlorothiazide . 3 year follow - up.883 patients with hypertensionno difference in the rate of progression of mean maximum imt ( p = 0.68 ) between treatment groups . higher ( but non - significant , p = 0.07 ) incidence of major vascular events ( mi , stroke , heart failure , angina , sudden death ) in the isradipine versus the hydrochlorothiazide group ( 25 vs 14 events).accomplish avoiding cardiovascular events through combination therapy in patients living with systolic hypertension study ; ascot - bpla anglo - scandinavian cardiac outcomes trial blood pressure lowering arm ; camelot comparison of amlodipine versus enalapril to limit occurrences of thrombosis study ; value , valsartan antihypertensive long - term use evaluation ; invest , the international verapamil - trandolapril study ; convince , controlled onset verapamil investigation of cardiovascular end points ; allhat , the antihypertensive and lipid - lowering treatment to prevent heart attack trial ; nordil , the nordic diltiazem study ; insight , intervention as a goal in hypertension treatment ; prevent prospective randomized evaluation of the vascular effects of norvasc trial ; sys - eur , systolic hypertension in europe ; midas , multicenter isradipine diuretic atherosclerosis study.cad coronary artery disease ; chd coronary heart disease ; ci confidence interval ; cv cardiovascular ; dbp diastolic blood pressure ; hr hazard ratio ; imt intimal - media thickness ; ivus intravascular ultrasound ; mi myocardial infarction ; sbp , systolic blood pressure outcome trials using calcium channel blockers accomplish avoiding cardiovascular events through combination therapy in patients living with systolic hypertension study ; ascot - bpla anglo - scandinavian cardiac outcomes trial blood pressure lowering arm ; camelot comparison of amlodipine versus enalapril to limit occurrences of thrombosis study ; value , valsartan antihypertensive long - term use evaluation ; invest , the international verapamil - trandolapril study ; convince , controlled onset verapamil investigation of cardiovascular end points ; allhat , the antihypertensive and lipid - lowering treatment to prevent heart attack trial ; nordil , the nordic diltiazem study ; insight , intervention as a goal in hypertension treatment ; prevent prospective randomized evaluation of the vascular effects of norvasc trial ; sys - eur , systolic hypertension in europe ; midas , multicenter isradipine diuretic atherosclerosis study . cad coronary artery disease ; chd coronary heart disease ; ci confidence interval ; cv cardiovascular ; dbp diastolic blood pressure ; hr hazard ratio ; imt intimal - media thickness ; ivus intravascular ultrasound ; mi myocardial infarction ; sbp , systolic blood pressure hypertension commonly clusters with other cardiovascular risk factors , including dyslipidemia and diabetes , greatly increasing an individual s risk of an event [ 49 , 50 ] . from a mechanistic viewpoint , these risk factors act synergistically to exacerbate endothelial dysfunction , oxidative stress , and inflammation , thereby accelerating the atherosclerotic process . although controversy remains over the optimal choice of antihypertensive drug therapy , hypertension management guidelines agree that most patients may require at least two antihypertensive drugs to reach the currently recommended blood pressure targets [ 5254 ] . in addition , many patients with hypertension also benefit from concurrent statin therapy [ 5254 ] . thus most patients with hypertension require multiple drug treatment regimens to manage their cardiovascular risk . a greater understanding of the mechanisms of actions of antihypertensive and lipid - lowering drugs may lead to more effective drug combinations with respect to clinical outcomes . this review focuses on the combination of ccbs with statins and ccbs in combination with drugs that affect the renin - angiotensin system ( ras ) . both ccbs and statins have been shown to reduce cardiovascular events in large clinical outcome trials [ 45 , 46 , 55 , 56 , 47 , 48 ] . ascot highlighted a potential synergy between co - administered amlodipine and atorvastatin in terms of the cardio - protective effect of this drug combination . in ascot , those randomized to amlodipine plus atorvastatin had a 53 % reduction in coronary heart disease events compared with a 16 % reduction among those randomized to atenolol plus atorvastatin . this could not be explained by differences in blood pressure and lipid parameters between the two treatment regimens . furthermore , the significant benefits of the amlodipine plus atorvastatin combination were observed within the first 3 months of treatment ( p = 0.02 ) . the findings from ascot may be explained , in part , by observations from the avalon arterial wall compliance ( awc ) trial . in the awc trial , 668 patients ( 61 % male , mean age 55 years ) with concomitant hypertension and dyslipidemia were randomized to one of four treatment groups ( placebo , amlodipine 5 mg , atorvastatin 10 mg , co - administered amlodipine 5 mg and atorvastatin 10 mg ) . after 8 weeks of treatment , there was a 19.3 % improvement in small artery compliance in the co - administered amlodipine and atorvastatin group compared with 11.7 % in the amlodipine alone group ( p = 0.03 ) , 3.1 % in the atorvastatin alone group ( p < 0.001 ) , and 1.3 % in the placebo group ( p < 0.0001 ) . after 28 weeks of treatment , the greatest improvement in small artery compliance remained among those taking co - administered amlodipine and atorvastatin ( p < 0.05 ) . the observation that combination therapy had a more than additive effect on small artery compliance is consistent with an improvement in the mechanisms that control vascular function [ 59 ] . indeed , the authors of the awc trial proposed that their study provided evidence that the synergistic effects of co - administered amlodipine and atorvastatin may be mediated by endothelial function . this hypothesis is supported by the results of the encore i study that compared no - mediated endothelial function among 343 patients with coronary artery disease . patients were randomized to 6 months treatment with placebo , cerivastatin 0.4 mg / day , nifedipine 30 to 60 mg / day , or their combination . endothelial function was assessed by infusing acetylcholine into a coronary segment and measuring luminal diameter by quantitative angiography . in the most constricted segment , nifedipine but not cerivastatin , significantly reduced acetylcholine - induced vasoconstriction by 18.8 % ( p < 0.05 , compared with placebo ) . the combination of nifedipine and cerivastatin also reduced acetylcholine - induced vasoconstriction by 11 % , but this only reached statistical significance when all coronary segments were analyzed together . after cerivastatin was withdrawn from the market in 2001 , the encore study design was modified to compare nifedipine with placebo . in encore ii , all patients were eligible for statin therapy at the discretion of their physician . in addition to measuring changes in luminal diameter , intravascular ultrasound ( ivus ) was used to assess change in plaque volume . overall , 454 patients were randomized and followed for 1824 months . compared with placebo , nifedipine plus background statin therapy significantly improved coronary endothelial function in the most constricted segment ( difference in luminal diameter 6.3 % , 95 % ci 1.6 to 10.9 ; p = 0.0088 ) . however , compared with placebo , nifedipine plus background statin therapy did not have a significant effect on coronary plaque volume as measured by ivus . together the encore studies add to the evidence that ccb plus statin therapy improves no - mediated endothelial function [ 35 , 36 ] . from a mechanistic perspective , synergy between ccbs and statins is plausible , as these drugs have complementary chemical structures . atorvastatin has negative polarity associated with its heptanoic side chain , whereas amlodipine is distinct among the dhps in having a positive charge on its aminoethoxy side chain [ 31 , 60 , 61 ] . indeed , studies have demonstrated that the concentration of these drugs is much higher in the cell membrane than in the surrounding aqueous environment [ 31 , 60 , 61 ] . these properties may facilitate interactions with novel receptor sites in vascular cell membranes , and contribute to explaining the superior clinical outcomes observed when these drugs are co - administered . this hypothesis was evaluated in a study using human umbilical vein endothelial cells ( huvec ) [ 59 ] . the combination of amlodipine and atorvastatin directly stimulated no release , which was about twofold greater than the sum of their separate effects ( p < 0.05 ) . this was attributed to enhanced enos function and expression along with decreased levels of cytotoxic onoo . following low density lipoprotein ( ldl ) enrichment there was a 60 % reduction in no production in the huvec and an almost twofold increase in onoo . treatment with the combination of amlodipine and atorvastatin partially reversed the adverse effects of ldl , including a 90 % increase in no and a 50 % reduction in onoo . small angle x - ray diffraction analysis indicated that both amlodipine and atorvastatin are lipophilic and share a common membrane location [ 59 ] . this study provides evidence to suggest the observed synergy between these drugs may be explained by electron transport mechanisms that facilitate antioxidant activity in the cell membrane related to their complementary locations . the ras has an important role in blood pressure control by regulating blood volume and peripheral vascular resistance . in brief , in response to the release of renin from the kidneys , the circulating substrate angiotensinogen is converted to angiotensin i. the angiotensin - converting enzyme ( ace ) in the vascular endothelium cleaves off two amino acids to form the octapeptide , angiotensin ii ( ang ii ) . ang ii binds to angiotensin ii type-1 ( at1 ) receptors in vascular smooth muscle cells , promoting vasoconstriction and increasing peripheral vascular resistance . ang ii also stimulates the release of various hormones including aldosterone and vasopressin , which act on the kidneys to increase sodium and fluid retention . ang ii also facilitates norepinephrine release from sympathetic nerve endings and inhibits its reuptake , thereby enhancing sympathetic adrenergic function . the interaction between ang ii and at1 receptors also activates signal transduction mechanisms that promote oxidative stress , inflammation , cell proliferation , and fibrosis . studies have shown that ang ii activates nad(p)h oxidase in endothelial and vascular smooth muscle cells . this results in the production of o2 and other ros , and contributes directly to reduced no bioavailability and endothelial dysfunction . blocking the ras with ace inhibitors and angiotensin receptor blockers ( arbs ) has been demonstrated to improve endothelial function and increase no bioavailability in mechanistic [ 6467 ] and clinical [ 6870 ] studies . in an animal model using infarcted adult male sprague dawley rats , treatment with the arb candesartan , enhanced vasorelaxation by increasing no bioavailability via an at2 receptor mediated upregulation of enos . a complementary in vitro study , using the arbs losartan and valsartan , stimulated no release from both platelets and human umbilical vein endothelial cells in a dose - dependent manner . however , there was more than 70 % greater potency in no release in platelets than endothelial cells . furthermore , the degree of inhibition of platelet adhesion and aggregation by losartan and valsartan was closely correlated with the level of no production . in an animal model using isolated coronary arterioles from pigs , ang ii was shown to evoke at1 receptor - mediated vasoconstriction and at2 receptor - mediated vasodilation . at the vascular level , ang ii was shown to impair endothelium - dependent no - mediated dilation attributable to elevated o2 production via at1 receptor activation of nad(p)h oxidase . the authors of this study commented that their results may partly explain why impaired coronary flow is associated with upregulation of the ras . the long - term effect of the arb valsartan on endothelial function and vascular structural changes in the aorta was explored in hypercholesterolemic rabbits . treatments with 3 and 10 mg / kg per day of valsartan reduced intimal lesion to 2.4 0.7 % and 2.7 0.9 % , respectively ( p < 0.05 ) and increased lumen area . the authors suggested that at1 receptor antagonists , besides their antihypertensive effects , could also have a role in reducing the development of atherosclerosis . the results of these mechanistic studies were consistent with the results from clinical studies [ 6870 ] . in an active - controlled , randomized trial , 35 patients with coronary artery disease received 4 weeks of treatment with either an ace inhibitor ( ramipril 10 mg / day ) or arb ( losartan 100 mg / day ) . no - mediated vasodilation of the radial artery was determined before and after intra - arterial l - nmma infusion . no - mediated vasodilation was increased by more than 75 % after treatment with ramipril and losartan ( each group p < 0.01 ) . these results were consistent with a double - blind , placebo - controlled trial among 60 patients with essential hypertension who received 6 weeks of treatment with either an arb ( valsartan 80 mg / day ) , a diuretic ( hydrochlorothiazide 25 mg / day ) , or placebo . there were similar reductions in brachial blood pressure in both active treatment arms ( p < 0.001 ) . however , patients in the arb group had significantly improved vasoconstrictive response to l - nmma , whereas no effect was seen in the diuretic and placebo groups . together , these clinical studies suggest that agents that block the ras may improve endothelial function by increasing no bioavailability . a further study , among 53 hypertensive patients with and without type 2 diabetes , showed that treatment with valsartan 80 mg / day for 8 weeks significantly decreased monocyte and endothelial cell activation markers among those with diabetes , but not among those without diabetes . this study highlights the importance of using complex disease models to explore mechanisms of actions , because focusing on single - risk - factor models may miss important biological pathways . there is a growing body of evidence to suggest that dhp - type ccbs and ras inhibitors have additional beneficial effects when used in combination . for example , in an animal model using dahl salt - sensitive rats , the combination of ccb plus ace inhibitor was more effective than either monotherapy in normalizing systolic blood pressure and proteinuria . this study was complimented by a rat myocardial infarction model that showed that treatment with the ccb amlodipine plus ace inhibitor benazepril increased no production and decreased inflammatory markers . in contrast , treatment with the diuretic hydrochlorothiazide had no effect on these indicators of endothelial dysfunction . in clinical studies , the combination of ccbs with an ace inhibitor versus a ccb alone has been shown to increase flow - mediated dilation , a surrogate biomarker of endothelial function . as previously described , the cafe study [ 6 ] demonstrated that the benefits of the ccb plus ace inhibitor combination may extend beyond brachial blood pressure lowering and have a positive impact on central blood pressure hemodynamics [ 6 ] . furthermore , outcome trials have reported that these beneficial effects may translate into reduced cardiovascular endpoints . the ascot - bpla study showed that treatment with an amlodipine plus perindopril - based regimen prevented more major cardiovascular events than an atenolol plus bendroflumethiazide - based regimen [ 47 ] . the accomplish study showed that treatment with benazepril plus amlodipine was more effective at preventing cardiovascular events than treatment with benazepril plus hydrochlorothiazide [ 48 ] . together , these mechanistic and clinical studies demonstrate the pleiotropic effects of the combination of ccb plus ras blockade on enhanced no bioavailability and reduced oxidative stress . understanding the complementary mechanisms of actions of these drugs forms the rationale for combining different antihypertensive agents to enhance their therapeutic effects . this review has explored the pleiotropic effects of ccbs alone and in combination with statins and ras inhibitors . the mechanisms by which these drugs exert their beneficial effects on endothelial and vascular function have been described , with a particular focus on the effects on no - mediated vasodilation and oxidative stress . by further elucidating the biological pathways by which these drugs exert their effects , we considered the rationale for choosing optimal drug combinations that will benefit patients at risk . the differential effect of ccbs on brachial and central blood pressure has been explored , along with the future role of central blood pressure as a prognostic marker for cardiovascular disease events . indeed , clinical hypertension trials have demonstrated the importance of incorporating measurements of central aortic pressure in their study designs . another important aspect of future hypertension research is the shift towards studying complex disease models . from a mechanistic viewpoint , multiple cardiovascular risk factors act synergistically to exacerbate endothelial dysfunction , oxidative stress and inflammation , thereby accelerating the atherosclerotic process . it is therefore logical that they should be considered together in global risk assessment . by further elucidating the underlying biological pathways and mechanisms that lead to target organ damage in multiple cardiovascular risk factor models such approaches will continue to require the scientific community to work together to carry out large - scale studies and to link the findings from experimental models with human populations .

clinical trials have reported reduced cardiovascular events with certain antihypertensive agents at a rate that could not be predicted by changes in brachial arterial pressure alone . these findings may be explained , in part , by pleiotropic effects of these agents and modulation of central blood pressures . this review focuses on the mechanisms by which calcium channel blockers exert pleiotropic effects , both alone and in combination with statins and inhibitors of the renin - angiotensin system . the essential role of nitric oxide ( no ) in maintaining endothelial function and the relationship between no and reactive oxygen species are discussed in the context of the etiology of hypertension . the importance of managing global cardiovascular risk is emphasized , as hypertension commonly clusters with dyslipidemia and loss of glucose control . from a mechanistic viewpoint , these risk factors contribute to endothelial dysfunction , oxidative stress , and inflammation in a synergistic fashion . a greater understanding of the mechanisms of actions of these cardiovascular agents may lead to more effective drug combinations , to the benefit of individual patients . furthermore , by elucidating the biological mechanisms by which cardiovascular risk factors lead to vascular injury , we may highlight common pathways and identify novel therapeutic targets .

Introduction Endothelial Dysfunction and the Role of NO Effect of Calcium Channel Blockers on Endothelial Function Role of Combination Therapy in Cardiovascular Risk Management Rationale for Combining Calcium Channel Blockers and Statins Rationale for Combining CCBs and Drugs that Affect the RAS Conclusions

over the last 10 years , several antihypertensive drug treatment trials have shown an unexpected discrepancy between reduction in brachial blood pressure and observed clinical outcomes . in the heart outcomes prevention evaluation ( hope ) , the losartan intervention for endpoint reduction ( life ) in hypertension , and the australian national blood pressure 2 ( anbp2 ) trials , the observed clinical benefits tended to be greater than those expected from the decrease in brachial blood pressure . this may be explained by the pleiotropic effects of the antihypertensive drugs used in these trials . in particular , the beneficial effects of these agents on endothelial function and its vascular manifestations , such as changes in central aortic pressure and atheroma development . furthermore , the state of the vasculature in the peripheral circulation also affects the proportion of the incident wave that is reflected , thus affecting central pressure . this may , in part , explain why central aortic pressure has been shown to have superior prognostic value with respect to cardiovascular events than brachial blood pressure in clinical studies [ 6 , 1113 ] . in ascot , patients with hypertension and at least three additional cardiovascular risk factors were randomized to an atenolol plus bendroflumethiazide - based treatment regimen or an amlodipine plus perindopril - based regimen . furthermore , central pulse pressure ( pp ) was associated with total cardiovascular events and procedures and the development of renal impairment ( unadjusted p < 0.0001 ; adjusted for baseline values p < 0.05 ) . the authors of the cafe study proposed that their study elucidated a plausible mechanism to explain the superior clinical outcomes observed in the amlodipine - based treatment arm of ascot . the findings of the cafe study [ 6 ] supported those of the preterax in regression of arterial stiffness in a controlled double - blind study ( reason ) , which reported that peripheral blood pressure measurements did not accurately reflect changes in central aortic pressure following treatment with different antihypertensive drugs . 2,199 patients with hypertension and 3 additional cv risk factors previously randomized to an amlodipine or atenolol - based regimen followed for up to 4 yearscentral aortic systolic bp was lower in the amlodipine versus atenolol arm throughout follow - up ( auc difference 4.3 mm hg , 3.3 to 5.4 , p < 0.0001 ) . in the perindopril + indapamide group the difference between brachial and central systolic bp was 8.28 1.53 mm hg versus 0.29 1.61 mm hg in the atenolol groupsafar ( 2002 ) , london ( 2001 ) aortic pulse wave velocity measurement and determination of arterial wave reflection by applanation tonometry on the common carotid artery180 patients with end - stage renal failure followed for a mean of 4.3 years . 40 cv and 30 non - cv events occurredaortic pulse wave velocity , increased augmentation index and carotid pp , were independent predictors of all - cause and cv mortalityascot anglo - scandinavian cardiac outcomes trial ; auc area under the curve ; bp blood pressure ; cafe conduit artery function evaluation ; cv cardiovascular ; pp pulse pressure ; reason preterax in regression of arterial stiffness in a controlled double - blind study studies reporting differences in measured brachial and central blood pressure that may explain cardiovascular outcomes ascot anglo - scandinavian cardiac outcomes trial ; auc area under the curve ; bp blood pressure ; cafe conduit artery function evaluation ; cv cardiovascular ; pp pulse pressure ; reason preterax in regression of arterial stiffness in a controlled double - blind study these findings are complemented by the results of the strong heart study [ 7 , 14 ] , a population - based , longitudinal study among 3,520 american indians ( mean age 58 years ) followed for a mean of 4.8 years . furthermore , central pp was strongly associated with carotid intima - media thickness , plaque score , and vascular mass , and was a stronger predictor of cardiovascular events than brachial blood pressure . together , these studies provide evidence to support the hypothesis that central aortic pressure may more accurately reflect the load on the central vasculature than brachial blood pressure ( table 1 ) [ 1113 , 6 , 7 ] . this has led to the suggestion that central and not brachial blood pressure should be a treatment target for cardiovascular disease risk reduction strategies . the mechanisms by which some antihypertensive drugs , including calcium channel blockers , affect the vasculature and lead to differential lowering of central and brachial blood pressure may be because of their effects on endothelial function , as will now be discussed in more detail . endothelial dysfunction , a key feature of hypertension , is primarily caused by enhanced oxidative stress , but other important contributors include age , vascular injury , metabolic disorders , deficiencies in essential substrates ( e.g. endothelial dysfunction is characterized by reduced nitric oxide ( no ) bioavailability resulting in increased vascular resistance and reduced sensitivity to normal stimuli of vasodilation , such as shear stress and acetylcholine . in addition , no has atheroprotective effects by reducing smooth muscle cell proliferation and migration , adhesion of leukocytes to the endothelium , and platelet aggregation . the net concentration of no in the circulation is dependent on a balance between the enzymatic production of no through the activity of enos and the production of superoxide ( o2 ) . enos , endothelial nitric oxide synthase ; bh4 , tetrahydrobiopterin ; fad , flavin adenine dinucleotide ; fmn , flavin mononucleotide ; ca , calcium ; o2 , oxygen ; o2 , superoxide , no , nitric oxide ; onoo , peroxynitrite targeting mechanisms a global approach to cardiovascular risk management . enos , endothelial nitric oxide synthase ; bh4 , tetrahydrobiopterin ; fad , flavin adenine dinucleotide ; fmn , flavin mononucleotide ; ca , calcium ; o2 , oxygen ; o2 , superoxide , no , nitric oxide ; onoo , peroxynitrite reactive oxygen species ( ros ) are also generated in the vasculature by oxidases such as nad(p)h oxidase that contribute to oxidative stress . furthermore , onoo molecules themselves are highly reactive and oxidize lipids , cause cellular injury , and enhanced arterial contraction ( fig . the findings of reduced no bioavailability , despite increased levels of enos observed in these studies , may be explained by the increased production of o2 by uncoupled enos . the finding of a relationship between reduced endothelial - derived no and increased oxidative stress led to the design of the a - heft study ( african american heart failure trial ) . in summary , the relationship between no and ros is strongly implicated in the etiology of hypertension , and drugs that have both antihypertensive and antioxidant properties may be more effective at reducing blood pressure and subsequent pathology . indeed , improving no bioavailability may be an important treatment goal in the management of hypertension ( fig . dihydropyridine ( dhp)-type calcium channel blockers ( ccbs ) reversibly inhibit calcium entry into cardiac and vascular smooth muscle cells by binding to l - type voltage - sensitive calcium channels . studies have also suggested that some of these drugs increase the antioxidant capacity of the endothelium by scavenging o2 [ 29 , 30 ] . the effects of amlodipine on both nitrite release and the no - dependent regulation of cardiac oxygen consumption were inhibited with specific antagonists of enos such as l - n - monomethylarginine ( l - nmma ) . in the elevation of nifedipine and cerivastatin on recovery of endothelial function ( encore ) i and encore ii studies , nifedipine significantly improved no - mediated coronary endothelial function in patients with coronary artery disease [ 35 , 36 ] . the safety and efficacy of ccb therapy and its role in reducing cardiovascular events and procedures has been demonstrated in several large clinical studies among patients with and without established cardiovascular disease [ 3746 , 47 , 48 ] . mean 5.5 year follow - up.19,257 patients with hypertension and 3 additional cv risk factors.compared with the atenolol - based regimen , fewer individuals on the amlodipine - based regimen had a primary endpoint ( nonfatal mi and fatal chd ) hr , 0.90 ; 95 % ci , 0.79 to 1.02 ; p = 0.1052 ; fatal and nonfatal stroke , hr , 0.77 ; 95 % ci , 0.66 to 0.89 ; p = 0.0003 ; total cv events and procedures , hr , 0.84 ; 95 % ci , 0.78 to 0.90 ; p < 0.0001 ; all - cause mortality , hr , 0.89 ; 95 % ci , 0.81 to 0.99 ; p = 0.025.camelot ( 2004 ) double - blind , placebo - controlled , randomized trial . follow - up 3 years after recruitment of the last patient.6,321 patients with hypertension and 1 additional cv risk factor.no difference in the primary outcome ( cv death , mi , heart failure , or stroke ) between the 2 groups , rr , 1.10 ; 95 % ci , 0.91 to 1.34 ; p = 0.35.prevent ( 2000 ) double - blind , placebo - controlled , randomized trial . higher ( but non - significant , p = 0.07 ) incidence of major vascular events ( mi , stroke , heart failure , angina , sudden death ) in the isradipine versus the hydrochlorothiazide group ( 25 vs 14 events).accomplish avoiding cardiovascular events through combination therapy in patients living with systolic hypertension study ; ascot - bpla anglo - scandinavian cardiac outcomes trial blood pressure lowering arm ; camelot comparison of amlodipine versus enalapril to limit occurrences of thrombosis study ; value , valsartan antihypertensive long - term use evaluation ; invest , the international verapamil - trandolapril study ; convince , controlled onset verapamil investigation of cardiovascular end points ; allhat , the antihypertensive and lipid - lowering treatment to prevent heart attack trial ; nordil , the nordic diltiazem study ; insight , intervention as a goal in hypertension treatment ; prevent prospective randomized evaluation of the vascular effects of norvasc trial ; sys - eur , systolic hypertension in europe ; midas , multicenter isradipine diuretic atherosclerosis study.cad coronary artery disease ; chd coronary heart disease ; ci confidence interval ; cv cardiovascular ; dbp diastolic blood pressure ; hr hazard ratio ; imt intimal - media thickness ; ivus intravascular ultrasound ; mi myocardial infarction ; sbp , systolic blood pressure outcome trials using calcium channel blockers accomplish avoiding cardiovascular events through combination therapy in patients living with systolic hypertension study ; ascot - bpla anglo - scandinavian cardiac outcomes trial blood pressure lowering arm ; camelot comparison of amlodipine versus enalapril to limit occurrences of thrombosis study ; value , valsartan antihypertensive long - term use evaluation ; invest , the international verapamil - trandolapril study ; convince , controlled onset verapamil investigation of cardiovascular end points ; allhat , the antihypertensive and lipid - lowering treatment to prevent heart attack trial ; nordil , the nordic diltiazem study ; insight , intervention as a goal in hypertension treatment ; prevent prospective randomized evaluation of the vascular effects of norvasc trial ; sys - eur , systolic hypertension in europe ; midas , multicenter isradipine diuretic atherosclerosis study . cad coronary artery disease ; chd coronary heart disease ; ci confidence interval ; cv cardiovascular ; dbp diastolic blood pressure ; hr hazard ratio ; imt intimal - media thickness ; ivus intravascular ultrasound ; mi myocardial infarction ; sbp , systolic blood pressure hypertension commonly clusters with other cardiovascular risk factors , including dyslipidemia and diabetes , greatly increasing an individual s risk of an event [ 49 , 50 ] . from a mechanistic viewpoint , these risk factors act synergistically to exacerbate endothelial dysfunction , oxidative stress , and inflammation , thereby accelerating the atherosclerotic process . a greater understanding of the mechanisms of actions of antihypertensive and lipid - lowering drugs may lead to more effective drug combinations with respect to clinical outcomes . this review focuses on the combination of ccbs with statins and ccbs in combination with drugs that affect the renin - angiotensin system ( ras ) . ascot highlighted a potential synergy between co - administered amlodipine and atorvastatin in terms of the cardio - protective effect of this drug combination . this could not be explained by differences in blood pressure and lipid parameters between the two treatment regimens . furthermore , the significant benefits of the amlodipine plus atorvastatin combination were observed within the first 3 months of treatment ( p = 0.02 ) . the findings from ascot may be explained , in part , by observations from the avalon arterial wall compliance ( awc ) trial . after 8 weeks of treatment , there was a 19.3 % improvement in small artery compliance in the co - administered amlodipine and atorvastatin group compared with 11.7 % in the amlodipine alone group ( p = 0.03 ) , 3.1 % in the atorvastatin alone group ( p < 0.001 ) , and 1.3 % in the placebo group ( p < 0.0001 ) . the observation that combination therapy had a more than additive effect on small artery compliance is consistent with an improvement in the mechanisms that control vascular function [ 59 ] . indeed , the authors of the awc trial proposed that their study provided evidence that the synergistic effects of co - administered amlodipine and atorvastatin may be mediated by endothelial function . this hypothesis is supported by the results of the encore i study that compared no - mediated endothelial function among 343 patients with coronary artery disease . compared with placebo , nifedipine plus background statin therapy significantly improved coronary endothelial function in the most constricted segment ( difference in luminal diameter 6.3 % , 95 % ci 1.6 to 10.9 ; p = 0.0088 ) . together the encore studies add to the evidence that ccb plus statin therapy improves no - mediated endothelial function [ 35 , 36 ] . from a mechanistic perspective , synergy between ccbs and statins is plausible , as these drugs have complementary chemical structures . indeed , studies have demonstrated that the concentration of these drugs is much higher in the cell membrane than in the surrounding aqueous environment [ 31 , 60 , 61 ] . these properties may facilitate interactions with novel receptor sites in vascular cell membranes , and contribute to explaining the superior clinical outcomes observed when these drugs are co - administered . treatment with the combination of amlodipine and atorvastatin partially reversed the adverse effects of ldl , including a 90 % increase in no and a 50 % reduction in onoo . this study provides evidence to suggest the observed synergy between these drugs may be explained by electron transport mechanisms that facilitate antioxidant activity in the cell membrane related to their complementary locations . in brief , in response to the release of renin from the kidneys , the circulating substrate angiotensinogen is converted to angiotensin i. the angiotensin - converting enzyme ( ace ) in the vascular endothelium cleaves off two amino acids to form the octapeptide , angiotensin ii ( ang ii ) . the interaction between ang ii and at1 receptors also activates signal transduction mechanisms that promote oxidative stress , inflammation , cell proliferation , and fibrosis . this results in the production of o2 and other ros , and contributes directly to reduced no bioavailability and endothelial dysfunction . blocking the ras with ace inhibitors and angiotensin receptor blockers ( arbs ) has been demonstrated to improve endothelial function and increase no bioavailability in mechanistic [ 6467 ] and clinical [ 6870 ] studies . the long - term effect of the arb valsartan on endothelial function and vascular structural changes in the aorta was explored in hypercholesterolemic rabbits . together , these clinical studies suggest that agents that block the ras may improve endothelial function by increasing no bioavailability . this study highlights the importance of using complex disease models to explore mechanisms of actions , because focusing on single - risk - factor models may miss important biological pathways . for example , in an animal model using dahl salt - sensitive rats , the combination of ccb plus ace inhibitor was more effective than either monotherapy in normalizing systolic blood pressure and proteinuria . as previously described , the cafe study [ 6 ] demonstrated that the benefits of the ccb plus ace inhibitor combination may extend beyond brachial blood pressure lowering and have a positive impact on central blood pressure hemodynamics [ 6 ] . furthermore , outcome trials have reported that these beneficial effects may translate into reduced cardiovascular endpoints . the accomplish study showed that treatment with benazepril plus amlodipine was more effective at preventing cardiovascular events than treatment with benazepril plus hydrochlorothiazide [ 48 ] . together , these mechanistic and clinical studies demonstrate the pleiotropic effects of the combination of ccb plus ras blockade on enhanced no bioavailability and reduced oxidative stress . understanding the complementary mechanisms of actions of these drugs forms the rationale for combining different antihypertensive agents to enhance their therapeutic effects . this review has explored the pleiotropic effects of ccbs alone and in combination with statins and ras inhibitors . the mechanisms by which these drugs exert their beneficial effects on endothelial and vascular function have been described , with a particular focus on the effects on no - mediated vasodilation and oxidative stress . by further elucidating the biological pathways by which these drugs exert their effects , we considered the rationale for choosing optimal drug combinations that will benefit patients at risk . the differential effect of ccbs on brachial and central blood pressure has been explored , along with the future role of central blood pressure as a prognostic marker for cardiovascular disease events . indeed , clinical hypertension trials have demonstrated the importance of incorporating measurements of central aortic pressure in their study designs . from a mechanistic viewpoint , multiple cardiovascular risk factors act synergistically to exacerbate endothelial dysfunction , oxidative stress and inflammation , thereby accelerating the atherosclerotic process . by further elucidating the underlying biological pathways and mechanisms that lead to target organ damage in multiple cardiovascular risk factor models such approaches will continue to require the scientific community to work together to carry out large - scale studies and to link the findings from experimental models with human populations .

today s world is a world of organizations , and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives . without people , the concepts of organization and management would become meaningless ( 1 ) . therefore , professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations . the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s , and , according to many experts , it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness ( 2 ) . health care organizations and treatment centers are responsible for maintaining the health of all citizens in a society , and nurses have a key role . their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction ( 1 ) . previous studies have shown that nurses should demonstrate a passion for their work and have the necessary proficiency and expertise to achieve the highest level of efficiency ( 2 ) . it could be argued that any person would provide better services if he or she were motivated and enthusiastic about the work . despite the great significance of the professional satisfaction of nurses due to its considerable impact on them , their patients , their organizations , and the very profession of nursing ( 3 ) , most previous studies have reported the satisfaction level to be low among nurses ( 4 ) . the results from the study conducted by lou et al . indicated that only half of the nurses in the study were moderately or highly satisfied with their work ( 5 ) . another study was conducted to compare the level of professional satisfaction among different groups of people in korea , and it was found that only 41.5% of nurses were satisfied with their work , whereas the percentages for other experts working at hospitals , social workers , and middle school teachers were 50.1 , 58.2 , and 89% , respectively . these statistics indicate that there is a lower level of satisfaction among nurses than other professionals ( 6 ) . various studies have been conducted regarding the extent of professional satisfaction among the nurses , and , sadly , all have indicated low degrees of satisfaction ( 7 ) . most nurses who work in internal medicine and surgical departments ( 54.4% ) demonstrated low levels of professional satisfaction ( 8) . overall , these studies suggested that the situation concerning nurses professional satisfaction is unacceptable . it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run . it could give rise to rebellion , diminished feelings of responsibility , and eventually resignation from the job ( 9 ) . professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and ineffectiveness ( 10 ) . according to a qualitative study done in 2010 in india , the reasons nurses quit their jobs were determined to be professional exhaustion , stress at work , and lack of a friendly environment , all of which contributed to their diminished professional satisfaction . in addition , it was indicated that a strong relationship exists between professional dissatisfaction and quitting work ( 11 ) . according to released statistics , the insufficient number of available nurses around the world is a tangible phenomenon . in australia , for instance furthermore , according to estimations , by 2010 , canada will need an additional 113,000 nurses , and , by 2020 , the united states would incur a 20% shortage in the number of nurses ( approximately 581,500 nurses ) ( 12 , 13 ) . in iran , one of the most problematic issues in nursing is the shortage of nursing staff . the shortage of nursing staff has different causes , including job dissatisfaction and organizational and sociocultural factors ( 14 ) . in iran , many studies have reported that nurses are not happy because of the shortage of nursing staff , high numbers of patients , the lack of independence and authority , the lack of appreciation and acknowledgment , low income , heavy workloads , low level of involvement in management , disagreements with physicians , managerial issues , and a lack of appropriate work conditions in hospitals ( 15 ) . iran regret having become nurses , whereas 43.9% of them feel disappointed and dissatisfied that they became nurses . the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation , and , unfortunately , most nurses in iran would like to change their work ( 14 ) . because satisfaction with nursing is related to improving the health system and patient satisfaction , this study was conducted to assess how the satisfaction of nurses with their work can be improved , their situation in hospitals can be improved , and also how patients satisfaction could be increased ( 15 ) . the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction . today s world is a world of organizations , and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives . without people , the concepts of organization and management would become meaningless ( 1 ) . therefore , professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations . the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s , and , according to many experts , it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness ( 2 ) . health care organizations and treatment centers are responsible for maintaining the health of all citizens in a society , and nurses have a key role . their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction ( 1 ) . previous studies have shown that nurses should demonstrate a passion for their work and have the necessary proficiency and expertise to achieve the highest level of efficiency ( 2 ) . it could be argued that any person would provide better services if he or she were motivated and enthusiastic about the work . despite the great significance of the professional satisfaction of nurses due to its considerable impact on them , their patients , their organizations , and the very profession of nursing ( 3 ) , most previous studies have reported the satisfaction level to be low among nurses ( 4 ) . the results from the study conducted by lou et al . indicated that only half of the nurses in the study were moderately or highly satisfied with their work ( 5 ) . another study was conducted to compare the level of professional satisfaction among different groups of people in korea , and it was found that only 41.5% of nurses were satisfied with their work , whereas the percentages for other experts working at hospitals , social workers , and middle school teachers were 50.1 , 58.2 , and 89% , respectively . these statistics indicate that there is a lower level of satisfaction among nurses than other professionals ( 6 ) . various studies have been conducted regarding the extent of professional satisfaction among the nurses , and , sadly , all have indicated low degrees of satisfaction ( 7 ) . most nurses who work in internal medicine and surgical departments ( 54.4% ) demonstrated low levels of professional satisfaction ( 8) . overall , these studies suggested that the situation concerning nurses professional satisfaction is unacceptable . it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run . it could give rise to rebellion , diminished feelings of responsibility , and eventually resignation from the job ( 9 ) . professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and ineffectiveness ( 10 ) . according to a qualitative study done in 2010 in india , the reasons nurses quit their jobs were determined to be professional exhaustion , stress at work , and lack of a friendly environment , all of which contributed to their diminished professional satisfaction . in addition , it was indicated that a strong relationship exists between professional dissatisfaction and quitting work ( 11 ) . according to released statistics , the insufficient number of available nurses around the world is a tangible phenomenon . in australia , for instance furthermore , according to estimations , by 2010 , canada will need an additional 113,000 nurses , and , by 2020 , the united states would incur a 20% shortage in the number of nurses ( approximately 581,500 nurses ) ( 12 , 13 ) . in iran , one of the most problematic issues in nursing is the shortage of nursing staff . the shortage of nursing staff has different causes , including job dissatisfaction and organizational and sociocultural factors ( 14 ) . in iran , many studies have reported that nurses are not happy because of the shortage of nursing staff , high numbers of patients , the lack of independence and authority , the lack of appreciation and acknowledgment , low income , heavy workloads , low level of involvement in management , disagreements with physicians , managerial issues , and a lack of appropriate work conditions in hospitals ( 15 ) . approximately 45% of nurses in iran regret having become nurses , whereas 43.9% of them feel disappointed and dissatisfied that they became nurses . the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation , and , unfortunately , most nurses in iran would like to change their work ( 14 ) . because satisfaction with nursing is related to improving the health system and patient satisfaction , this study was conducted to assess how the satisfaction of nurses with their work can be improved , their situation in hospitals can be improved , and also how patients satisfaction could be increased ( 15 ) . the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction . this was a qualitative study that was conducted with a targeted sampling of 10 nurses ( 4 men and 6 women ) , with a minimum of one year and a maximum of 25 years of work experience . all participants had earned a bachelor s degree and cared for patients directly as staff members in general or intensive care units at public or private hospitals in tehran . in this study , data were collected using semi - structured interviews . before conducting the interviews , the objective of the study was explained to the participants , and their consent to participate in the study was obtained in writing . all interviewees were ensured that their identities would not be disclosed in the reports and that they could have access to the results . the interviews were conducted in a place where the participants felt comfortable , such as in the break room in the hospital where they worked . in addition to covering the general questions of the study , attempts were made to personalize each interview depending on its flow . the qca is an analytical procedure that is utilized to provide a subjective interpretation of textual data contents ( 16 ) . in this method , codes and categories are extracted from raw data directly and inductively through a systematic categorization process ( 17 ) . content analysis is much more than the extraction of objective contents out of textual data , i.e. , key concepts and hidden patterns may be revealed from the content of the participant - generated data through this method ( 16 ) . in the qca , data collection and analysis are done simultaneously ( 17 ) . in this method , the researcher collects and analyzes data back and forth in order to collect new data to be able to provide answers to the research questions ( 16 ) . an analytical unit is a part of text that can be analyzed to help achieve the research objectives . initial codes were extracted from meaning units , which are important and reliable parts of the analytical units . initial codes could contain either the exact content of the participant s interview or the abstract of the content . based on their similarities and differences , the initial codes were reduced to sub - categories , which , in turn , were used to abstract categories and key concepts ( 17 ) . in the first step of this study , the researchers listened to the interviews , and then they wrote the results of the interviews word for word . in the next step , then , based on similarities and differences , the initial codes were reduced to sub - categories , abstracts were prepared , and the key codes were identified . in order to achieve reliability in this research , the data were controlled by the participants and investigated by an investigation team . in addition to restoring the speech and experiences of the participants during the interviews by replaying them , the researcher gave them full typed texts of the first four interviews together with their initial codes to those with whom he or she had conducted the initial interviews for verified or revision . in addition , coding and initial categories with respect to content analysis were submitted to the researcher s supervisor , and verification was received on her / his part regarding the implementation , coding , and initial categories . in order to increase the reliability of the data , the researcher attempted to establish a deeper relationship with the participants and also allocated sufficient time for the interviews . the researcher also attempted to provide a friendly atmosphere in order to gain the participants trust , so that more accurate data and information could be elicited . the participants were willing to participate in the interviews , and they express their feelings , experiences , and thoughts with no fear of censorship or pressure . also , to increase the degree of reliability , the researcher attempted to avoid including his personal preferences in the study . from the qualitative analysis of the data collected from the interviewees , fair conduct was extracted as the turning point and basis upon which professional satisfaction of nurses could be achieved , which , in turn , consisted of three sub - categories , i.e. , 1- expectation of fairness in social - professional settings , 2- expectation of fairness in receiving professional benefits , and 3- expectation of fairness in the area of professional interactions . many of the participants maintained that there was a gross distinction between what the society said and how people reacted , in that , even though nurses were academically educated , cared for patients , and dealt with their problems in a never - ending manner , they were not regarded by patients , their families , or even society as having an equal status to those engaged in other professions , including medicine . one of the nurses expressed her feelings by saying , nursing is a difficult job . you study for four years at university just like any other discipline , you get a bachelor degree , yet at the end , people regard nurses as a bunch of illiterates with no independence or literacy whatsoever , and who merely obey what doctors command them to do . society regards nurses as mere observers who tend their patients , but view doctors as even those engaged in other fields such as accounting , engineering , etc . with equivalent bachelor degrees enjoy a better salary and are held in greater esteem than the people employed in nursing . yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do , are referred to ( e.g. the engineer man / lady ) , or when i compare the level of respect shown to doctors with that shown to nurses . people do not have high regards for the community of nurses ; they view them as ill - humored or bad people . , nurses are shown wearing dressed with all kinds of colors running after doctors . yet , in those same movies , doctors are displayed as distinguished people helping patients . most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction , in such a way that their attempts were directed towards quitting their job or finding a new one with better salary . concerning this matter , one of the nurses stated , in today s world my basic needs have to be fulfilled , and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors . on lack of a financial fairness , another nurse maintained , it might be true that a doctor s education is higher than that of a nurse , but a nurse devotes more of his / her time to patients , in return . the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy . in advanced countries , nursing has a high status , while this is not the case for iran , and that is exactly the reason behind many dissatisfactions . a male nurse expressed his discontent of nursing as follows , with 35 years of age , i suffer from diabetes due to the stress at work , which i try to handle with strict regiments and such . i am trying to change my job , since no matter how hard i work i end up having nothing . i have to hold two or three jobs to be able to buy a house . yet , this is not the case for those engaging in other professions , with little or no difference than nursing from an academic point of view ? another male nurse expressed his discontent of nursing as follows , i know for a fact that , nurses in the great britain earn an equal amount of salary compared to surgeons , which is something to reflect upon . that how the monthly income of a nurse is equal to that of a surgeon is something that requires serious contemplation . furthermore , they merely work one shift and tend two patients all night long , unlike in iran , where two nurses tend 45 patients simultaneously . another nurse with two years experience said , with rising inflation , doctors salaries and fees increase day by day , whether officially or unofficially , and the authorities regard this as their inalienable right . however , nurses live off of a petty salary and then they complain about the few number of working nurses , and that why it is that they continually quit their job ! most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job , the most important of which was the viewpoint and attitude of hospital managers towards nurses . one of the participants in this study voiced her / his opinion on this matter as such , the treatment received by nurses from hospital management is unfair . they do not show them the level of respect they deserve , yet always support doctors rights . one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction , during all these years that i have worked as a nurse , i have seen with my own eyes that many decisions are made for us by those in the medical department . they are just not fair ! another participant stated , justice is not observed at iranian hospitals . one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction , since , as you know , working with mentally - challenged patients drives one to exhaustion . yet , despite all these hardships , our relationship with our managers is at a very high level . they act very reasonably , and this brings about satisfaction and makes us care for patients with greater motivation . the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place . the following statement could be brought to attention in this regard , despite the high level of physical and mental exhaustion that our work brings about , a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job , including lack of sleep , intensity of work , low salary , etc . many of the participants maintained that there was a gross distinction between what the society said and how people reacted , in that , even though nurses were academically educated , cared for patients , and dealt with their problems in a never - ending manner , they were not regarded by patients , their families , or even society as having an equal status to those engaged in other professions , including medicine . one of the nurses expressed her feelings by saying , nursing is a difficult job . you study for four years at university just like any other discipline , you get a bachelor degree , yet at the end , people regard nurses as a bunch of illiterates with no independence or literacy whatsoever , and who merely obey what doctors command them to do . society regards nurses as mere observers who tend their patients , but view doctors as even those engaged in other fields such as accounting , engineering , etc . with equivalent bachelor degrees enjoy a better salary and are held in greater esteem than the people employed in nursing . yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do , are referred to ( e.g. the engineer man / lady ) , or when i compare the level of respect shown to doctors with that shown to nurses . people do not have high regards for the community of nurses ; they view them as ill - humored or bad people . , nurses are shown wearing dressed with all kinds of colors running after doctors . yet , in those same movies , doctors are displayed as distinguished people helping patients . most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction , in such a way that their attempts were directed towards quitting their job or finding a new one with better salary . concerning this matter , one of the nurses stated , in today s world my basic needs have to be fulfilled , and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors . on lack of a financial fairness , another nurse maintained , it might be true that a doctor s education is higher than that of a nurse , but a nurse devotes more of his / her time to patients , in return . the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy . in advanced countries , nursing has a high status , while this is not the case for iran , and that is exactly the reason behind many dissatisfactions . a male nurse expressed his discontent of nursing as follows , with 35 years of age , i suffer from diabetes due to the stress at work , which i try to handle with strict regiments and such . i am trying to change my job , since no matter how hard i work i end up having nothing . i have to hold two or three jobs to be able to buy a house . yet , this is not the case for those engaging in other professions , with little or no difference than nursing from an academic point of view ? another male nurse expressed his discontent of nursing as follows , i know for a fact that , nurses in the great britain earn an equal amount of salary compared to surgeons , which is something to reflect upon . that how the monthly income of a nurse is equal to that of a surgeon is something that requires serious contemplation . furthermore , they merely work one shift and tend two patients all night long , unlike in iran , where two nurses tend 45 patients simultaneously . another nurse with two years experience said , with rising inflation , doctors salaries and fees increase day by day , whether officially or unofficially , and the authorities regard this as their inalienable right . however , nurses live off of a petty salary and then they complain about the few number of working nurses , and that why it is that they continually quit their job ! most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job , the most important of which was the viewpoint and attitude of hospital managers towards nurses . one of the participants in this study voiced her / his opinion on this matter as such , the treatment received by nurses from hospital management is unfair . they do not show them the level of respect they deserve , yet always support doctors rights . one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction , during all these years that i have worked as a nurse , i have seen with my own eyes that many decisions are made for us by those in the medical department . they are just not fair ! another participant stated , justice is not observed at iranian hospitals . one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction , since , as you know , working with mentally - challenged patients drives one to exhaustion . yet , despite all these hardships , our relationship with our managers is at a very high level . they act very reasonably , and this brings about satisfaction and makes us care for patients with greater motivation . the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place . the following statement could be brought to attention in this regard , despite the high level of physical and mental exhaustion that our work brings about , a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job , including lack of sleep , intensity of work , low salary , etc . more bearable . as was discussed , one of the main components of professional satisfaction is fair conduct , which consists of three sub - categories , i.e. , expectation of fairness in social - professional settings , expectation of fairness in receiving professional benefits , and expectation of fairness in the area of professional interactions . the subject of fairness is one of the few fundamental topics relevant to various spheres of science and philosophy , and it can be applied to any situation in terms of people s needs and expectations . the concept of fairness is integrated with moral and social values and within the sphere of morality , in particular social morality , supporting fairness and equity is of the most fundamental moral values . researchers regard the establishment of justice as one of the most important factors affecting professional satisfaction ( 18 ) . one of the long - term challenges of the nursing profession is its public image ( 19 ) . there are various ups and downs to nursing due to the challenging nature of this profession from the initial education at university until retirement . dropping out of university , although low salaries and benefits and professional exhaustion are among the factors causing discontent among nurses , according to conducted studies , lack of consideration towards the status and public image of the nursing profession is responsible for 70% of professional dissatisfaction ( 20 ) . generally , people form opinions or mental images of other individuals based on their look , way of clothing , reaction , communication , behavior and attitude ( 21 ) . the image is the perception or assumption that the general public makes about an individual , profession , or organization . the people within those organizations use this image as a tool to communicate with people , and they , in turn , interact with those organizations based on the projected image , whether positive or negative ( 20 ) . the social status of a profession and the society s perspective towards it are of effective motivational factors influencing the extent of professional satisfaction , which , in turn , leaves an impact on the attitude shown by individuals in fulfilling their professional functions . these statuses include placing a high value on a profession and its required proficiencies and the value and status of a profession in a particular society . whenever the status is damaged on a societal scale , the status of the profession is also diminished ( 22 ) . in nursing , one of the most important concerns with regards to professional status is lack of a high value and social status within society . with regards to the importance of this issue , stevens stated that public opinion acts very strongly in determining the social structure and norms of a society ( 23 ) . a positive public image will leave a considerable effect on gaining social and situational support . as was understood from the findings of this study , most nurses in this research regarded professional satisfaction as manifested through expectation of fairness in social - professional settings . regarding the second important finding of this study , i.e. , the expectation of fairness in receiving professional benefits , the participants stated that unequal salaries among the personnel of the medical department and a gross distinction between the payments to the medical staff have been sources of disappointment and dissatisfaction . markovsky was among the first theoreticians to recognize the significance of the issue of salary satisfaction in observing fairness ( 24 ) . therefore , the findings of this study , that is , feeling a sense of fairness in receiving professional benefits has an intermediary role in bringing professional satisfaction , is in line with markovsky s theory . furthermore , expectation of fairness in receiving professional benefits gives the indication that , if individuals deem a condition to be unfair , they will have an emotional response to it ( 25 ) . yet another important finding of this study was the expectation of fairness in professional interactions . the participants of this study stated that the treatment they receive from managers is unfair and disrespectful compared to other health teams . defined interactional justice as the feeling experienced by employees when interacting with their supervisor and state ; cooperation with supervisors and other members of the work team and respectful behavior , and providing the required information would result in a high satisfaction level among employees ( 25 ) . kim et al . maintained that interactional fairness , fair treatment by managers towards employees , and instituting behavioral justice significantly affect professional satisfaction ( 26 ) . regarding the issue of fairness , the results of the present study were in line with those obtained by lou et al . ( 21 ) , in which it was demonstrated that british nurses were more displeased compared to australian nurses . the british nurses felt that their professional statuses were lower , their relationships with hospital managers were weaker , and their work conditions were less favorable than those of australian nurses . the results of this study confirm the point that the existence of a relative social status to profession , and of justice within the work environment , and also respectful treatment towards nurses by management . generally speaking , fair conduct is an integral part of professional satisfaction in the nursing profession , which could encourage nurses to employ the related measures and interventions with a higher degree of motivation ( 18 ) . furthermore , a review of the previous studies indicated that management and administrative experiences , which refer to organizational structures , such as ( de)centralization and fair and transparent organizational policies and procedures are among the factors affecting professional satisfaction . researchers have found out that decentralization and all kinds of cooperative management models are more to nurses liking , and that they resent being looked at with indifference , and this leads to a lower professional satisfaction level in their eyes ( 12 ) . the study conducted by farsi et al . indicated that the low public image of nursing in the iranian society has caused nurses to feel unappreciated and disrespected ( 16 ) . as was demonstrated in other studies conducted in iran , the motivation level of iranian nurses in fulfilling their professional tasks , which to a large degree is dependent upon the public image , is not acceptable ( 20 ) . in general , since professional satisfaction is a single concept that can not be deconstructed , it is possible to be defined by individuals in vague manners . therefore , professional satisfaction can be viewed as a general feeling , or as the attitude taken by an individual with respect to part of her or his job . overall , it is possible to improve the professional satisfaction of nurses through emphasizing the ( in)visible aspects of professional satisfaction , such as fair conduct , removing tension - generating factors , improving the attitude of society towards the nursing profession , establishing justice among all hospital staff , improving communication , organizing the nursing profession , and enhancing the payment system and making it fair .

introductionthe professional satisfaction of staff is one of the most challenging organizational concepts that can enhance the efficiency level of organizations . in a similar vein , the professional satisfaction of nurses is of considerable importance , in that , professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and inefficacy and would negatively impact the organization s work rate . the aim of this study was to understand iranian nurses experiences of the concept of professional satisfaction.methodsthis was a qualitative study conducted with a targeted sampling of 10 nurses ( 4 men and 6 women ) in 2015 . the data were collected through conducting in - depth interviews , and textual data were analyzed subsequently using the qualitative content analysis ( qca ) method.resultsthe findings of this study pointed to fair conduct , which was comprised of three sub - categories , i.e. , expectation of fairness in social - professional settings , expectation of fairness in receiving professional benefits , and expectation of fairness in the area of professional interactions.conclusionsthere are various ups and downs in nursing due to the challenging nature of the profession , from the initial education at the university until retirement . according to the findings of this study , a lack of fairness in social - professional settings , a lack of fairness in receiving professional benefits , and a lack of fairness in the area of professional interactions were among the factors that have great impacts on the degree of professional dissatisfaction among nurses .

1. Introduction 1.1. Background 1.2. Statement of the problem and study logic 1.3. Objective 2. Material and Methods 3. Results 3.1. Expectation of fairness in social-professional settings 3.2. Expectation of fairness in receiving professional benefits 3.3. Expectation of fairness in the area of professional interactions 4. Discussion 5. Conclusions

therefore , professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations . the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s , and , according to many experts , it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness ( 2 ) . their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction ( 1 ) . despite the great significance of the professional satisfaction of nurses due to its considerable impact on them , their patients , their organizations , and the very profession of nursing ( 3 ) , most previous studies have reported the satisfaction level to be low among nurses ( 4 ) . the results from the study conducted by lou et al . another study was conducted to compare the level of professional satisfaction among different groups of people in korea , and it was found that only 41.5% of nurses were satisfied with their work , whereas the percentages for other experts working at hospitals , social workers , and middle school teachers were 50.1 , 58.2 , and 89% , respectively . various studies have been conducted regarding the extent of professional satisfaction among the nurses , and , sadly , all have indicated low degrees of satisfaction ( 7 ) . it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run . professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and ineffectiveness ( 10 ) . according to a qualitative study done in 2010 in india , the reasons nurses quit their jobs were determined to be professional exhaustion , stress at work , and lack of a friendly environment , all of which contributed to their diminished professional satisfaction . according to released statistics , the insufficient number of available nurses around the world is a tangible phenomenon . in australia , for instance furthermore , according to estimations , by 2010 , canada will need an additional 113,000 nurses , and , by 2020 , the united states would incur a 20% shortage in the number of nurses ( approximately 581,500 nurses ) ( 12 , 13 ) . in iran , one of the most problematic issues in nursing is the shortage of nursing staff . in iran , many studies have reported that nurses are not happy because of the shortage of nursing staff , high numbers of patients , the lack of independence and authority , the lack of appreciation and acknowledgment , low income , heavy workloads , low level of involvement in management , disagreements with physicians , managerial issues , and a lack of appropriate work conditions in hospitals ( 15 ) . the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation , and , unfortunately , most nurses in iran would like to change their work ( 14 ) . because satisfaction with nursing is related to improving the health system and patient satisfaction , this study was conducted to assess how the satisfaction of nurses with their work can be improved , their situation in hospitals can be improved , and also how patients satisfaction could be increased ( 15 ) . the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction . today s world is a world of organizations , and their main operators are the people who invigorate them and prepare the grounds for the realization of their objectives . therefore , professional satisfaction on the part of manpower is one of the most important factors in the efficacy of organizations . the issue of professional satisfaction has been studied repeatedly with respect to various organizations since the 1920s , and , according to many experts , it is one of the most challenging organizational concepts and the basis behind the many management policies adopted to improve organizational effectiveness ( 2 ) . their success in fulfilling this role depends on the measures taken by competent managers who recognize the prominent factors involved in the activities of nurses and seek to create favorable work conditions in order to enhance their professional satisfaction ( 1 ) . despite the great significance of the professional satisfaction of nurses due to its considerable impact on them , their patients , their organizations , and the very profession of nursing ( 3 ) , most previous studies have reported the satisfaction level to be low among nurses ( 4 ) . another study was conducted to compare the level of professional satisfaction among different groups of people in korea , and it was found that only 41.5% of nurses were satisfied with their work , whereas the percentages for other experts working at hospitals , social workers , and middle school teachers were 50.1 , 58.2 , and 89% , respectively . various studies have been conducted regarding the extent of professional satisfaction among the nurses , and , sadly , all have indicated low degrees of satisfaction ( 7 ) . most nurses who work in internal medicine and surgical departments ( 54.4% ) demonstrated low levels of professional satisfaction ( 8) . it should be noted that a lack of sufficient concern towards professional satisfaction will result in serious consequences in the long run . professional dissatisfaction among nurses could result in emotional detachment , depression , anger , evasion from work , and ineffectiveness ( 10 ) . according to a qualitative study done in 2010 in india , the reasons nurses quit their jobs were determined to be professional exhaustion , stress at work , and lack of a friendly environment , all of which contributed to their diminished professional satisfaction . according to released statistics , the insufficient number of available nurses around the world is a tangible phenomenon . in australia , for instance furthermore , according to estimations , by 2010 , canada will need an additional 113,000 nurses , and , by 2020 , the united states would incur a 20% shortage in the number of nurses ( approximately 581,500 nurses ) ( 12 , 13 ) . in iran , one of the most problematic issues in nursing is the shortage of nursing staff . in iran , many studies have reported that nurses are not happy because of the shortage of nursing staff , high numbers of patients , the lack of independence and authority , the lack of appreciation and acknowledgment , low income , heavy workloads , low level of involvement in management , disagreements with physicians , managerial issues , and a lack of appropriate work conditions in hospitals ( 15 ) . the most challenging problem in nursing in iran is the shortage of nurses working in hospitals because of their dissatisfaction and lack of motivation , and , unfortunately , most nurses in iran would like to change their work ( 14 ) . because satisfaction with nursing is related to improving the health system and patient satisfaction , this study was conducted to assess how the satisfaction of nurses with their work can be improved , their situation in hospitals can be improved , and also how patients satisfaction could be increased ( 15 ) . the objective of this research was to determine iranian nurses experiences of the concept of professional satisfaction . this was a qualitative study that was conducted with a targeted sampling of 10 nurses ( 4 men and 6 women ) , with a minimum of one year and a maximum of 25 years of work experience . in this study , data were collected using semi - structured interviews . before conducting the interviews , the objective of the study was explained to the participants , and their consent to participate in the study was obtained in writing . content analysis is much more than the extraction of objective contents out of textual data , i.e. based on their similarities and differences , the initial codes were reduced to sub - categories , which , in turn , were used to abstract categories and key concepts ( 17 ) . in the first step of this study , the researchers listened to the interviews , and then they wrote the results of the interviews word for word . in the next step , then , based on similarities and differences , the initial codes were reduced to sub - categories , abstracts were prepared , and the key codes were identified . in order to achieve reliability in this research , the data were controlled by the participants and investigated by an investigation team . in addition to restoring the speech and experiences of the participants during the interviews by replaying them , the researcher gave them full typed texts of the first four interviews together with their initial codes to those with whom he or she had conducted the initial interviews for verified or revision . in addition , coding and initial categories with respect to content analysis were submitted to the researcher s supervisor , and verification was received on her / his part regarding the implementation , coding , and initial categories . in order to increase the reliability of the data , the researcher attempted to establish a deeper relationship with the participants and also allocated sufficient time for the interviews . the participants were willing to participate in the interviews , and they express their feelings , experiences , and thoughts with no fear of censorship or pressure . also , to increase the degree of reliability , the researcher attempted to avoid including his personal preferences in the study . from the qualitative analysis of the data collected from the interviewees , fair conduct was extracted as the turning point and basis upon which professional satisfaction of nurses could be achieved , which , in turn , consisted of three sub - categories , i.e. , 1- expectation of fairness in social - professional settings , 2- expectation of fairness in receiving professional benefits , and 3- expectation of fairness in the area of professional interactions . many of the participants maintained that there was a gross distinction between what the society said and how people reacted , in that , even though nurses were academically educated , cared for patients , and dealt with their problems in a never - ending manner , they were not regarded by patients , their families , or even society as having an equal status to those engaged in other professions , including medicine . yet another participant stressed the significance of professional satisfaction in making nurses continue to do their jobs ; i become very upset when i see the respectful way by which people of other professions who have bachelor degrees just like we do , are referred to ( e.g. most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction , in such a way that their attempts were directed towards quitting their job or finding a new one with better salary . concerning this matter , one of the nurses stated , in today s world my basic needs have to be fulfilled , and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors . on lack of a financial fairness , another nurse maintained , it might be true that a doctor s education is higher than that of a nurse , but a nurse devotes more of his / her time to patients , in return . the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy . a male nurse expressed his discontent of nursing as follows , with 35 years of age , i suffer from diabetes due to the stress at work , which i try to handle with strict regiments and such . another male nurse expressed his discontent of nursing as follows , i know for a fact that , nurses in the great britain earn an equal amount of salary compared to surgeons , which is something to reflect upon . most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job , the most important of which was the viewpoint and attitude of hospital managers towards nurses . one of the participants in this study voiced her / his opinion on this matter as such , the treatment received by nurses from hospital management is unfair . one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction , during all these years that i have worked as a nurse , i have seen with my own eyes that many decisions are made for us by those in the medical department . one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction , since , as you know , working with mentally - challenged patients drives one to exhaustion . the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place . the following statement could be brought to attention in this regard , despite the high level of physical and mental exhaustion that our work brings about , a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job , including lack of sleep , intensity of work , low salary , etc . many of the participants maintained that there was a gross distinction between what the society said and how people reacted , in that , even though nurses were academically educated , cared for patients , and dealt with their problems in a never - ending manner , they were not regarded by patients , their families , or even society as having an equal status to those engaged in other professions , including medicine . most of the participants stated that unfair practices at work with respect to financial matters had left them disappointed and lowered their professional satisfaction , in such a way that their attempts were directed towards quitting their job or finding a new one with better salary . concerning this matter , one of the nurses stated , in today s world my basic needs have to be fulfilled , and currently a nurse s salary is not in any sense proportionate to that earned by those working in other medical sectors . the thing that bothers the iranian nurses the most is their awareness of all professional benefits that nurses working in other countries enjoy . a male nurse expressed his discontent of nursing as follows , with 35 years of age , i suffer from diabetes due to the stress at work , which i try to handle with strict regiments and such . another male nurse expressed his discontent of nursing as follows , i know for a fact that , nurses in the great britain earn an equal amount of salary compared to surgeons , which is something to reflect upon . most of the participants viewed a number of factors as influencing the satisfaction they felt in doing their job , the most important of which was the viewpoint and attitude of hospital managers towards nurses . one of the participants in this study voiced her / his opinion on this matter as such , the treatment received by nurses from hospital management is unfair . one of the nurses pointed to decision making by doctors or nurses as one of the factors causing professional dissatisfaction , during all these years that i have worked as a nurse , i have seen with my own eyes that many decisions are made for us by those in the medical department . one of the nurses working in the department of psychiatry pointed to the following as one of the reasons behind his / her professional satisfaction , since , as you know , working with mentally - challenged patients drives one to exhaustion . the belief in establishing a kind of justice on the part of some of the participants was so strong that they viewed the existing difficulties in the nursing profession to be more bearable if some sense of justice was in place . the following statement could be brought to attention in this regard , despite the high level of physical and mental exhaustion that our work brings about , a fair treatment by managers and equality with respect to observing the rules would make the hardships of this job , including lack of sleep , intensity of work , low salary , etc . as was discussed , one of the main components of professional satisfaction is fair conduct , which consists of three sub - categories , i.e. , expectation of fairness in social - professional settings , expectation of fairness in receiving professional benefits , and expectation of fairness in the area of professional interactions . the subject of fairness is one of the few fundamental topics relevant to various spheres of science and philosophy , and it can be applied to any situation in terms of people s needs and expectations . the concept of fairness is integrated with moral and social values and within the sphere of morality , in particular social morality , supporting fairness and equity is of the most fundamental moral values . researchers regard the establishment of justice as one of the most important factors affecting professional satisfaction ( 18 ) . one of the long - term challenges of the nursing profession is its public image ( 19 ) . there are various ups and downs to nursing due to the challenging nature of this profession from the initial education at university until retirement . dropping out of university , although low salaries and benefits and professional exhaustion are among the factors causing discontent among nurses , according to conducted studies , lack of consideration towards the status and public image of the nursing profession is responsible for 70% of professional dissatisfaction ( 20 ) . the people within those organizations use this image as a tool to communicate with people , and they , in turn , interact with those organizations based on the projected image , whether positive or negative ( 20 ) . the social status of a profession and the society s perspective towards it are of effective motivational factors influencing the extent of professional satisfaction , which , in turn , leaves an impact on the attitude shown by individuals in fulfilling their professional functions . whenever the status is damaged on a societal scale , the status of the profession is also diminished ( 22 ) . in nursing , one of the most important concerns with regards to professional status is lack of a high value and social status within society . as was understood from the findings of this study , most nurses in this research regarded professional satisfaction as manifested through expectation of fairness in social - professional settings . regarding the second important finding of this study , i.e. , the expectation of fairness in receiving professional benefits , the participants stated that unequal salaries among the personnel of the medical department and a gross distinction between the payments to the medical staff have been sources of disappointment and dissatisfaction . markovsky was among the first theoreticians to recognize the significance of the issue of salary satisfaction in observing fairness ( 24 ) . therefore , the findings of this study , that is , feeling a sense of fairness in receiving professional benefits has an intermediary role in bringing professional satisfaction , is in line with markovsky s theory . furthermore , expectation of fairness in receiving professional benefits gives the indication that , if individuals deem a condition to be unfair , they will have an emotional response to it ( 25 ) . yet another important finding of this study was the expectation of fairness in professional interactions . the participants of this study stated that the treatment they receive from managers is unfair and disrespectful compared to other health teams . defined interactional justice as the feeling experienced by employees when interacting with their supervisor and state ; cooperation with supervisors and other members of the work team and respectful behavior , and providing the required information would result in a high satisfaction level among employees ( 25 ) . maintained that interactional fairness , fair treatment by managers towards employees , and instituting behavioral justice significantly affect professional satisfaction ( 26 ) . regarding the issue of fairness , the results of the present study were in line with those obtained by lou et al . the results of this study confirm the point that the existence of a relative social status to profession , and of justice within the work environment , and also respectful treatment towards nurses by management . generally speaking , fair conduct is an integral part of professional satisfaction in the nursing profession , which could encourage nurses to employ the related measures and interventions with a higher degree of motivation ( 18 ) . furthermore , a review of the previous studies indicated that management and administrative experiences , which refer to organizational structures , such as ( de)centralization and fair and transparent organizational policies and procedures are among the factors affecting professional satisfaction . as was demonstrated in other studies conducted in iran , the motivation level of iranian nurses in fulfilling their professional tasks , which to a large degree is dependent upon the public image , is not acceptable ( 20 ) . in general , since professional satisfaction is a single concept that can not be deconstructed , it is possible to be defined by individuals in vague manners . therefore , professional satisfaction can be viewed as a general feeling , or as the attitude taken by an individual with respect to part of her or his job . overall , it is possible to improve the professional satisfaction of nurses through emphasizing the ( in)visible aspects of professional satisfaction , such as fair conduct , removing tension - generating factors , improving the attitude of society towards the nursing profession , establishing justice among all hospital staff , improving communication , organizing the nursing profession , and enhancing the payment system and making it fair .

nationwide interest in the cost to society of drug abuse has been on the rise in recent years , particularly among those groups responsible for national , state , and local policy , as well as among leaders of industry confronted by soaring health insurance costs and diminished worker productivity . a recent estimate of the overall cost is $ 58 billion dollars ( rice et al . , methods used to estimate these costs have varied considerably , and have been consistently hampered by the meager availability of data on both the distribution and dynamics of drug disorders in society and the ways and means to most effectively treat those afflicted . prevention efforts are also hampered by the lack of data delineating the associated costs , risk factors , and determinants of individual susceptibility . the investment in drug treatment has increased , as has a recognition of the need for additional data on the effects of drug disorders on hospital costs . current research relies on selected samples of the population , such as those referred for treatment by the criminal justice system , or national representative samples , such as those used in the national hospital discharge survey ( nhds ) . such samples are limited in their generalizability to the rest of the national population or even to smaller demographic groups of interest . in the case of a national sample , furthermore , in a discharge survey such as nhds , there is no way for researchers to identify and combine records to access information on patient history , because the sampling is done on discharges . it would be impossible to link all records to do a patient - based analysis because the sampling scheme will eliminate some of these records . thus , the great advantage of using 1 fiscal year of administrative records , such as the discharge records of the medicare hospital part a billing record , is the ability to assemble discharges into one patient record . patient - based studies to date have been limited in scope both because of the difficulties in obtaining a patient 's complete medical administrative records and the great expense of following patients in prospective studies . opportunities to examine the medicare population are available within the large reimbursement system data base . these data comprise about 96 percent of the population 65 years of age or over . the data on the medicare disabled population represent a much smaller proportion of the total disabled population because of the stringent process to obtain eligibility . although medicare data include actual yearly charges for essentially the entire medicare population , the reported charges are very limited in scope ( in this case , to short - stay , inpatient , hospital facilities ) and do not represent the total spectrum of incurred expenses in the aged population . the availability of data for the disabled medicare population provides an opportunity to study a younger population . little is known about the adm disorder comorbidity of this group . a recent study ( cartwright and ingster , to be published ) conducted on medicare discharge data indicated that the expectation of higher charges associated with hospital stays involving drug disorders is not necessarily borne out . evidence from this study showed that , on a per discharge basis , drug disorder diagnosed patients incur lower charges than patients not diagnosed with drug disorders . this finding motivated us to change the unit of analysis from discharge to the patient . we speculated that these patients may initially be healthier in order to support the underlying drug abuse or dependency that leads to the drug disorder , may be discharged earlier , or may simply require less of the acute , intensive , and costly care normally associated with short - stay hospital visits , and incur the lower costs associated with constant observation or convalescent care . economic factors associated with the medicare reimbursement system itself , particularly the prospective payment system ( pps ) , may also influence or require hospitals and physicians to discharge drug disorder diagnosed patients sooner than they might otherwise . another possible reason for the observed lower charges may lie with the diagnosis - related group ( drg ) codes . prior to fy 1988 , certain types of comorbidities were not reimbursable , which may have resulted in an underestimate of the true prevalence of patients with drug disorders by their international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) discharge codes . in this article , annual charges for patients are aggregated from a year of discharge data so that comparisons may be made between patients with diagnoses of drug disorder and patients with no drug disorder . we hypothesize that patients with drug disorder diagnoses might be more frequent users of inpatient hospital resources . in practice therefore , the patients with identified icd-9-cm drug disorder diagnoses would cost more on an annual basis than would patients without this disorder . this hypothesis would then be reflected in longer average loss , higher average annual hospital charges , and a greater number of average annual discharges . the entire universe of medicare discharges submitted for reimbursement during fy 1987 was utilized to create a patient file . these data were collected by the health care financing administration ( hcfa ) and can be found in the part a medpar file . the discharge records were sorted and matched by beneficiary identification code , provider number , and social security number codes , which would identify all records belonging to one individual . the analysis file is limited to individuals who were inpatients in short - stay hospitals . medicare patients classified with end stage renal disease ( esrd ) were excluded because of the uncertain interpretation of drug disorder effects for a terminal renal disease , and those discharges whose principal diagnosis would fall into the icd-9-cm category of pregnancy , childbirth , and puerperium were also excluded because of the unique nature of this medical event . the variables selected for examination were race , gender , age , medicare status ( aged , disabled ) , los ( days ) , total hospital charges , and the five discharge diagnoses per record . these were used to create several broad drug disorder diagnostic groups and to identify tobacco use disorder , and alcohol and psychiatric disorders . statistics presented by race were limited to white and black persons and do not cover the remaining population . the other race categories did not have sufficient numbers to allow for meaningful population comparisons across the desired categories . the medicare status category of aged is comprised of individuals 65 years of age or over , and the disabled category is comprised of those individuals either physically or mentally disabled , who are under 65 years of age and qualify for medicare support . summary files were created that provide information on a variety of cross - categories demographically . demographic summary statistics were created for patients identified by the presence or absence of any drug - disorder diagnosis ( either principal or comorbid ) during any hospitalization occurring in fy 1987 and , correspondingly , for alcohol and psychiatric disorders . alcohol and mental disorders were identified by icd-9-cm codes in major diagnostic categories ( mdcs ) 19 and 20 from the diagnosis - related groups , fourth revision definitions manual . table 1 indicates the icd-9-cm codes used to define the drug disorders and subsequently specify six categories . within these categories , icd-9-cm code 305 is non - specific and could be possibly either alcohol or drug . therefore , it was not included in the non - dependent drug abuse category . principal and comorbid diagnoses were taken from the five discharge diagnoses available on the discharge record , denoting the first diagnosis as principal and the remaining four as comorbid . six drug disorder diagnostic categories were created : adverse reactions ; psychoses ; dependence ; tobacco ; non - dependent drug abuse ; and poisoning . in order to estimate the effects of a drug diagnosis on resource utilization at the patient level , several annual estimates were created . these were average annual los ( the sum of days in the hospital during fy 1987 for all patients in the same category divided by the number of patients ) , average annual hospital charges ( the sum of all hospital charges during fy 1987 divided by the number of patients in the same category ) , and the average number of discharges per patient during fy 1987 per category . the summary statistics were then disaggregated to see if any of the drug disorder diagnostic categories were associated with longer loss , higher hospital charges , or higher rates of hospital discharges per year . patients with multiple drug disorder diagnoses were also stratified by the number of different drug diagnoses they had , and summary statistics were separately calculated . the interpretation of comorbidity in a patient - based analysis varies from the interpretation of comorbidity in a discharge record analysis . in a patient - based data analysis , an individual who has both a primary diagnosis of alcohol abuse in one hospital discharge and a primary diagnosis of drug dependence in a second hospital discharge in the same year would be considered comorbid or overlapping , even though the diagnoses were not co - occurring . in a discharge data analysis , the individual would not count as comorbid because this record did not indicate a co - occurring condition . approximately 6 million medicare beneficiaries were hospitalized in fy 1987 and included in this study ( table 2 ) . the distribution of these patients by race , gender , and medicare status can be seen in tables 2 and 3 . the average annual discharge rate is slightly higher for the disabled population than for the aged population . the discharge rate for the black population is higher than that for the white population . a higher percent of the black medicare population is disabled ( 18.6 percent ) compared with the white population ( 8.4 percent ) . there are more females than males among the aged population , and there are more males among the disabled population . table 4 presents the percent of the patient base stratified by the presence of any diagnoses identified as adm disorders during fy 1987 . patients with any combination of two or more diagnoses were classified as having adm disorders . as expected , the largest percent of any race , gender , or medicare status group was attributed to the mental disorder category . the disabled medicare population was substantially more afflicted by these disorders than was the aged medicare population . alcohol disorder diagnoses were more prevalent among the black medicare population compared with the white population , and a similar observation was made for males versus females . focusing on the drug disorder diagnosed population , a higher prevalence of disabled persons versus aged persons were identified with the disorder . there appears to be an increased rate of white persons with drug diagnoses over black persons , and there is higher prevalence among males than among females . table 5 shows the distribution of the aged and disabled medicare population by the presence or absence of drug or alcohol or mental disorders ( principal or comorbid ) on any discharge during fy 1987 . the percent of the medicare aged population with these disorders reported is displayed alongside the percent of the medicare disabled population with the same disorders reported . the prevalence rate for drug disorder diagnoses is four times higher for the disabled population than for the aged . the highest concentration of the drug diagnosed is split between patients with mental disorders only , and patients without either mental or alcohol disorders . among the aged and disabled drug diagnosed patients , 30 and 37 percent , respectively , were also diagnosed with a mental disorder . among the aged and disabled with no drug diagnosis , 9 and 18 percent , respectively , were diagnosed with a mental disorder . among the aged and disabled drug diagnosed , 4 and 11 percent , respectively , had an alcohol diagnosis , as compared with 1 and 4 percent , respectively , in the non - drug diagnosis group . among the disabled drug diagnosed patients , 15 percent had both an alcohol and a mental disorder . there are substantially higher costs of health care incurred by the drug disorder diagnosed population , as seen in table 6 . regardless of the type of cost measurement shown , whether number of discharges per year , loss , or hospital charges , the medicare population diagnosed with drug disorders consistently averages higher values . when the patient 's health was also compromised by mental disorders , the cost of inpatient care rose markedly . the breakdown of the medicare population by aged and disabled definitions shows a slight increase in these measurements for the disabled . the same information was generated for comparisons between gender and race : the patterns are very similar ( data not shown ) . thus , patients diagnosed with drug disorders were heavier users of inpatient hospital resources during a 1-year period than were patients without these disorders . when disaggregated by gender , there are no exceptions among the variables of hospital charges or annual number of discharges . there is less of a pattern witnessed in los . in comparing white and black beneficiaries , there is a rise in hospital charges and los for patients with drug disorders , but no discernible pattern for annual number of discharges . stratifying by race and gender ( table 7 ) , patients with drug diagnoses have longer annual loss and higher annual hospital charges than do patients without drug diagnoses . the one observation that appears contrary to the overall pattern is the marginal increase in hospital charges for aged black males with alcohol or mental disorders . this difference is rather small , and los does move in the direction observed for the other data . the same increase in charges and los for patients with alcohol or mental disorders is also maintained . as previously demonstrated , it is the addition of these complications ( and not medicare status ) that plays the key role in inflating the statistics . table 8 presents patterns existing among the different types of drug disorder diagnoses , under the premise that one or more particular categories of drug disorders may be responsible for most of the observed increase in cost attributed to patients with these diagnoses . table 8 has been stratified by the medicare aged and disabled populations into those patients diagnosed with only one type of drug disorder . although initially the different combinations of categories were analyzed , there seemed to be no patterns for the various combinations that could not simply be ascribed to how many categories of drug disorders a patient was diagnosed as having . both drug psychoses and drug dependence are associated with the highest resource utilization for the aged and the disabled medicare populations . poisonings appeared to be more severe for the aged medicare population in terms of hospital discharges and los . the disabled medicare population display greater effects when presented with drug abuse or adverse drug reactions . all patients with diagnoses of drug disorders are nonetheless associated with longer loss and higher hospital charges than patients without diagnoses of drug disorders . for patients with multiple drug disorders , analyses were conducted with multiple drug disorder diagnoses , excluding tobacco use disorder , but little difference was found in comparison with the data presented . there were no patients with a history of five different drug disorders . patients with four reported drug disorders , however , did have the largest cost and los , and they were also the smallest group . when the data were stratified by race and gender ( not presented ) , there were few observable differences . among the black population , a diagnosis of adverse drug reactions appeared to be as costly as diagnoses of drug psychoses or drug dependence . this patient - based analysis differs in significant ways from an analysis of discharge data . in a discharge data analysis , the investigator is presented with a record structured to indicate a first diagnosis that is considered primary and possible second to fifth diagnoses that are considered secondary or comorbid . when assembling discharge data into an annual patient record , the structure of this information ( primary and secondary ) loses much of its meaning . drug diagnosed patients are identified using both the principal and secondary diagnoses of the relevant discharges for the entire year . within the drug diagnoses , although the elderly are not generally targeted as a population suffering from a substantial amount of drug disorders , the growing size of this population and the demonstrated additional burden that drug disorders place on medicare resource consumption warrant further investigation into the distribution and dynamics of drug disorders in this population . according to rice and kelman ( 1989 ) , 22 percent of average annual short - stay hospital costs attributed to adm disorders were ascribed to persons 65 years of age or over . of the $ 1.453 billion expended for psychotropic prescription drugs , $ 265 million went to the medicare population 65 years of age or over . these figures indicate the potential for future growth in drug disorders among the elderly . for the disabled , drug abuse disorders occur more frequently , reflecting the younger age of this group . from the medicare data , we demonstrate an increase among measures of cost on an annual basis for patients identified with any drug disorder diagnoses . average los per year for the medicare aged without any drug disorders was 13 days . this figure increased to 16.3 days for the aged with drug disorders and peaked at 30.4 days for patients with adm disorders . similar patterns were observed for the disabled , even though they are a much more uniquely selected population . the difference in average hospital discharges per year for a patient with adm disorders amounts to a $ 5,293 increase over an aged patient with no drug disorders . a similar increase of $ 3,413 can be seen for the disabled . considering the expected growth of the elderly population base , this pattern of increasing burden holds regardless of the many cross - tabulations that were examined among race and gender . although previous examination ( cartwright and ingster , to be published ) of the data by discharge indicated reduced los and hospital charges , when annual estimates were made , the results altered dramatically and unequivocally in this patient - based examination of the same data . patients with drug disorders were more frequent users of short - stay hospital resources , even though they averaged shorter stays . ( 1987 ) led to the authors ' independent conclusions that the drg - based pps would systematically redistribute revenues to facilities with shorter lengths of stay . this phenomenon , and the increases in readmission rates found in studies of drg impact on department of veterans affairs hospitals , lend support to the pattern of discharge versus annual costs that is found for both the disabled and aged medicare populations in this study . the distribution of average annual discharges , hospital charges , and los for the six different drug disorder categories has physiologic support in the known progression of the severity of drug disorders . drug dependence ( the stage following drug abuse ) and drug psychoses are the most severe manifestations of the six categories and , correspondingly , exhibit the highest number of hospital visits , inpatient days per year , and annual hospital charges . tobacco use disorder , however , a relatively mild form of drug disorder , is nearly indistinguishable in cost from the category of no drug disorders . similarly , patients with multiple drug disorders diagnosed throughout the year can be assumed to be sicker than those with only single disorders diagnosed . it comes as no surprise that increased severity of ill health would be manifest in demonstrated higher costs and utilization of services . this examination of patient drug - related costs and los varies considerably from epidemiologic studies of similar comorbidity phenomena . for example , aged medicare population estimates vary substantially for race and age from comparable u.s . 1990 ) , although both sources of data are comparable for total elderly population counts . second , these data are restricted to hospital inpatient short stays that are recovered in the administrative records and are obtained from the process of diagnostic coding . an alternative approach to case finding is represented in the work of regier et al . ( 1990 ) , which utilized a survey instrument to determine prevalence rates and associated resource demands attributable to adm comorbidity . there are no companion studies that verify the accuracy of the coding process , such as iezzoni ( 1988 ) did for the coding of myocardial infarction . such research would be vital to an understanding of the clinical and policy ramifications of resource use generated by drug comorbidity . this article provides additional information on overlap of alcohol and mental disorders for drug diagnosed patients which may complement two previous studies ( rice and kelman , 1989 ; regier et al . , care must be taken in comparing results of the many differences in both study populations and comorbidity concepts . the disabled medicare population had higher rates of adm overlap than the aged medicare population . given the nature of the disability population , there are various definitions of comorbidity used in analysis of adm . a lifetime prevalence approach ( regier et al . , 1990 ) begins with an identification of the one or more adm conditions for which the individual has ever met a diagnostic criterion . a statistical association between the disorders indicates either causation or some underlying third factor which may jointly cause the disorders ( e.g. , physiological or psychological vulnerability ) . such an approach would be difficult to further refine so that temporal order may be established and causation determined . on the other hand , discharge data of adm comorbidity essentially compress the temporal aspect towards a short interval in time , the medical episode . these data are more directly related to what a clinician would actually see and diagnose . comorbidity rates from discharge data would be smaller than the rates from a lifetime prevalence approach . for example , prevalence rates tend to monotonically increase from 1 month to 6 months to lifetime periods and , similarly , so would comorbidity rates . this article shows the value of examining data in a longitudinal framework where multiple discharges may be captured . in a discharge - based analysis , drug abuse comorbidity may lead to a reduction in measured costs ; on a patient - level based analysis , costs are shown to increase for those with a drug diagnosis . results from the inclusion and exclusion of tobacco abuse indicate that this facet is unimportant in this type of drug abuse study . of course , tobacco use is indicated as a cost - increasing factor in many other health outcomes . further work on the validity and reliability of diagnostic coding is also warranted and may be easily implemented in community sites . for the drug diagnosed , negative cost overlap with mental and alcohol disorders was found . additional research is required on various subgroup disorders where there may be even greater cost problems . finally , if drug treatment can be associated with the patient in the administrative records , readmissions can be easily examined as an outcome with a resulting greater potential for measuring cost savings to the health care system . reducing the numbers of drug diagnosed for example , a primary intervention would be a referral to substance abuse treatment services . it is not possible to infer the adequacy of the referral made by clinicians in this study , and such a study should be encouraged . referrals may be adequate but patients may not be willing to enter treatment , or patients may not be able to gain access to the treatment system , for any number of financial or capacity constraints . work in this area would be fruitful , with potential savings available from reducing the high rate of readmission found in this study .

this article utilizes the part a medicare provider analysis and review ( medpar ) file for fiscal year ( fy ) 1987 . the discharge records were organized into a patient - based record that included alcohol , drug , and mental ( adm ) disorder diagnoses as well as measures of resource use . the authors find that there are substantially higher costs of health care incurred by the drug disorder diagnosed population . those of the medicare population diagnosed with drug disorders had longer lengths of stay ( loss ) , higher hospital charges , and more discharges . costs increased monotonically as the number of drug diagnoses increased . overlap of mental and alcohol problems is presented for the drug disorder diagnosed population .

Introduction Methods Results Discussion Conclusion

nationwide interest in the cost to society of drug abuse has been on the rise in recent years , particularly among those groups responsible for national , state , and local policy , as well as among leaders of industry confronted by soaring health insurance costs and diminished worker productivity . a recent estimate of the overall cost is $ 58 billion dollars ( rice et al . , methods used to estimate these costs have varied considerably , and have been consistently hampered by the meager availability of data on both the distribution and dynamics of drug disorders in society and the ways and means to most effectively treat those afflicted . prevention efforts are also hampered by the lack of data delineating the associated costs , risk factors , and determinants of individual susceptibility . the investment in drug treatment has increased , as has a recognition of the need for additional data on the effects of drug disorders on hospital costs . current research relies on selected samples of the population , such as those referred for treatment by the criminal justice system , or national representative samples , such as those used in the national hospital discharge survey ( nhds ) . such samples are limited in their generalizability to the rest of the national population or even to smaller demographic groups of interest . in the case of a national sample , furthermore , in a discharge survey such as nhds , there is no way for researchers to identify and combine records to access information on patient history , because the sampling is done on discharges . it would be impossible to link all records to do a patient - based analysis because the sampling scheme will eliminate some of these records . thus , the great advantage of using 1 fiscal year of administrative records , such as the discharge records of the medicare hospital part a billing record , is the ability to assemble discharges into one patient record . patient - based studies to date have been limited in scope both because of the difficulties in obtaining a patient 's complete medical administrative records and the great expense of following patients in prospective studies . opportunities to examine the medicare population are available within the large reimbursement system data base . these data comprise about 96 percent of the population 65 years of age or over . the data on the medicare disabled population represent a much smaller proportion of the total disabled population because of the stringent process to obtain eligibility . although medicare data include actual yearly charges for essentially the entire medicare population , the reported charges are very limited in scope ( in this case , to short - stay , inpatient , hospital facilities ) and do not represent the total spectrum of incurred expenses in the aged population . the availability of data for the disabled medicare population provides an opportunity to study a younger population . a recent study ( cartwright and ingster , to be published ) conducted on medicare discharge data indicated that the expectation of higher charges associated with hospital stays involving drug disorders is not necessarily borne out . evidence from this study showed that , on a per discharge basis , drug disorder diagnosed patients incur lower charges than patients not diagnosed with drug disorders . we speculated that these patients may initially be healthier in order to support the underlying drug abuse or dependency that leads to the drug disorder , may be discharged earlier , or may simply require less of the acute , intensive , and costly care normally associated with short - stay hospital visits , and incur the lower costs associated with constant observation or convalescent care . economic factors associated with the medicare reimbursement system itself , particularly the prospective payment system ( pps ) , may also influence or require hospitals and physicians to discharge drug disorder diagnosed patients sooner than they might otherwise . another possible reason for the observed lower charges may lie with the diagnosis - related group ( drg ) codes . prior to fy 1988 , certain types of comorbidities were not reimbursable , which may have resulted in an underestimate of the true prevalence of patients with drug disorders by their international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) discharge codes . in this article , annual charges for patients are aggregated from a year of discharge data so that comparisons may be made between patients with diagnoses of drug disorder and patients with no drug disorder . we hypothesize that patients with drug disorder diagnoses might be more frequent users of inpatient hospital resources . in practice therefore , the patients with identified icd-9-cm drug disorder diagnoses would cost more on an annual basis than would patients without this disorder . this hypothesis would then be reflected in longer average loss , higher average annual hospital charges , and a greater number of average annual discharges . the entire universe of medicare discharges submitted for reimbursement during fy 1987 was utilized to create a patient file . these data were collected by the health care financing administration ( hcfa ) and can be found in the part a medpar file . the discharge records were sorted and matched by beneficiary identification code , provider number , and social security number codes , which would identify all records belonging to one individual . the analysis file is limited to individuals who were inpatients in short - stay hospitals . medicare patients classified with end stage renal disease ( esrd ) were excluded because of the uncertain interpretation of drug disorder effects for a terminal renal disease , and those discharges whose principal diagnosis would fall into the icd-9-cm category of pregnancy , childbirth , and puerperium were also excluded because of the unique nature of this medical event . the variables selected for examination were race , gender , age , medicare status ( aged , disabled ) , los ( days ) , total hospital charges , and the five discharge diagnoses per record . these were used to create several broad drug disorder diagnostic groups and to identify tobacco use disorder , and alcohol and psychiatric disorders . statistics presented by race were limited to white and black persons and do not cover the remaining population . the medicare status category of aged is comprised of individuals 65 years of age or over , and the disabled category is comprised of those individuals either physically or mentally disabled , who are under 65 years of age and qualify for medicare support . summary files were created that provide information on a variety of cross - categories demographically . demographic summary statistics were created for patients identified by the presence or absence of any drug - disorder diagnosis ( either principal or comorbid ) during any hospitalization occurring in fy 1987 and , correspondingly , for alcohol and psychiatric disorders . alcohol and mental disorders were identified by icd-9-cm codes in major diagnostic categories ( mdcs ) 19 and 20 from the diagnosis - related groups , fourth revision definitions manual . table 1 indicates the icd-9-cm codes used to define the drug disorders and subsequently specify six categories . principal and comorbid diagnoses were taken from the five discharge diagnoses available on the discharge record , denoting the first diagnosis as principal and the remaining four as comorbid . six drug disorder diagnostic categories were created : adverse reactions ; psychoses ; dependence ; tobacco ; non - dependent drug abuse ; and poisoning . in order to estimate the effects of a drug diagnosis on resource utilization at the patient level , several annual estimates were created . these were average annual los ( the sum of days in the hospital during fy 1987 for all patients in the same category divided by the number of patients ) , average annual hospital charges ( the sum of all hospital charges during fy 1987 divided by the number of patients in the same category ) , and the average number of discharges per patient during fy 1987 per category . the summary statistics were then disaggregated to see if any of the drug disorder diagnostic categories were associated with longer loss , higher hospital charges , or higher rates of hospital discharges per year . patients with multiple drug disorder diagnoses were also stratified by the number of different drug diagnoses they had , and summary statistics were separately calculated . the interpretation of comorbidity in a patient - based analysis varies from the interpretation of comorbidity in a discharge record analysis . in a patient - based data analysis , an individual who has both a primary diagnosis of alcohol abuse in one hospital discharge and a primary diagnosis of drug dependence in a second hospital discharge in the same year would be considered comorbid or overlapping , even though the diagnoses were not co - occurring . approximately 6 million medicare beneficiaries were hospitalized in fy 1987 and included in this study ( table 2 ) . the distribution of these patients by race , gender , and medicare status can be seen in tables 2 and 3 . the average annual discharge rate is slightly higher for the disabled population than for the aged population . the discharge rate for the black population is higher than that for the white population . a higher percent of the black medicare population is disabled ( 18.6 percent ) compared with the white population ( 8.4 percent ) . there are more females than males among the aged population , and there are more males among the disabled population . table 4 presents the percent of the patient base stratified by the presence of any diagnoses identified as adm disorders during fy 1987 . as expected , the largest percent of any race , gender , or medicare status group was attributed to the mental disorder category . the disabled medicare population was substantially more afflicted by these disorders than was the aged medicare population . alcohol disorder diagnoses were more prevalent among the black medicare population compared with the white population , and a similar observation was made for males versus females . focusing on the drug disorder diagnosed population , a higher prevalence of disabled persons versus aged persons were identified with the disorder . there appears to be an increased rate of white persons with drug diagnoses over black persons , and there is higher prevalence among males than among females . table 5 shows the distribution of the aged and disabled medicare population by the presence or absence of drug or alcohol or mental disorders ( principal or comorbid ) on any discharge during fy 1987 . the percent of the medicare aged population with these disorders reported is displayed alongside the percent of the medicare disabled population with the same disorders reported . the prevalence rate for drug disorder diagnoses is four times higher for the disabled population than for the aged . the highest concentration of the drug diagnosed is split between patients with mental disorders only , and patients without either mental or alcohol disorders . among the aged and disabled drug diagnosed patients , 30 and 37 percent , respectively , were also diagnosed with a mental disorder . among the aged and disabled with no drug diagnosis , 9 and 18 percent , respectively , were diagnosed with a mental disorder . among the aged and disabled drug diagnosed , 4 and 11 percent , respectively , had an alcohol diagnosis , as compared with 1 and 4 percent , respectively , in the non - drug diagnosis group . there are substantially higher costs of health care incurred by the drug disorder diagnosed population , as seen in table 6 . regardless of the type of cost measurement shown , whether number of discharges per year , loss , or hospital charges , the medicare population diagnosed with drug disorders consistently averages higher values . the breakdown of the medicare population by aged and disabled definitions shows a slight increase in these measurements for the disabled . thus , patients diagnosed with drug disorders were heavier users of inpatient hospital resources during a 1-year period than were patients without these disorders . when disaggregated by gender , there are no exceptions among the variables of hospital charges or annual number of discharges . in comparing white and black beneficiaries , there is a rise in hospital charges and los for patients with drug disorders , but no discernible pattern for annual number of discharges . stratifying by race and gender ( table 7 ) , patients with drug diagnoses have longer annual loss and higher annual hospital charges than do patients without drug diagnoses . the one observation that appears contrary to the overall pattern is the marginal increase in hospital charges for aged black males with alcohol or mental disorders . this difference is rather small , and los does move in the direction observed for the other data . the same increase in charges and los for patients with alcohol or mental disorders is also maintained . table 8 presents patterns existing among the different types of drug disorder diagnoses , under the premise that one or more particular categories of drug disorders may be responsible for most of the observed increase in cost attributed to patients with these diagnoses . table 8 has been stratified by the medicare aged and disabled populations into those patients diagnosed with only one type of drug disorder . although initially the different combinations of categories were analyzed , there seemed to be no patterns for the various combinations that could not simply be ascribed to how many categories of drug disorders a patient was diagnosed as having . both drug psychoses and drug dependence are associated with the highest resource utilization for the aged and the disabled medicare populations . poisonings appeared to be more severe for the aged medicare population in terms of hospital discharges and los . the disabled medicare population display greater effects when presented with drug abuse or adverse drug reactions . all patients with diagnoses of drug disorders are nonetheless associated with longer loss and higher hospital charges than patients without diagnoses of drug disorders . for patients with multiple drug disorders , analyses were conducted with multiple drug disorder diagnoses , excluding tobacco use disorder , but little difference was found in comparison with the data presented . there were no patients with a history of five different drug disorders . patients with four reported drug disorders , however , did have the largest cost and los , and they were also the smallest group . when the data were stratified by race and gender ( not presented ) , there were few observable differences . among the black population , a diagnosis of adverse drug reactions appeared to be as costly as diagnoses of drug psychoses or drug dependence . this patient - based analysis differs in significant ways from an analysis of discharge data . in a discharge data analysis , the investigator is presented with a record structured to indicate a first diagnosis that is considered primary and possible second to fifth diagnoses that are considered secondary or comorbid . when assembling discharge data into an annual patient record , the structure of this information ( primary and secondary ) loses much of its meaning . drug diagnosed patients are identified using both the principal and secondary diagnoses of the relevant discharges for the entire year . within the drug diagnoses , although the elderly are not generally targeted as a population suffering from a substantial amount of drug disorders , the growing size of this population and the demonstrated additional burden that drug disorders place on medicare resource consumption warrant further investigation into the distribution and dynamics of drug disorders in this population . according to rice and kelman ( 1989 ) , 22 percent of average annual short - stay hospital costs attributed to adm disorders were ascribed to persons 65 years of age or over . of the $ 1.453 billion expended for psychotropic prescription drugs , $ 265 million went to the medicare population 65 years of age or over . these figures indicate the potential for future growth in drug disorders among the elderly . for the disabled , drug abuse disorders occur more frequently , reflecting the younger age of this group . from the medicare data , we demonstrate an increase among measures of cost on an annual basis for patients identified with any drug disorder diagnoses . average los per year for the medicare aged without any drug disorders was 13 days . this figure increased to 16.3 days for the aged with drug disorders and peaked at 30.4 days for patients with adm disorders . similar patterns were observed for the disabled , even though they are a much more uniquely selected population . the difference in average hospital discharges per year for a patient with adm disorders amounts to a $ 5,293 increase over an aged patient with no drug disorders . a similar increase of $ 3,413 can be seen for the disabled . considering the expected growth of the elderly population base , this pattern of increasing burden holds regardless of the many cross - tabulations that were examined among race and gender . although previous examination ( cartwright and ingster , to be published ) of the data by discharge indicated reduced los and hospital charges , when annual estimates were made , the results altered dramatically and unequivocally in this patient - based examination of the same data . patients with drug disorders were more frequent users of short - stay hospital resources , even though they averaged shorter stays . ( 1987 ) led to the authors ' independent conclusions that the drg - based pps would systematically redistribute revenues to facilities with shorter lengths of stay . this phenomenon , and the increases in readmission rates found in studies of drg impact on department of veterans affairs hospitals , lend support to the pattern of discharge versus annual costs that is found for both the disabled and aged medicare populations in this study . the distribution of average annual discharges , hospital charges , and los for the six different drug disorder categories has physiologic support in the known progression of the severity of drug disorders . drug dependence ( the stage following drug abuse ) and drug psychoses are the most severe manifestations of the six categories and , correspondingly , exhibit the highest number of hospital visits , inpatient days per year , and annual hospital charges . tobacco use disorder , however , a relatively mild form of drug disorder , is nearly indistinguishable in cost from the category of no drug disorders . similarly , patients with multiple drug disorders diagnosed throughout the year can be assumed to be sicker than those with only single disorders diagnosed . it comes as no surprise that increased severity of ill health would be manifest in demonstrated higher costs and utilization of services . for example , aged medicare population estimates vary substantially for race and age from comparable u.s . 1990 ) , although both sources of data are comparable for total elderly population counts . an alternative approach to case finding is represented in the work of regier et al . ( 1990 ) , which utilized a survey instrument to determine prevalence rates and associated resource demands attributable to adm comorbidity . there are no companion studies that verify the accuracy of the coding process , such as iezzoni ( 1988 ) did for the coding of myocardial infarction . such research would be vital to an understanding of the clinical and policy ramifications of resource use generated by drug comorbidity . this article provides additional information on overlap of alcohol and mental disorders for drug diagnosed patients which may complement two previous studies ( rice and kelman , 1989 ; regier et al . , care must be taken in comparing results of the many differences in both study populations and comorbidity concepts . the disabled medicare population had higher rates of adm overlap than the aged medicare population . given the nature of the disability population , there are various definitions of comorbidity used in analysis of adm . a lifetime prevalence approach ( regier et al . , 1990 ) begins with an identification of the one or more adm conditions for which the individual has ever met a diagnostic criterion . a statistical association between the disorders indicates either causation or some underlying third factor which may jointly cause the disorders ( e.g. these data are more directly related to what a clinician would actually see and diagnose . comorbidity rates from discharge data would be smaller than the rates from a lifetime prevalence approach . for example , prevalence rates tend to monotonically increase from 1 month to 6 months to lifetime periods and , similarly , so would comorbidity rates . this article shows the value of examining data in a longitudinal framework where multiple discharges may be captured . in a discharge - based analysis , drug abuse comorbidity may lead to a reduction in measured costs ; on a patient - level based analysis , costs are shown to increase for those with a drug diagnosis . results from the inclusion and exclusion of tobacco abuse indicate that this facet is unimportant in this type of drug abuse study . further work on the validity and reliability of diagnostic coding is also warranted and may be easily implemented in community sites . for the drug diagnosed , negative cost overlap with mental and alcohol disorders was found . finally , if drug treatment can be associated with the patient in the administrative records , readmissions can be easily examined as an outcome with a resulting greater potential for measuring cost savings to the health care system . reducing the numbers of drug diagnosed for example , a primary intervention would be a referral to substance abuse treatment services . it is not possible to infer the adequacy of the referral made by clinicians in this study , and such a study should be encouraged . referrals may be adequate but patients may not be willing to enter treatment , or patients may not be able to gain access to the treatment system , for any number of financial or capacity constraints .

highly eusocial bees vary greatly in their social structure , with some species typically consisting of one single - mated mother queen and her worker offspring ( peters et al . , 1999 ; strassmann , 2001 ) . in this case , the queen lays most of the eggs and the sterile workers increase their overall fitness by helping to rear the queens offspring ( hamilton , 1964 , 1972 ) . in contrast , other species form colonies with multiple laying queens or multiple mated queens . polygyny and/or polyandry affect genetic diversity within the colony , decrease the average progeny relatedness , promote interactions among individuals with different levels of relatedness and generate within - colony genetic conflicts ( bourke and franks , 1995 ; keller and chapuisat , 1999 ) . supersedure of the queen and the transitional stages of colonies ( one or multiple queens in different periods of the year , depending on the colony ) can also change the social structure of colonies to a more complex one ( herbers and stuart , 1996 ; hastings et al . several studies have demonstrated that queens in polygynous colonies may be completely unrelated or full sisters ( seppa , 1996 ; banschbach and herbers , 1996 ; satoh et al . , 1997 ; goodisman and ross , 1998 ; hastings et al . , 1998 ; pedersen and boomsma , 1999 ; heinze and keller , 2000 ) . consequently , the average genetic relatedness among nestmates in polygynous species can remain relatively close to the value of 0.75 expected with a single once - mated queen ( queller et al . , 1988 ; strassmann et al . , 1991 ; herbers , 1993 ; rosengren et al . , 1993 ; crozier and pamilo , 1996 ; bourke et al . , 1997 ; fournier et al . , stingless bee colonies typically consist of one single - mated mother queen and her worker offspring ( camargo , 1972 ; peters et al . , 1999 ; strassmann , 2001 ; palmer et al . , 2002 ; tth et al . , these two castes are morphologically distinct and the workers are unable to mate , although in many species they can produce haploid eggs ( sakagami et al . , 1963 ) . however , the process of worker egg laying is very diverse among different species and the frequency of males that are sons of workers varies from species to species ( sommeijer and van buren , 1992 ; koedam et al . , 1996 ; sommeijer et al . , 1999 ; for example , frieseomelitta silvestri workers are physiologically incapable of laying eggs ( boleli et al . , 2000 ) while in melipona scutellaris and m. favosa , 22.88% and 94.5% , respectively , of the males are sons of workers ( sommeijer et al . , 1999 ; witter and wittmann ( 1997 ) described a polygynic colony in plebeia wittmanni in which a new queen started oviposition while the older one was still alive and laying eggs . carvalho - zilse and kerr ( 2004 ) reported a similar case for m. scutellaris , and recently alves et al . ( 2010 ) reported on a polygynic colony of m. quadrifasciata in which eight egg - laying queens coexisted in a colony for ca . melipona bicolor ( hymenoptera : apidae ) is a stingless bee species that naturally displays facultative and long - lasting polygyny , with the number of queens varying among populations and even among colonies of the same population . some colonies are headed by a single queen , whereas others have more than one queen living together for considerable periods of time ( cepeda , 2006 ; velthuis et al . , 2006 ) . ( 2001 ) observed up to five physogastric queens laying eggs in a colony of m. bicolor and noted that workers did not show preference or aggressiveness to foreign queens . in m. bicolor polygynous colonies , queens do not show antagonism , territoriality , aggressiveness or competition ( bego , 1989 ) and workers do not distinguish among the physogastric queens , to which they have different degrees of relatedness ( alonso et al . , 1998 ) . additionally , more than one queen is frequently seen inspecting the provisioning process ( velthuis et al . , the workers construct and mass provision the cells with a mixture of regurgitated nectar and pollen . thereafter , one of the queens lay her eggs on top of this liquid and , finally , the workers seal the cells ( sakagami , 1982 ) . within each cell , polygynous colonies are not larger or more productive than monogynous ones ( velthuis et al . , 2006 ) . like other stingless bees , m. bicolor colonies are long lived , persisting perhaps for decades , while queens may live for 13 years ( velthuis et al . , 2006 ) . the variation in social structure of m. bicolor affects the genetic diversity within the colony , the relatedness among nestmates , and the architecture of potential kinconflicts and kin cooperation ( bourke and franks , 1995 ; crozier and pamilo , 1996 ) . hence , understanding the dynamics of variation in queen number is fundamental for a better understanding of the social evolution in this species . data on the dynamics of queen number and on variation in social structure over time are also needed to obtain a clearer picture of how complex social structures appear and are maintained . since the number of queens and their genetic relatedness , as well as their relative reproductive success , can vary among colonies , we investigated the social structure and genetic relatedness among workers from 14 colonies of m. bicolor over one year in order to test the theoretical expectations concerning social behavior . eight natural colonies ( six from caet and two from cataguases in the southeastern brazilian state of minas gerais , mg ) and six manipulated colonies of m. bicolor ( maintained in artificial hives at the central apiary of the universidade federal de viosa , viosa , mg ) were sampled monthly from march 2007 to january 2008 . natural colonies were maintained in their original shelter and the workers were sampled directly from the colony entrance , without opening the colony . in contrast , manipulated colonies were opened to collect the workers . in these cases , the social structure of the colonies was monitored over a one year period by analyzing ten workers / colony that were collected in march , may , june , september and november 2007 and january 2008 ( total of 60 workers / colony ) . four microsatellite loci ( mbi11 , 201 , 233 and 278 ) were analyzed using the amplification conditions described by peters et al . the polymerase chain reaction ( pcr ) amplifications were done in reaction volumes of 10 l containing 12.5 ng of genomic dna , 1x taq pcr buffer , 1 u taq dna polymerase ( promega ) , 0.5 m of each forward and reverse primer , 0.1 mm dntp and 1.5 mm mgcl2 . the conditions for the pcr were the following : 3 min at 94 c followed by 40 cycles of 30 s at 92 c , 1 min at the specific primer pairing temperature and 30 s at 72 c , with a final extension of 5 min at 72 c . the pcr products were separated on 8% denaturing polyacrylamide gels and visualized by staining with 0.2% silver nitrate . since the aim of this study was to analyze the dynamics of queen numbers and variation in the social structure over time , the queens were not collected or genotyped . instead , assuming that queens mated with a single male ( peters et al . , 1999 ; strassmann , 2001 ) , queen and fathering male genotypes , as well as the social structure ( polygyny or monogyny ) of each colony , were determined indirectly from worker genotypes . when all workers were heterozygous ( a / b , for example ) for a determined locus , alleles from queens and the males that mated with them could not be determined with precision . in these cases , queen and male genotypes were considered as a / a or b / b and b or a , respectively . the non - detection error due to paternal males displaying identical genotypes by chance was estimated for each population as : ( qi2)jwhere qi denotes the allele frequencies at each of j loci ( foster et al . , 1999 ) . the monthly genetic relatedness among nestmate workers , the social structure of colonies and the genetic relatedness among queens in the polygynous colonies were estimated according to queller and goodnight ( 1989 ) by using the software relatedness 4.2 ( goodnight and queller , 1994 ) . relatedness and the genetic relationships among queens from polygynous colonies were also checked by inspecting their multilocus genotypes or the inferred multilocus genotypes of the original pair ( the original queen and her mate ) and that of the new queen , in cases of queen replacement or queen adoption . the allelic frequencies of each locus and the observed ( ho ) and expected ( he ) heterozygosities ( table s1 supplementary online material ) were calculated using popgene version 1.32 ( yeh et al . , 1999 ) . eight natural colonies ( six from caet and two from cataguases in the southeastern brazilian state of minas gerais , mg ) and six manipulated colonies of m. bicolor ( maintained in artificial hives at the central apiary of the universidade federal de viosa , viosa , mg ) were sampled monthly from march 2007 to january 2008 . natural colonies were maintained in their original shelter and the workers were sampled directly from the colony entrance , without opening the colony . in contrast , manipulated colonies were opened to collect the workers . in these cases , the social structure of the colonies was monitored over a one year period by analyzing ten workers / colony that were collected in march , may , june , september and november 2007 and january 2008 ( total of 60 workers / colony ) . four microsatellite loci ( mbi11 , 201 , 233 and 278 ) were analyzed using the amplification conditions described by peters et al . the polymerase chain reaction ( pcr ) amplifications were done in reaction volumes of 10 l containing 12.5 ng of genomic dna , 1x taq pcr buffer , 1 u taq dna polymerase ( promega ) , 0.5 m of each forward and reverse primer , 0.1 mm dntp and 1.5 mm mgcl2 . the conditions for the pcr were the following : 3 min at 94 c followed by 40 cycles of 30 s at 92 c , 1 min at the specific primer pairing temperature and 30 s at 72 c , with a final extension of 5 min at 72 c . the pcr products were separated on 8% denaturing polyacrylamide gels and visualized by staining with 0.2% silver nitrate . since the aim of this study was to analyze the dynamics of queen numbers and variation in the social structure over time , the queens were not collected or genotyped . instead , assuming that queens mated with a single male ( peters et al . , 1999 ; strassmann , 2001 ) , queen and fathering male genotypes , as well as the social structure ( polygyny or monogyny ) of each colony , were determined indirectly from worker genotypes . when all workers were heterozygous ( a / b , for example ) for a determined locus , alleles from queens and the males that mated with them could not be determined with precision . in these cases , queen and male genotypes were considered as a / a or b / b and b or a , respectively . the non - detection error due to paternal males displaying identical genotypes by chance was estimated for each population as : ( qi2)jwhere qi denotes the allele frequencies at each of j loci ( foster et al . , 1999 ) . the monthly genetic relatedness among nestmate workers , the social structure of colonies and the genetic relatedness among queens in the polygynous colonies were estimated according to queller and goodnight ( 1989 ) by using the software relatedness 4.2 ( goodnight and queller , 1994 ) . relatedness and the genetic relationships among queens from polygynous colonies were also checked by inspecting their multilocus genotypes or the inferred multilocus genotypes of the original pair ( the original queen and her mate ) and that of the new queen , in cases of queen replacement or queen adoption . the allelic frequencies of each locus and the observed ( ho ) and expected ( he ) heterozygosities ( table s1 supplementary online material ) were calculated using popgene version 1.32 ( yeh et al . , 1999 ) . direct observation of worker genotypes showed that eight of the 14 colonies analyzed ( cataguases 1 , 2 , caet 2 , 3 , 7 , viosa 916 , 921 and 934 ) had genotypes consistent with a single queen mated to a single male . on the other hand , colonies caet 1 , 5 and 6 , and viosa 814 , 905 and 915 were polygynous . the number of queens in each of these colonies was two , except for caet 5 , the workers of which had genotypes consistent with three queens . in the manipulated colony viosa 915 , the two queens were seen throughout the period of sampling and relatedness estimates confirmed their polygynic status . in the other manipulated colonies ( viosa 814 and 905 ) , there was no visual confirmation of the presence of multiple queens . this lack of visual confirmation partly reflected the minimal manipulation of the colonies in order to avoid altering their organization and the fact that the queens generally hide during manipulation . the presence of multiple queens in natural colonies could not be confirmed visually because the colonies were not opened during sampling . the estimated genetic relatedness among workers from monogynous and polygynous colonies was 0.75 0.12 ( mean sem ; range : 0.490.96 ) and 0.53 0.16 ( mean sem ; range : 0.210.90 ) , respectively ( table 1 ) . the high genetic relatedness ( r = 0.90 0.15 ) detected among workers from the polygynous colony caet 1 in september resulted from the sampling of workers from a single matriline . the effects of non - detection error at the population level , which can lead to an overestimation of relatedness ( boomsma and ratnieks , 1996 ; foster et al . , 1999 ) , were relatively low ( 0.08 ; range : 0.017 to 0.22 ) and exerted only a minor additional impact on the mean nestmate relatedness . the estimated relatedness among queens from polygynous colonies revealed that queens from colonies viosa 814 , viosa 905 , caet 6 and two out of the three queens from colony caet 5 were close relatives ( r = 0.76 0.008 , 0.56 0.009 , 0.74 0.008 and 0.81 0.15 , respectively ) , while the third queen of colony caet 5 was less related to the others ( r = 0.32 0.03 ) . similarly , queens from colonies caet 1 and viosa 915 were also not closely related to each other ( r = 0.08 0.04 and 0.29 0.02 , respectively ) . at the colony level , the social structure was very stable throughout the study ( 12 consecutive months ) : queen numbers did not change in 12 out of the 14 analyzed colonies . a case of queen replacement was detected in colony viosa 921 in july , and a case of queen adoption was detected in colony caet 6 in may ( in march a single queen was detected in this colony ) . the two queens detected in colony caet 6 in may persisted in the colony until the end of the sampling period ; this finding strengthened the hypothesis that polygyny is stable in m. bicolor . the allelic variation and heterozygosity indices observed in the 14 colonies , the inferred genotypes of queens and drones and the number of workers attributed to each queen in the polygynous colonies are summarized in tables s1 , s2 and s3 , respectively , of the supplementary material . the genetic analysis of m. bicolor workers confirmed that monogynous and polygynous colonies are widespread in natural ( caet ) and manipulated ( viosa ) populations . in monogynous colonies , the progeny had a maximum of three alleles at each locus this being consistent with one paternal and up to two maternal alleles ; this finding also confirmed that in these colonies workers are the progeny of single - mated queens . although the estimated genetic relatedness among workers from monogynous and polygynous colonies varied considerably throughout the year and from colony to colony , the average relatedness estimates for these colonies ( 0.75 and 0.53 , respectively ) were in good agreement with theoretical expectations for these social structures ( bourke and franks , 1995 ) . additionally , the average relatedness estimated for monogynous colonies was very similar to the mean of 0.739 reported by peters et al . ( 1999 ) for 12 species of single - queen stingless bees . in the case of monogynous colonies , the results demonstrated that parental genotypes have a significant effect on relatedness in this species . for example , workers from the monogynous colony viosa 916 had an estimated genetic relatedness ranging from 0.49 to 0.58 . this probably occurred because the queen was heterozygous for all loci analyzed and the paternal alleles were different from the queen s alleles at all of these loci . consequently , in these two colonies the estimated genetic relatedness among workers differed considerable from the expected value of 0.75 for full sisters . our results also demonstrated that the estimated genetic relatedness among workers from the monogynous colony viosa 921 was relatively low in march and may compared to the other months . in this case , the workers were found to be the progeny of two queens . in may , however , the progeny of one of these queens decreased substantially and in the subsequent months the workers sampled represented the progeny of a single queen . this finding suggested that an older queen was replaced by a new one , with the samples from march and may representing the transitional phase of this otherwise monogynous colony . comparisons between the inferred multilocus genotypes of the original pair ( the original queen and her mate ) and that of the new queen and the genetic relatedness between queens ( r = 0.86 0.14 ) revealed that both queens were very closely related ( probably mother and daughter ) . analysis of the worker genotypes from colony caet 5 showed that they were more related to each other than were workers from viosa 915 . this occurred because two of the three queens from colony caet 5 were closely related , while the two queens from viosa 915 were unrelated . worker genotypes and the inferred queen / male genotypes further confirmed that the queens from colony caet 1 were unrelated . in these cases , the unrelated queens may have been adopted secondarily . in contrast , the two queens present in each of the colonies viosa 814 , viosa 905 and caet 6 were full sisters . these findings support the view that polygyny in m. bicolor may arise by two mechanisms . first , since , as in other melipona species , m. bicolor queens are produced all year long and may persist in the colony , polygyny may arise from the adoption of a related queen ( mother - daughter or full sisters ) , thus confirming observations of velthuis et al . in such cases , variation in the number of queens may have no detectable effect on relatedness . in other cases , however , unrelated queens may be secondarily adopted by established colonies of m. bicolor , thereby diminishing the relatedness among nestmate workers . overall , polygyny markedly reduced the relatedness in m. bicolor ( from 0.75 to 0.53 ) . the limited variation in the number of queens in monogynous and polygynous colonies over a one year period agreed with the common view that queen replacement is rare in stingless bee colonies ( queen replacement was detected in only one of the 14 analyzed colonies here ) and that queens have long life spans . queens of melipona compressipes and m. scutellaris , for example , have a maximum longevity of 84 months ( 7 years ) ( carvalho - zilse and kerr , 2004 ) . this reduced turnover of queens could help to maintain the relatedness among nestmate workers stable over time . the single - mating behavior of m. bicolor means that workers from monogynous colonies are highly related and this seems to be a key factor in the evolution of eusociality and the maintenance of their altruistic behavior ( cole , 1983 ; boomsma , 2007 ) . in contrast , in polygynous colonies , the degree of relatedness among workers will decrease according to the queen - queen relatedness . kin selection theory predicts conflicts between queens and workers over male production in monandrous / monogynous colonies of m. bicolor because workers are more related to their own eggs ( r = 0.5 ) and to other workers eggs ( r = 0.375 ) than they are to the eggs of the queens ( r = 0.25 ) ( ratnieks , 1988 ) . in these cases , workers could attempt to monopolize male production . however , in polyandrous / polygynous colonies in which workers are on average more related to sons of the queens ( r = 0.25 ) than to the sons of other workers ( r = 0.125 ) they may prefer the queen - laid eggs , as already verified for some polyandrous species such as apis mellifera ( ratnieks , 1988 ; ratnieks and visscher , 1989 ) and vespula vulgaris ( foster and ratnieks , 2001 ) . evidence for queen - worker conflict can be found in the cell provisioning and oviposition process of most stingless bee species . m. bicolor queens , in particular , undertake elaborate , ritualized interactions with workers before and during egg - laying ( velthuis et al . , 2006 ) . additionally , as with many stingless bees , m. bicolor workers in monogynous and polygynous colonies have been observed to lay reproductive and/or trophic eggs ( koedam et al . , 2001 ) . these authors observed that after laying a trophic egg , the worker generally leaves the cell , giving the queen the opportunity of eating this egg . in the case of a reproductive egg , competing reproductive workers , however , frequently eat and replace this egg by their own ( velthuis et al . , 2002 ; koedam et al . , 2007 ) . in polygynous colonies with highly related queens ( such as observed in several colonies in this study ) , the reasonably high levels of relatedness among workers could lower the costs of sharing reproduction ( giraud et al . , , kin selection may inhibit the workers ability to become reproductive since they can gain greater inclusive fitness by functioning as helpers of close relatives ( hamilton , 1964 ; queller and strassmann , 1998 ) . on the other hand , in polygynous colonies with queens that are not closely related , workers would lay most of the male progeny because of the lower intra - colony relatedness ( koedam et al . , 2007 ) . accordingly , in a polygynous colony of m. bicolor containing three unrelated queens ( two of them introduced experimentally ) , 27%82% of the males were workers sons . in this colony , workers replaced reproductive eggs laid by other workers with their own , but they tended to avoid eating the queen s eggs ; this behavior was considered a form of policing by reproductive workers ( koedam et al . , 2007 ) . in conclusion , the results described here provide additional insights into the social structure and relatedness in m. bicolor . however , further research is needed in order to clarify the conflict over male parentage in monogynous and polygynous colonies , and to address other aspects related to worker oviposition , such as worker policing by egg eating . such studies will lead to a better understanding of the development of social behavior in melipona . the following online material is available for this article : allelic frequencies and observed ( ho ) and expected ( he ) heterozygosities in colonies of melipona bicolor .

stingless bee colonies typically consist of one single - mated mother queen and her worker offspring . the stingless bee melipona bicolor ( hymenoptera : apidae ) shows facultative polygyny , which makes this species particularly suitable for testing theoretical expectations concerning social behavior . in this study , we investigated the social structure and genetic relatedness among workers from eight natural and six manipulated colonies of m. bicolor over a period of one year . the populations of m. bicolor contained monogynous and polygynous colonies . the estimated genetic relatedness among workers from monogynous and polygynous colonies was 0.75 0.12 and 0.53 0.16 ( mean sem ) , respectively . although the parental genotypes had significant effects on genetic relatedness in monogynous and polygynous colonies , polygyny markedly decreased the relatedness among nestmate workers . our findings also demonstrate that polygyny in m. bicolor may arise from the adoption of related or unrelated queens .

Introduction Materials and Methods Samples DNA extraction and microsatellite analysis Genetic data analysis Results Discussion Supplementary Material

highly eusocial bees vary greatly in their social structure , with some species typically consisting of one single - mated mother queen and her worker offspring ( peters et al . in this case , the queen lays most of the eggs and the sterile workers increase their overall fitness by helping to rear the queens offspring ( hamilton , 1964 , 1972 ) . in contrast , other species form colonies with multiple laying queens or multiple mated queens . supersedure of the queen and the transitional stages of colonies ( one or multiple queens in different periods of the year , depending on the colony ) can also change the social structure of colonies to a more complex one ( herbers and stuart , 1996 ; hastings et al . several studies have demonstrated that queens in polygynous colonies may be completely unrelated or full sisters ( seppa , 1996 ; banschbach and herbers , 1996 ; satoh et al . , 1997 ; goodisman and ross , 1998 ; hastings et al . , 1998 ; pedersen and boomsma , 1999 ; heinze and keller , 2000 ) . consequently , the average genetic relatedness among nestmates in polygynous species can remain relatively close to the value of 0.75 expected with a single once - mated queen ( queller et al . , stingless bee colonies typically consist of one single - mated mother queen and her worker offspring ( camargo , 1972 ; peters et al . , these two castes are morphologically distinct and the workers are unable to mate , although in many species they can produce haploid eggs ( sakagami et al . , 1963 ) . however , the process of worker egg laying is very diverse among different species and the frequency of males that are sons of workers varies from species to species ( sommeijer and van buren , 1992 ; koedam et al . , 2000 ) while in melipona scutellaris and m. favosa , 22.88% and 94.5% , respectively , of the males are sons of workers ( sommeijer et al . ( 2010 ) reported on a polygynic colony of m. quadrifasciata in which eight egg - laying queens coexisted in a colony for ca . melipona bicolor ( hymenoptera : apidae ) is a stingless bee species that naturally displays facultative and long - lasting polygyny , with the number of queens varying among populations and even among colonies of the same population . ( 2001 ) observed up to five physogastric queens laying eggs in a colony of m. bicolor and noted that workers did not show preference or aggressiveness to foreign queens . in m. bicolor polygynous colonies , queens do not show antagonism , territoriality , aggressiveness or competition ( bego , 1989 ) and workers do not distinguish among the physogastric queens , to which they have different degrees of relatedness ( alonso et al . additionally , more than one queen is frequently seen inspecting the provisioning process ( velthuis et al . , the workers construct and mass provision the cells with a mixture of regurgitated nectar and pollen . thereafter , one of the queens lay her eggs on top of this liquid and , finally , the workers seal the cells ( sakagami , 1982 ) . within each cell , polygynous colonies are not larger or more productive than monogynous ones ( velthuis et al . , 2006 ) . like other stingless bees , m. bicolor colonies are long lived , persisting perhaps for decades , while queens may live for 13 years ( velthuis et al . the variation in social structure of m. bicolor affects the genetic diversity within the colony , the relatedness among nestmates , and the architecture of potential kinconflicts and kin cooperation ( bourke and franks , 1995 ; crozier and pamilo , 1996 ) . hence , understanding the dynamics of variation in queen number is fundamental for a better understanding of the social evolution in this species . data on the dynamics of queen number and on variation in social structure over time are also needed to obtain a clearer picture of how complex social structures appear and are maintained . since the number of queens and their genetic relatedness , as well as their relative reproductive success , can vary among colonies , we investigated the social structure and genetic relatedness among workers from 14 colonies of m. bicolor over one year in order to test the theoretical expectations concerning social behavior . eight natural colonies ( six from caet and two from cataguases in the southeastern brazilian state of minas gerais , mg ) and six manipulated colonies of m. bicolor ( maintained in artificial hives at the central apiary of the universidade federal de viosa , viosa , mg ) were sampled monthly from march 2007 to january 2008 . natural colonies were maintained in their original shelter and the workers were sampled directly from the colony entrance , without opening the colony . in contrast , manipulated colonies were opened to collect the workers . in these cases , the social structure of the colonies was monitored over a one year period by analyzing ten workers / colony that were collected in march , may , june , september and november 2007 and january 2008 ( total of 60 workers / colony ) . the polymerase chain reaction ( pcr ) amplifications were done in reaction volumes of 10 l containing 12.5 ng of genomic dna , 1x taq pcr buffer , 1 u taq dna polymerase ( promega ) , 0.5 m of each forward and reverse primer , 0.1 mm dntp and 1.5 mm mgcl2 . since the aim of this study was to analyze the dynamics of queen numbers and variation in the social structure over time , the queens were not collected or genotyped . , 1999 ; strassmann , 2001 ) , queen and fathering male genotypes , as well as the social structure ( polygyny or monogyny ) of each colony , were determined indirectly from worker genotypes . in these cases , queen and male genotypes were considered as a / a or b / b and b or a , respectively . the monthly genetic relatedness among nestmate workers , the social structure of colonies and the genetic relatedness among queens in the polygynous colonies were estimated according to queller and goodnight ( 1989 ) by using the software relatedness 4.2 ( goodnight and queller , 1994 ) . relatedness and the genetic relationships among queens from polygynous colonies were also checked by inspecting their multilocus genotypes or the inferred multilocus genotypes of the original pair ( the original queen and her mate ) and that of the new queen , in cases of queen replacement or queen adoption . , 1999 ) . eight natural colonies ( six from caet and two from cataguases in the southeastern brazilian state of minas gerais , mg ) and six manipulated colonies of m. bicolor ( maintained in artificial hives at the central apiary of the universidade federal de viosa , viosa , mg ) were sampled monthly from march 2007 to january 2008 . natural colonies were maintained in their original shelter and the workers were sampled directly from the colony entrance , without opening the colony . in contrast , manipulated colonies were opened to collect the workers . in these cases , the social structure of the colonies was monitored over a one year period by analyzing ten workers / colony that were collected in march , may , june , september and november 2007 and january 2008 ( total of 60 workers / colony ) . the polymerase chain reaction ( pcr ) amplifications were done in reaction volumes of 10 l containing 12.5 ng of genomic dna , 1x taq pcr buffer , 1 u taq dna polymerase ( promega ) , 0.5 m of each forward and reverse primer , 0.1 mm dntp and 1.5 mm mgcl2 . since the aim of this study was to analyze the dynamics of queen numbers and variation in the social structure over time , the queens were not collected or genotyped . , 1999 ; strassmann , 2001 ) , queen and fathering male genotypes , as well as the social structure ( polygyny or monogyny ) of each colony , were determined indirectly from worker genotypes . in these cases , queen and male genotypes were considered as a / a or b / b and b or a , respectively . the non - detection error due to paternal males displaying identical genotypes by chance was estimated for each population as : ( qi2)jwhere qi denotes the allele frequencies at each of j loci ( foster et al . the monthly genetic relatedness among nestmate workers , the social structure of colonies and the genetic relatedness among queens in the polygynous colonies were estimated according to queller and goodnight ( 1989 ) by using the software relatedness 4.2 ( goodnight and queller , 1994 ) . relatedness and the genetic relationships among queens from polygynous colonies were also checked by inspecting their multilocus genotypes or the inferred multilocus genotypes of the original pair ( the original queen and her mate ) and that of the new queen , in cases of queen replacement or queen adoption . the allelic frequencies of each locus and the observed ( ho ) and expected ( he ) heterozygosities ( table s1 supplementary online material ) were calculated using popgene version 1.32 ( yeh et al . , 1999 ) . the number of queens in each of these colonies was two , except for caet 5 , the workers of which had genotypes consistent with three queens . in the manipulated colony viosa 915 , the two queens were seen throughout the period of sampling and relatedness estimates confirmed their polygynic status . in the other manipulated colonies ( viosa 814 and 905 ) , there was no visual confirmation of the presence of multiple queens . this lack of visual confirmation partly reflected the minimal manipulation of the colonies in order to avoid altering their organization and the fact that the queens generally hide during manipulation . the estimated genetic relatedness among workers from monogynous and polygynous colonies was 0.75 0.12 ( mean sem ; range : 0.490.96 ) and 0.53 0.16 ( mean sem ; range : 0.210.90 ) , respectively ( table 1 ) . the high genetic relatedness ( r = 0.90 0.15 ) detected among workers from the polygynous colony caet 1 in september resulted from the sampling of workers from a single matriline . the effects of non - detection error at the population level , which can lead to an overestimation of relatedness ( boomsma and ratnieks , 1996 ; foster et al . , 1999 ) , were relatively low ( 0.08 ; range : 0.017 to 0.22 ) and exerted only a minor additional impact on the mean nestmate relatedness . the estimated relatedness among queens from polygynous colonies revealed that queens from colonies viosa 814 , viosa 905 , caet 6 and two out of the three queens from colony caet 5 were close relatives ( r = 0.76 0.008 , 0.56 0.009 , 0.74 0.008 and 0.81 0.15 , respectively ) , while the third queen of colony caet 5 was less related to the others ( r = 0.32 0.03 ) . similarly , queens from colonies caet 1 and viosa 915 were also not closely related to each other ( r = 0.08 0.04 and 0.29 0.02 , respectively ) . at the colony level , the social structure was very stable throughout the study ( 12 consecutive months ) : queen numbers did not change in 12 out of the 14 analyzed colonies . a case of queen replacement was detected in colony viosa 921 in july , and a case of queen adoption was detected in colony caet 6 in may ( in march a single queen was detected in this colony ) . the two queens detected in colony caet 6 in may persisted in the colony until the end of the sampling period ; this finding strengthened the hypothesis that polygyny is stable in m. bicolor . the allelic variation and heterozygosity indices observed in the 14 colonies , the inferred genotypes of queens and drones and the number of workers attributed to each queen in the polygynous colonies are summarized in tables s1 , s2 and s3 , respectively , of the supplementary material . the genetic analysis of m. bicolor workers confirmed that monogynous and polygynous colonies are widespread in natural ( caet ) and manipulated ( viosa ) populations . in monogynous colonies , the progeny had a maximum of three alleles at each locus this being consistent with one paternal and up to two maternal alleles ; this finding also confirmed that in these colonies workers are the progeny of single - mated queens . although the estimated genetic relatedness among workers from monogynous and polygynous colonies varied considerably throughout the year and from colony to colony , the average relatedness estimates for these colonies ( 0.75 and 0.53 , respectively ) were in good agreement with theoretical expectations for these social structures ( bourke and franks , 1995 ) . additionally , the average relatedness estimated for monogynous colonies was very similar to the mean of 0.739 reported by peters et al . ( 1999 ) for 12 species of single - queen stingless bees . in the case of monogynous colonies , the results demonstrated that parental genotypes have a significant effect on relatedness in this species . for example , workers from the monogynous colony viosa 916 had an estimated genetic relatedness ranging from 0.49 to 0.58 . this probably occurred because the queen was heterozygous for all loci analyzed and the paternal alleles were different from the queen s alleles at all of these loci . consequently , in these two colonies the estimated genetic relatedness among workers differed considerable from the expected value of 0.75 for full sisters . our results also demonstrated that the estimated genetic relatedness among workers from the monogynous colony viosa 921 was relatively low in march and may compared to the other months . in this case , the workers were found to be the progeny of two queens . in may , however , the progeny of one of these queens decreased substantially and in the subsequent months the workers sampled represented the progeny of a single queen . comparisons between the inferred multilocus genotypes of the original pair ( the original queen and her mate ) and that of the new queen and the genetic relatedness between queens ( r = 0.86 0.14 ) revealed that both queens were very closely related ( probably mother and daughter ) . analysis of the worker genotypes from colony caet 5 showed that they were more related to each other than were workers from viosa 915 . in these cases , the unrelated queens may have been adopted secondarily . these findings support the view that polygyny in m. bicolor may arise by two mechanisms . first , since , as in other melipona species , m. bicolor queens are produced all year long and may persist in the colony , polygyny may arise from the adoption of a related queen ( mother - daughter or full sisters ) , thus confirming observations of velthuis et al . in other cases , however , unrelated queens may be secondarily adopted by established colonies of m. bicolor , thereby diminishing the relatedness among nestmate workers . overall , polygyny markedly reduced the relatedness in m. bicolor ( from 0.75 to 0.53 ) . the limited variation in the number of queens in monogynous and polygynous colonies over a one year period agreed with the common view that queen replacement is rare in stingless bee colonies ( queen replacement was detected in only one of the 14 analyzed colonies here ) and that queens have long life spans . queens of melipona compressipes and m. scutellaris , for example , have a maximum longevity of 84 months ( 7 years ) ( carvalho - zilse and kerr , 2004 ) . this reduced turnover of queens could help to maintain the relatedness among nestmate workers stable over time . the single - mating behavior of m. bicolor means that workers from monogynous colonies are highly related and this seems to be a key factor in the evolution of eusociality and the maintenance of their altruistic behavior ( cole , 1983 ; boomsma , 2007 ) . in contrast , in polygynous colonies , the degree of relatedness among workers will decrease according to the queen - queen relatedness . kin selection theory predicts conflicts between queens and workers over male production in monandrous / monogynous colonies of m. bicolor because workers are more related to their own eggs ( r = 0.5 ) and to other workers eggs ( r = 0.375 ) than they are to the eggs of the queens ( r = 0.25 ) ( ratnieks , 1988 ) . however , in polyandrous / polygynous colonies in which workers are on average more related to sons of the queens ( r = 0.25 ) than to the sons of other workers ( r = 0.125 ) they may prefer the queen - laid eggs , as already verified for some polyandrous species such as apis mellifera ( ratnieks , 1988 ; ratnieks and visscher , 1989 ) and vespula vulgaris ( foster and ratnieks , 2001 ) . evidence for queen - worker conflict can be found in the cell provisioning and oviposition process of most stingless bee species . m. bicolor queens , in particular , undertake elaborate , ritualized interactions with workers before and during egg - laying ( velthuis et al . , 2006 ) . additionally , as with many stingless bees , m. bicolor workers in monogynous and polygynous colonies have been observed to lay reproductive and/or trophic eggs ( koedam et al . in the case of a reproductive egg , competing reproductive workers , however , frequently eat and replace this egg by their own ( velthuis et al . , 2007 ) . in polygynous colonies with highly related queens ( such as observed in several colonies in this study ) , the reasonably high levels of relatedness among workers could lower the costs of sharing reproduction ( giraud et al . on the other hand , in polygynous colonies with queens that are not closely related , workers would lay most of the male progeny because of the lower intra - colony relatedness ( koedam et al . , 2007 ) . accordingly , in a polygynous colony of m. bicolor containing three unrelated queens ( two of them introduced experimentally ) , 27%82% of the males were workers sons . in this colony , workers replaced reproductive eggs laid by other workers with their own , but they tended to avoid eating the queen s eggs ; this behavior was considered a form of policing by reproductive workers ( koedam et al . , 2007 ) . in conclusion , the results described here provide additional insights into the social structure and relatedness in m. bicolor . however , further research is needed in order to clarify the conflict over male parentage in monogynous and polygynous colonies , and to address other aspects related to worker oviposition , such as worker policing by egg eating . such studies will lead to a better understanding of the development of social behavior in melipona . the following online material is available for this article : allelic frequencies and observed ( ho ) and expected ( he ) heterozygosities in colonies of melipona bicolor .

in most cases , standard techniques of implant placement can not be applied in the posterior maxilla due to poor bone quality and quantity . insufficient bone volume as a result of maxillary sinus enlargement leads to a decrease in bone height in the posterior area . various vertical bone augmentation procedures have been introduced to date . the most common technique for elevating the sinus floor is the lateral window technique first introduced by tatum in 1977 and then published by boyne and james in 1980 . the long - term success of this technique has been reported even with the use of different types of graft materials and implants . in 1994 , summers introduced a new technique for elevating the sinus floor with the use of special instruments called osteotomes . in this technique , the schneiderian membrane is lifted from the alveolar crest by applying an osteotome . both the above - mentioned techniques can be applied in one- or two - stage protocols . however , the height of the residual bone is a factor which determines the protocol that should be applied . if the residual bone height is more than 5 mm and primary stability of the implant is achievable , one - stage protocol is adopted and sinus lifting is implemented at the same time as the implant placement ; otherwise , it is essential to use the delayed approach and place the implants after the healing period . based on the results of studies on sinus augmentation techniques , the osteotome technique is a predictable and efficacious technique for simultaneous or delayed implant placement [ 1,57 ] . in this technique , the average amount of sinus lifting has been reported to be 4.40.2 mm without bone grafting and 37 mm with bone graft materials . studies have shown that the crestal method using an osteotome in comparison to the conventional technique ( lateral window ) is less invasive and has some advantages including a shorter surgical duration and minimum post - surgical complications . furthermore , in the osteotome technique it is possible to condense the weak maxillary bone ( bone types 3 and 4 ) and increase the implant - to - bone contact area , which leads to more primary stability . moreover , it has been noted that accessing the sinus from the crestal zone requires a smaller socket , which has a much shorter healing period . numerous clinical investigations have been carried out to compare osteotome with lateral window techniques with immediate and delayed implant placements for sinus lifting [ 1,4,1117 ] . however , in the author s review of literature , no histological study comparing these two techniques can be found . the effect of the osteotome technique on the healing process has been poorly understood . here the question is whether or not the force and the temperature produced by tapping of the osteotome with the surgical mallet have any effects on factors such as the healing process , quantity of the new bone and severity of inflammation in the surgical zone . the osteotome technique is more convenient compared to the conventional technique due to the simplicity of application , less post - surgical complications and the shorter healing period . however , it is obvious that in order to confirm and validate the benefits of applying the osteotome technique as an alternative to the conventional technique ( lateral window ) , it is essential for the comparative histological evaluations of the two methods to demonstrate similar results . such a comparative study has not yet been conducted . the aim of this study was to evaluate and compare the osteotome and lateral window techniques using histological and histomorphometric methods in patients with less than 5 mm of posterior maxillary alveolar residual bone , who were referred to the dental implant research center of tehran university of medical sciences . in this research , which was a second - phase clinical trial ( pilot ) without blinding , 10 patients ( six females and four males ) were selected from patients needing implant treatment in the maxillary posterior region who were referred to the implant department of tehran university of medical sciences , school of dentistry . the selected patients suffered from severe atrophy in the posterior area ; consequently , the application of conventional methods for implant placement was impossible . in all the cases , the maximum bone height between the sinus floor and the crestal bone was less than 5 mm . uncontrollable systemic diseases such as diabetes , acute sinus infections , chemotherapy within 12 months before surgery , radiotherapy of more than 5000 rads in the head and neck region and psychological problems were listed as the excluding criteria . to evaluate the residual bone height , it was essential for the patients to complete the consent form for participating in this study . the study protocol was approved by the ethics committee of tehran university of medical sciences . prophylactic antibiotics , 2 gr of amoxicillin or 600 mgr of clindamycin were prescribed one or two hours before the surgery . natural randomization was performed to divide the patients into two groups so that in cases with 12 mm of residual bone height , the lateral technique was adopted and in patients with 34 mm of bone height the osteotome technique was applied . in both methods , bio - oss ( geistlish pharma ag / wolhousen , switzerland ) was used as the graft material . in the lateral technique , the lateral wall of the sinus was exposed by performing a crestal incision and a mucoperiosteal flap . when the bony window became removable , the surgeon started to separate the sinus membrane from the inferior edge of the osteotomy region and pushed the membrane upward . the sinus membrane was carefully separated from the inner and inferior walls . at the same time , the external wall was pushed inward and upward to form a new horizontal ceiling for the space created . care was exercised not to perforate the membrane ; however , in the cases of perforation a resorbable collagen membrane ( biogide geistlish pharma ag / wolhousen , switzerland ) was applied to cover the hole . the graft material ( bio - oss ) was mixed with normal saline solution and packed gently into the sinus in order to completely fill the cavity with bone substitute material and achieve the desired bone height . next , a resorbable membrane ( biogide ) was placed on the outer surface of the window and the flap was sutured as a primary closure . in the second group , the sinus floor elevation procedure was performed by applying the osteotome technique . in this technique , the buccal and palatal mucoperiosteal flaps were prepared by means of a crestal incision and the alveolar ridge was exposed after reflecting the flap . site positioning was marked using a small bur on the alveolar ridge and the osteotomy sites were prepared independently and separately relevant to the ultimate number of implants . the approximate location of the sinus membrane was determined based on initial radiographs . by applying a two - millimeter twist drill , the osteotomy was prepared so that the distance between the bottom of the osteotomy and the sinus floor was approximately 0.51.5 mm . the diameters of the osteotomy were gradually increased , using a combination of different drills and scaled osteotomes ( iti - straumann ) and osteotomy sites were prepared at a distance of 0.51.5 mm away from the sinus floor . the sinus floor was fractured with the final osteotome with the same diameter as the final drill . the odds of sinus perforations were assessed using valsalva maneuver immediately after fracturing the sinus floor . in case of sinus membrane perforation , the surgical method was converted to the lateral window technique and the site was covered with a resorbable collagen membrane ( biogide ) . in the absence of perforation , graft material ( bio - oss ) was placed in a multiple - step procedure in the osteotomy site and packed with an osteotome . graft material elevated the intact sinus membrane after displacing the sinus floor , which was lifted with the final osteotome to the desirable height . after filling the cavity completely with bone graft material , the sinus membrane was elevated to an appropriate level in order to provide sufficient space for placement of implants with a minimum length of 9 mm . all the patients were given 500 mg of amoxicillin , 4 times per day for 1 week . furthermore , an anti - inflammatory and a nasal decongestant drug were prescribed when required . all the patients were instructed in how to take care after the sinus graft surgery . any accidental sinus membrane perforation during the surgery and the possible occurrence of any post - surgical complications , such as acute infection and bleeding during the healing period were registered . after a 10-month healing period , biopsy specimens were obtained from the alveolar crest with a 3-mm trephine bur in all the cases , simultaneous with implant placement procedures . it should be noted that histological evaluations were performed by one pathologist ( pm ) who was blind to the surgical techniques using only coded samples . after 48 hours , when complete fixation was achieved , the fixation solution was replaced with 10% formic acid for decalcification . the decalcification process lasted for 5 days , during which the acid was renewed daily . after completion of the decalcification process , the cylindrical specimens were cut longitudinally along their axial direction and divided into two equal parts . the specimens were prepared for staining and microscopic sections using the conventional method . in this method , the specimens are placed in various solutions containing different concentrations of alcohol and xylene ( xilol ) in the preparation equipment to be cut and stained . after initial paraffinization , half - cylindrical specimens were embedded in paraffin blocks from their newly cut surfaces . subsequently , paraffin - embedded blocks were serially sectioned using a microtome device ( laica , germany ) to produce slices with 5-m thickness . at least five sections of each sample were placed on glass slides and stained by the conventional protocol for hematoxylin and eosin staining ( h&e ) . the slides were studied under a light microscope ( bx51 , olympus , japan ) at 100 magnification ( lens number 10 ) . the histological parameters evaluated in this study included the status of the residual biomaterial , the amount of the newly formed bone , the amount of connective tissue between the new bone and the residual biomaterial , the location of biomaterial in relation to the new bone , the lamellar - woven bone ratio , which is determined using polarized microscopy , the extent of inflammation in the connective tissue and the occurrence of a foreign body reaction . photographs were taken using a digital camera ( dp72 , olympus , japan ) from the central region of each slide and assessed by a histological evaluation software ( professional analysis , germany ) to determine and register the area of the newly formed bone , the area of the residual biomaterial and the area of connective tissue . the mean value of these measured areas was calculated for 5 slides , which were cut out of each specimen and ascribed to the relevant specimen . in addition to bone structure , residual biomaterial and connective tissue , other factors such as inflammatory infiltrates and foreign body reactions can be identified easily in these slides . the extent of inflammation was determined based on the number of lymphocytes existing in the tissue and based on the type of inflammation ( focal or diffuse inflammation ) . the quantitative parameters were reported as mean values and standard deviations ( mean sd ) and qualitative parameters were presented as crude and relative frequencies . comparison of variable distribution between the two groups was carried out by mann - whitney u test and fisher s exact test . prophylactic antibiotics , 2 gr of amoxicillin or 600 mgr of clindamycin were prescribed one or two hours before the surgery . natural randomization was performed to divide the patients into two groups so that in cases with 12 mm of residual bone height , the lateral technique was adopted and in patients with 34 mm of bone height the osteotome technique was applied . in both methods , bio - oss ( geistlish pharma ag / wolhousen , switzerland ) was used as the graft material . in the lateral technique , the lateral wall of the sinus was exposed by performing a crestal incision and a mucoperiosteal flap . when the bony window became removable , the surgeon started to separate the sinus membrane from the inferior edge of the osteotomy region and pushed the membrane upward . the sinus membrane was carefully separated from the inner and inferior walls . at the same time , the external wall was pushed inward and upward to form a new horizontal ceiling for the space created . care was exercised not to perforate the membrane ; however , in the cases of perforation a resorbable collagen membrane ( biogide geistlish pharma ag / wolhousen , switzerland ) was applied to cover the hole . the graft material ( bio - oss ) was mixed with normal saline solution and packed gently into the sinus in order to completely fill the cavity with bone substitute material and achieve the desired bone height . next , a resorbable membrane ( biogide ) was placed on the outer surface of the window and the flap was sutured as a primary closure . in the second group , the sinus floor elevation procedure was performed by applying the osteotome technique . in this technique , the buccal and palatal mucoperiosteal flaps were prepared by means of a crestal incision and the alveolar ridge was exposed after reflecting the flap . site positioning was marked using a small bur on the alveolar ridge and the osteotomy sites were prepared independently and separately relevant to the ultimate number of implants . the approximate location of the sinus membrane was determined based on initial radiographs . by applying a two - millimeter twist drill , the osteotomy was prepared so that the distance between the bottom of the osteotomy and the sinus floor was approximately 0.51.5 mm . the diameters of the osteotomy were gradually increased , using a combination of different drills and scaled osteotomes ( iti - straumann ) and osteotomy sites were prepared at a distance of 0.51.5 mm away from the sinus floor . the sinus floor was fractured with the final osteotome with the same diameter as the final drill . the odds of sinus perforations were assessed using valsalva maneuver immediately after fracturing the sinus floor . in case of sinus membrane perforation , the surgical method was converted to the lateral window technique and the site was covered with a resorbable collagen membrane ( biogide ) . in the absence of perforation , graft material ( bio - oss ) was placed in a multiple - step procedure in the osteotomy site and packed with an osteotome . graft material elevated the intact sinus membrane after displacing the sinus floor , which was lifted with the final osteotome to the desirable height . after filling the cavity completely with bone graft material , the sinus membrane was elevated to an appropriate level in order to provide sufficient space for placement of implants with a minimum length of 9 mm . all the patients were given 500 mg of amoxicillin , 4 times per day for 1 week . furthermore , an anti - inflammatory and a nasal decongestant drug were prescribed when required . all the patients were instructed in how to take care after the sinus graft surgery . any accidental sinus membrane perforation during the surgery and the possible occurrence of any post - surgical complications , such as acute infection and bleeding during the healing period were registered . after a 10-month healing period , biopsy specimens were obtained from the alveolar crest with a 3-mm trephine bur in all the cases , simultaneous with implant placement procedures . it should be noted that histological evaluations were performed by one pathologist ( pm ) who was blind to the surgical techniques using only coded samples . after 48 hours , when complete fixation was achieved , the fixation solution was replaced with 10% formic acid for decalcification . the decalcification process lasted for 5 days , during which the acid was renewed daily . after completion of the decalcification process , the cylindrical specimens were cut longitudinally along their axial direction and divided into two equal parts . the specimens were prepared for staining and microscopic sections using the conventional method . in this method , the specimens are placed in various solutions containing different concentrations of alcohol and xylene ( xilol ) in the preparation equipment to be cut and stained . after initial paraffinization , half - cylindrical specimens were embedded in paraffin blocks from their newly cut surfaces . subsequently , paraffin - embedded blocks were serially sectioned using a microtome device ( laica , germany ) to produce slices with 5-m thickness . at least five sections of each sample were placed on glass slides and stained by the conventional protocol for hematoxylin and eosin staining ( h&e ) . the slides were studied under a light microscope ( bx51 , olympus , japan ) at 100 magnification ( lens number 10 ) . the histological parameters evaluated in this study included the status of the residual biomaterial , the amount of the newly formed bone , the amount of connective tissue between the new bone and the residual biomaterial , the location of biomaterial in relation to the new bone , the lamellar - woven bone ratio , which is determined using polarized microscopy , the extent of inflammation in the connective tissue and the occurrence of a foreign body reaction . photographs were taken using a digital camera ( dp72 , olympus , japan ) from the central region of each slide and assessed by a histological evaluation software ( professional analysis , germany ) to determine and register the area of the newly formed bone , the area of the residual biomaterial and the area of connective tissue . the mean value of these measured areas was calculated for 5 slides , which were cut out of each specimen and ascribed to the relevant specimen . in addition to bone structure , residual biomaterial and connective tissue , other factors such as inflammatory infiltrates and foreign body reactions can be identified easily in these slides . the extent of inflammation was determined based on the number of lymphocytes existing in the tissue and based on the type of inflammation ( focal or diffuse inflammation ) . the quantitative parameters were reported as mean values and standard deviations ( mean sd ) and qualitative parameters were presented as crude and relative frequencies . comparison of variable distribution between the two groups was carried out by mann - whitney u test and fisher s exact test . in this study , a total of 14 sinuses in ten patients ( bilateral in four cases ) were augmented with bio - oss . lateral window and osteotome techniques were applied for augmentation in 8 and 6 sinuses , respectively . in one case , the osteotome technique was converted to the lateral window technique due to membrane perforation during the sinus augmentation procedure . therefore , finally , 5 sinuses were considered in the osteotome group and 9 sinuses in the lateral window group . during the ten - month healing period , no post - surgical complications such as sinus infection or bleeding was reported . newly formed bone around the residual biomaterial mass was observed in all the specimens in both groups . this viable bone consisted of lacunae containing osteocytes in all cases . in the osteotome group , 45% and 55% of the new bone consisted of lamellar bone and woven bone , respectively . in the lateral window group , the new bone consisted of 35% and 65% of lamellar bone and woven bone , respectively . the osteoid matrix / total bone ratios were estimated approximately 15% and 10% in the lateral window and osteotome techniques , respectively . the values of inflammation parameters as the number of inflammatory cells and distribution patterns are presented in table 2 . in all the cases , chronic inflammatory cells , including lymphocytes , plasma cells and low quantities of macrophages were observed . no statistically significant differences were observed in the number of inflammatory cells between the two groups and no foreign body reactions were reported . the mean values of the new bone in the lateral window and osteotome techniques were 306.0 and 25.25.2 , respectively , with no significant differences between the two groups . moreover , the average amounts of residual biomaterial and connective tissue were similar in both groups . newly formed bone around the residual biomaterial mass was observed in all the specimens in both groups . this viable bone consisted of lacunae containing osteocytes in all cases . in the osteotome group , 45% and 55% of the new bone consisted of lamellar bone and woven bone , respectively . in the lateral window group , the new bone consisted of 35% and 65% of lamellar bone and woven bone , respectively . the osteoid matrix / total bone ratios were estimated approximately 15% and 10% in the lateral window and osteotome techniques , respectively . the values of inflammation parameters as the number of inflammatory cells and distribution patterns are presented in table 2 . the inflammation severity was considered as mild . in all the cases , chronic inflammatory cells , including lymphocytes , plasma cells and low quantities of macrophages were observed . no statistically significant differences were observed in the number of inflammatory cells between the two groups and no foreign body reactions were reported . the mean values of the new bone in the lateral window and osteotome techniques were 306.0 and 25.25.2 , respectively , with no significant differences between the two groups . moreover , the average amounts of residual biomaterial and connective tissue were similar in both groups . however , the osteotome technique can be regarded as an alternative less invasive technique to augment the sinus floor and to improve bone density and quality . in the current study , histological and histomorphometric differences of osteotome and lateral window techniques were evaluated . to the best of our knowledge , such a comparative study does not exist in the literature . the rationale of the present research was the fact that it is essential for a secondary technique to demonstrate similar clinical and histological results similar to the conventional method to be regarded as an alternative technique . the results of this study did not reveal statistically significant differences in the amount of new viable bone between the osteotome ( 25.25.2 ) and lateral window ( 306.0 ) techniques . furthermore , the inflammation severity was almost similar in both groups without any significant differences . therefore , the controlled force and the temperature produced by tapping of the osteotome with the surgical mallet did not affect the healing process and the formation of the new bone . in this study , a modified version of summers osteotome technique was applied , in which the osteotome was directly tapped to the sinus floor without using any intermediate graft material . the reason for application of the modified summers technique was to reduce the forces produced by the surgical mallet . although the risk of membrane perforation is low in summers technique , due to utilization of graft material as an intermediate layer , severe tapping can be irritating for patients . on the other hand , tactile and auditory changes associated with sinus floor encroachment are considered warnings for the clinicians to change the mallet pressure . the clinician 's experience and skills are essential for achieving a controlled fracture without perforation . a deproteinized bovine bone mineral , known as bio - oss , was applied . since it is necessary to maintain the space produced by elevating the schneiderian membrane for the staged implant placement , the low resorption rate of bio - oss makes it advantageous as a graft material . in this study , bone materials which are not resorbable , such as ha , also function as space maintainers and preserve the space created by elevating the schneiderian membrane for sinus augmentation and also prevent the collapse of the schneiderian membrane . there are several studies comparing the effect of the techniques adopted for sinus augmentation on the clinical outcome of the implants . in all the cases , ( 2010 ) , through a three - year study , reported a 100% survival rate for implants placed after the sinus floor was elevated using the osteotome technique . santagata ( 2010 ) suggested that if a series of incrementally larger osteotomes were used to achieve improvement of bone density , sinus floor elevation and simultaneous implant placement and even immediate loading will be possible . in the literature review , sinus augmentation using osteotome technique and implant placement have been performed simultaneously , except for one case report , in which only clinical ( not histological ) evaluation of the osteotome technique was carried out . in this case report , implants were placed 6 months after sinus lifting ( staged implant placement ) due to the insufficient bone height of less than 5 mm from the sinus floor . it is suggested that in cases with more than 5 mm of residual bone height , implants can be placed simultaneously with the sinus augmentation procedure . however , if the residual bone height is less than 5 mm , staged approach should be undertaken ; otherwise , primary stability of the implant might be undermined . in the present study , a residual bone height of less than 5 mm was one of the including criteria ; therefore , the staged approach was necessary . moreover , a trephine bur was used to collect biopsy specimens from the alveolar crest at the same time as implant placement for histological evaluations . ( 2002 ) , revealed that applying the osteotome technique increases new bone formation and enhances osseointegration of the dental implants . they also suggested carrying out the same study on humans . according to the literature review conducted in this study , the only histological study evaluating the application of osteotome technique on humans is a case report by khatibloo ( 2011 ) , in which the sinus floor was elevated using the osteotome technique and after 45 months based on histometric analysis the new bone formation was estimated to be 24.8% , while the connective tissue and the bone marrow was 79.2% . however , the percentages of residual biomaterial in khatibloo s study and the current study are significantly different . as reported by khatibloo ( 2011 ) , there was no residual biomaterial after 45 months , while the mean value of residual biomaterial in the present study was 8.68.8 . this discrepancy might be attributed to the different biomaterials used in the two studies . the material used in khatibloo s study the presence of bio - oss particles in the graft area in humans was reported by avera ( 1997 ) after 44 months and even after 4 years by piattelli 1999 ) . dies ( 1996 ) observed that only a limited amount of particles was resorbed after 9 months . based on histomorphometric findings , new bone formation in the lateral window technique in the present study ( 306 ) was similar to that of a study by hans - dieter 2004 , in which bio - oss was the only material used for sinus augmentation ( 29.57 ) . the mean value of the residual biomaterial was reported to be 14.96 in a study by dieter 2004 , which is slightly higher in comparison to that ( 16.112 ) in the current research . in dieter s study , similar to the present study , the new bone was in direct contact with the biomaterial , without any gaps or connective tissue in between . in 2001 , yildirim observed bio - oss in biopsy specimens 9.5 months after sinus augmentation and histological analysis in his study revealed that bio - oss was in direct contact with the new bone which consisted of lamellar and woven bone . his findings were consistent with the observations in the current study . in 2008 , iezzi observed 122.9% of bio - oss and 402.4% of bone which consisted of 50% lamellar and 50% woven bone after five years . no foreign body reaction was observed in iezzi s study similar to the present study . however , in our study a limited number of osteoclasts were present . in cases of severe atrophic ridge ( 2 mm or less bone height ) in the posterior maxilla , the osteotome technique is not an appropriate treatment modality since it is time - consuming with relatively less predictable results . therefore , in the present study , natural randomization was used to divide patients into two study groups , i.e. the patient 's conditions determined the type of the treatment technique adopted . lateral window technique was chosen for patients who had 12 mm of residual bone height and the osteotome technique was applied for patients whose residual bone height was 34 mm . as the residual bone height does not affect the histological outcomes , dividing the patients into two study groups based on the natural randomization method does not lead to any biases in the study results . although this protocol decreases the sample size , it can lead to obtaining valuable statistical results and can be considered a positive aspect of this research . in this study , the osteotome technique was successfully applied for patients with less than 5 mm of residual bone height , which was a significant achievement of the current study . the volume of graft material used for one sinus augmentation procedure was 23 cc in the lateral window technique and less than 0.5 cc in the osteotome technique , enabling both the patient and the physician to economize . the small sample size , as a limitation of this study , can reduce the statistical power of the study . due to the small access region and the close proximity of the bony walls in the osteotome technique thus , it is recommended to perform histological evaluations 46 months after sinus augmentation in future studies . the nature and the amount of the newly formed bone did not exhibit any statistically significant differences between the lateral window and osteotome techniques . no statistically significant differences were observed between the two groups in terms of inflammation and edema . in the osteotome technique , hammering forces did not affect the healing process in the sinus . the osteotome technique can be applied in cases with less than 5 mm of residual bone height . it is suggested that the osteotome technique should be considered as an alternative for the lateral window technique , especially in cases in which a septum exists in the sinus or there is a single tooth in the posterior maxilla , where there is a high risk of membrane perforation due to limited access for window preparation .

objective : the aim of this study was to compare the lateral window and osteotome techniques for sinus lifting using histological and histomorphometric methods.materials and methods : in this clinical trial 10 patients ( a total number of 14 sinus areas ) who needed implant treatment in the atrophic posterior maxilla were enrolled . in all the cases the residual bone height between the sinus floor and the alveolar crest was less than 5 mm . sinus augmentation was performed . the treatment modality for a given residual bone height was selected randomly and bio - oss was applied in all the cases as the graft material . after a healing period of about 10 months , in all the cases , the implants were placed and biopsies of alveolar crestal bone were obtained at the same time ; biopsy specimens were evaluated using histological and histomorphometric methods . fisher s exact and mann - whitney u tests were used to compare distribution of variables in the two groups . statistical significance was defined at p<0.05.results : the new bone was located in direct contact with the biomaterial without any gaps . this viable bone consisted of lacunae containing osteocytes . infiltration of inflammatory cells did not exhibit any significant differences between the two techniques . foreign body reaction was not observed in any cases . histomorphometric evaluations demonstrated that the mean values of the new bone in the lateral window and osteotome techniques were 306.0 and 25.25.2 , respectively , with no significant differences between the two groups .. moreover , the average quantity of residual biomaterial and connective tissue were similar for the two groups.conclusion:the nature and the volume of the new bone in lateral window and osteotome techniques were the same .

INTRODUCTION MATERIALS AND METHODS Surgical Procedure Histological Analysis and Histomorphometry Statistical Analysis RESULT Histological Findings DISCUSSION CONCLUSION

in most cases , standard techniques of implant placement can not be applied in the posterior maxilla due to poor bone quality and quantity . the most common technique for elevating the sinus floor is the lateral window technique first introduced by tatum in 1977 and then published by boyne and james in 1980 . in this technique , the schneiderian membrane is lifted from the alveolar crest by applying an osteotome . however , the height of the residual bone is a factor which determines the protocol that should be applied . if the residual bone height is more than 5 mm and primary stability of the implant is achievable , one - stage protocol is adopted and sinus lifting is implemented at the same time as the implant placement ; otherwise , it is essential to use the delayed approach and place the implants after the healing period . in this technique , the average amount of sinus lifting has been reported to be 4.40.2 mm without bone grafting and 37 mm with bone graft materials . numerous clinical investigations have been carried out to compare osteotome with lateral window techniques with immediate and delayed implant placements for sinus lifting [ 1,4,1117 ] . here the question is whether or not the force and the temperature produced by tapping of the osteotome with the surgical mallet have any effects on factors such as the healing process , quantity of the new bone and severity of inflammation in the surgical zone . however , it is obvious that in order to confirm and validate the benefits of applying the osteotome technique as an alternative to the conventional technique ( lateral window ) , it is essential for the comparative histological evaluations of the two methods to demonstrate similar results . the aim of this study was to evaluate and compare the osteotome and lateral window techniques using histological and histomorphometric methods in patients with less than 5 mm of posterior maxillary alveolar residual bone , who were referred to the dental implant research center of tehran university of medical sciences . in this research , which was a second - phase clinical trial ( pilot ) without blinding , 10 patients ( six females and four males ) were selected from patients needing implant treatment in the maxillary posterior region who were referred to the implant department of tehran university of medical sciences , school of dentistry . in all the cases , the maximum bone height between the sinus floor and the crestal bone was less than 5 mm . to evaluate the residual bone height , it was essential for the patients to complete the consent form for participating in this study . natural randomization was performed to divide the patients into two groups so that in cases with 12 mm of residual bone height , the lateral technique was adopted and in patients with 34 mm of bone height the osteotome technique was applied . in both methods , bio - oss ( geistlish pharma ag / wolhousen , switzerland ) was used as the graft material . in the lateral technique , the lateral wall of the sinus was exposed by performing a crestal incision and a mucoperiosteal flap . when the bony window became removable , the surgeon started to separate the sinus membrane from the inferior edge of the osteotomy region and pushed the membrane upward . at the same time , the external wall was pushed inward and upward to form a new horizontal ceiling for the space created . care was exercised not to perforate the membrane ; however , in the cases of perforation a resorbable collagen membrane ( biogide geistlish pharma ag / wolhousen , switzerland ) was applied to cover the hole . the graft material ( bio - oss ) was mixed with normal saline solution and packed gently into the sinus in order to completely fill the cavity with bone substitute material and achieve the desired bone height . in the second group , the sinus floor elevation procedure was performed by applying the osteotome technique . in this technique , the buccal and palatal mucoperiosteal flaps were prepared by means of a crestal incision and the alveolar ridge was exposed after reflecting the flap . site positioning was marked using a small bur on the alveolar ridge and the osteotomy sites were prepared independently and separately relevant to the ultimate number of implants . by applying a two - millimeter twist drill , the osteotomy was prepared so that the distance between the bottom of the osteotomy and the sinus floor was approximately 0.51.5 mm . the diameters of the osteotomy were gradually increased , using a combination of different drills and scaled osteotomes ( iti - straumann ) and osteotomy sites were prepared at a distance of 0.51.5 mm away from the sinus floor . the sinus floor was fractured with the final osteotome with the same diameter as the final drill . in case of sinus membrane perforation , the surgical method was converted to the lateral window technique and the site was covered with a resorbable collagen membrane ( biogide ) . in the absence of perforation , graft material ( bio - oss ) was placed in a multiple - step procedure in the osteotomy site and packed with an osteotome . graft material elevated the intact sinus membrane after displacing the sinus floor , which was lifted with the final osteotome to the desirable height . after filling the cavity completely with bone graft material , the sinus membrane was elevated to an appropriate level in order to provide sufficient space for placement of implants with a minimum length of 9 mm . after a 10-month healing period , biopsy specimens were obtained from the alveolar crest with a 3-mm trephine bur in all the cases , simultaneous with implant placement procedures . the histological parameters evaluated in this study included the status of the residual biomaterial , the amount of the newly formed bone , the amount of connective tissue between the new bone and the residual biomaterial , the location of biomaterial in relation to the new bone , the lamellar - woven bone ratio , which is determined using polarized microscopy , the extent of inflammation in the connective tissue and the occurrence of a foreign body reaction . photographs were taken using a digital camera ( dp72 , olympus , japan ) from the central region of each slide and assessed by a histological evaluation software ( professional analysis , germany ) to determine and register the area of the newly formed bone , the area of the residual biomaterial and the area of connective tissue . in addition to bone structure , residual biomaterial and connective tissue , other factors such as inflammatory infiltrates and foreign body reactions can be identified easily in these slides . comparison of variable distribution between the two groups was carried out by mann - whitney u test and fisher s exact test . natural randomization was performed to divide the patients into two groups so that in cases with 12 mm of residual bone height , the lateral technique was adopted and in patients with 34 mm of bone height the osteotome technique was applied . in both methods , bio - oss ( geistlish pharma ag / wolhousen , switzerland ) was used as the graft material . in the lateral technique , the lateral wall of the sinus was exposed by performing a crestal incision and a mucoperiosteal flap . at the same time , the external wall was pushed inward and upward to form a new horizontal ceiling for the space created . care was exercised not to perforate the membrane ; however , in the cases of perforation a resorbable collagen membrane ( biogide geistlish pharma ag / wolhousen , switzerland ) was applied to cover the hole . the graft material ( bio - oss ) was mixed with normal saline solution and packed gently into the sinus in order to completely fill the cavity with bone substitute material and achieve the desired bone height . in the second group , the sinus floor elevation procedure was performed by applying the osteotome technique . in this technique , the buccal and palatal mucoperiosteal flaps were prepared by means of a crestal incision and the alveolar ridge was exposed after reflecting the flap . by applying a two - millimeter twist drill , the osteotomy was prepared so that the distance between the bottom of the osteotomy and the sinus floor was approximately 0.51.5 mm . the diameters of the osteotomy were gradually increased , using a combination of different drills and scaled osteotomes ( iti - straumann ) and osteotomy sites were prepared at a distance of 0.51.5 mm away from the sinus floor . the sinus floor was fractured with the final osteotome with the same diameter as the final drill . in case of sinus membrane perforation , the surgical method was converted to the lateral window technique and the site was covered with a resorbable collagen membrane ( biogide ) . in the absence of perforation , graft material ( bio - oss ) was placed in a multiple - step procedure in the osteotomy site and packed with an osteotome . graft material elevated the intact sinus membrane after displacing the sinus floor , which was lifted with the final osteotome to the desirable height . after a 10-month healing period , biopsy specimens were obtained from the alveolar crest with a 3-mm trephine bur in all the cases , simultaneous with implant placement procedures . the histological parameters evaluated in this study included the status of the residual biomaterial , the amount of the newly formed bone , the amount of connective tissue between the new bone and the residual biomaterial , the location of biomaterial in relation to the new bone , the lamellar - woven bone ratio , which is determined using polarized microscopy , the extent of inflammation in the connective tissue and the occurrence of a foreign body reaction . photographs were taken using a digital camera ( dp72 , olympus , japan ) from the central region of each slide and assessed by a histological evaluation software ( professional analysis , germany ) to determine and register the area of the newly formed bone , the area of the residual biomaterial and the area of connective tissue . in addition to bone structure , residual biomaterial and connective tissue , other factors such as inflammatory infiltrates and foreign body reactions can be identified easily in these slides . comparison of variable distribution between the two groups was carried out by mann - whitney u test and fisher s exact test . in this study , a total of 14 sinuses in ten patients ( bilateral in four cases ) were augmented with bio - oss . lateral window and osteotome techniques were applied for augmentation in 8 and 6 sinuses , respectively . in one case , the osteotome technique was converted to the lateral window technique due to membrane perforation during the sinus augmentation procedure . newly formed bone around the residual biomaterial mass was observed in all the specimens in both groups . this viable bone consisted of lacunae containing osteocytes in all cases . in the osteotome group , 45% and 55% of the new bone consisted of lamellar bone and woven bone , respectively . in the lateral window group , the new bone consisted of 35% and 65% of lamellar bone and woven bone , respectively . the osteoid matrix / total bone ratios were estimated approximately 15% and 10% in the lateral window and osteotome techniques , respectively . the values of inflammation parameters as the number of inflammatory cells and distribution patterns are presented in table 2 . in all the cases , chronic inflammatory cells , including lymphocytes , plasma cells and low quantities of macrophages were observed . no statistically significant differences were observed in the number of inflammatory cells between the two groups and no foreign body reactions were reported . the mean values of the new bone in the lateral window and osteotome techniques were 306.0 and 25.25.2 , respectively , with no significant differences between the two groups . moreover , the average amounts of residual biomaterial and connective tissue were similar in both groups . newly formed bone around the residual biomaterial mass was observed in all the specimens in both groups . this viable bone consisted of lacunae containing osteocytes in all cases . in the osteotome group , 45% and 55% of the new bone consisted of lamellar bone and woven bone , respectively . in the lateral window group , the new bone consisted of 35% and 65% of lamellar bone and woven bone , respectively . the osteoid matrix / total bone ratios were estimated approximately 15% and 10% in the lateral window and osteotome techniques , respectively . the values of inflammation parameters as the number of inflammatory cells and distribution patterns are presented in table 2 . in all the cases , chronic inflammatory cells , including lymphocytes , plasma cells and low quantities of macrophages were observed . no statistically significant differences were observed in the number of inflammatory cells between the two groups and no foreign body reactions were reported . the mean values of the new bone in the lateral window and osteotome techniques were 306.0 and 25.25.2 , respectively , with no significant differences between the two groups . moreover , the average amounts of residual biomaterial and connective tissue were similar in both groups . however , the osteotome technique can be regarded as an alternative less invasive technique to augment the sinus floor and to improve bone density and quality . in the current study , histological and histomorphometric differences of osteotome and lateral window techniques were evaluated . the results of this study did not reveal statistically significant differences in the amount of new viable bone between the osteotome ( 25.25.2 ) and lateral window ( 306.0 ) techniques . furthermore , the inflammation severity was almost similar in both groups without any significant differences . therefore , the controlled force and the temperature produced by tapping of the osteotome with the surgical mallet did not affect the healing process and the formation of the new bone . in this study , a modified version of summers osteotome technique was applied , in which the osteotome was directly tapped to the sinus floor without using any intermediate graft material . since it is necessary to maintain the space produced by elevating the schneiderian membrane for the staged implant placement , the low resorption rate of bio - oss makes it advantageous as a graft material . in this study , bone materials which are not resorbable , such as ha , also function as space maintainers and preserve the space created by elevating the schneiderian membrane for sinus augmentation and also prevent the collapse of the schneiderian membrane . there are several studies comparing the effect of the techniques adopted for sinus augmentation on the clinical outcome of the implants . in all the cases , ( 2010 ) , through a three - year study , reported a 100% survival rate for implants placed after the sinus floor was elevated using the osteotome technique . in the literature review , sinus augmentation using osteotome technique and implant placement have been performed simultaneously , except for one case report , in which only clinical ( not histological ) evaluation of the osteotome technique was carried out . in this case report , implants were placed 6 months after sinus lifting ( staged implant placement ) due to the insufficient bone height of less than 5 mm from the sinus floor . it is suggested that in cases with more than 5 mm of residual bone height , implants can be placed simultaneously with the sinus augmentation procedure . however , if the residual bone height is less than 5 mm , staged approach should be undertaken ; otherwise , primary stability of the implant might be undermined . in the present study , a residual bone height of less than 5 mm was one of the including criteria ; therefore , the staged approach was necessary . moreover , a trephine bur was used to collect biopsy specimens from the alveolar crest at the same time as implant placement for histological evaluations . according to the literature review conducted in this study , the only histological study evaluating the application of osteotome technique on humans is a case report by khatibloo ( 2011 ) , in which the sinus floor was elevated using the osteotome technique and after 45 months based on histometric analysis the new bone formation was estimated to be 24.8% , while the connective tissue and the bone marrow was 79.2% . however , the percentages of residual biomaterial in khatibloo s study and the current study are significantly different . as reported by khatibloo ( 2011 ) , there was no residual biomaterial after 45 months , while the mean value of residual biomaterial in the present study was 8.68.8 . the material used in khatibloo s study the presence of bio - oss particles in the graft area in humans was reported by avera ( 1997 ) after 44 months and even after 4 years by piattelli 1999 ) . based on histomorphometric findings , new bone formation in the lateral window technique in the present study ( 306 ) was similar to that of a study by hans - dieter 2004 , in which bio - oss was the only material used for sinus augmentation ( 29.57 ) . the mean value of the residual biomaterial was reported to be 14.96 in a study by dieter 2004 , which is slightly higher in comparison to that ( 16.112 ) in the current research . in dieter s study , similar to the present study , the new bone was in direct contact with the biomaterial , without any gaps or connective tissue in between . in 2001 , yildirim observed bio - oss in biopsy specimens 9.5 months after sinus augmentation and histological analysis in his study revealed that bio - oss was in direct contact with the new bone which consisted of lamellar and woven bone . in 2008 , iezzi observed 122.9% of bio - oss and 402.4% of bone which consisted of 50% lamellar and 50% woven bone after five years . no foreign body reaction was observed in iezzi s study similar to the present study . in cases of severe atrophic ridge ( 2 mm or less bone height ) in the posterior maxilla , the osteotome technique is not an appropriate treatment modality since it is time - consuming with relatively less predictable results . lateral window technique was chosen for patients who had 12 mm of residual bone height and the osteotome technique was applied for patients whose residual bone height was 34 mm . as the residual bone height does not affect the histological outcomes , dividing the patients into two study groups based on the natural randomization method does not lead to any biases in the study results . in this study , the osteotome technique was successfully applied for patients with less than 5 mm of residual bone height , which was a significant achievement of the current study . the volume of graft material used for one sinus augmentation procedure was 23 cc in the lateral window technique and less than 0.5 cc in the osteotome technique , enabling both the patient and the physician to economize . due to the small access region and the close proximity of the bony walls in the osteotome technique thus , it is recommended to perform histological evaluations 46 months after sinus augmentation in future studies . the nature and the amount of the newly formed bone did not exhibit any statistically significant differences between the lateral window and osteotome techniques . no statistically significant differences were observed between the two groups in terms of inflammation and edema . the osteotome technique can be applied in cases with less than 5 mm of residual bone height . it is suggested that the osteotome technique should be considered as an alternative for the lateral window technique , especially in cases in which a septum exists in the sinus or there is a single tooth in the posterior maxilla , where there is a high risk of membrane perforation due to limited access for window preparation .

there are numerous sources of suspended particulate matter ( pm ) including industrial sources , automobile traffic , natural disasters , such as forest fires and volcanic eruptions , and local sources generated either in the home or workplace [ 13 ] . urban air pollution originates from a variety of sources , of which the combustion of fossil fuel products is the principal source . air pollutants can be classified by their source , chemical composition , size , mode of release ( gaseous or particulate ) , and space ( indoor or outdoor ) . epidemiological studies show that air pollution exposure positively correlates with admissions for pneumonia , asthma , and chronic obstructive pulmonary disease ( copd ) . of all the pollutants , inhalable particles ( pm10 ) showed the strongest association with adverse respiratory health effects . in addition , data from large population cohorts have indicated an association between exposure to pm and cardiovascular morbidity and mortality [ 710 ] . mechanistically , this is thought to be due to the systemic inflammatory response induced by exposure to pm air pollution . this concept is supported by studies showing a positive association between long - term pm exposure and hematological markers of inflammation and diseases such as diabetes mellitus . inhalation of air pollution particles induces a local response in the lung that is initiated by alveolar macrophages ( ams ) and airway epithelial cells . the macrophages are several times more potent in producing proinflammatory mediators that contribute to the local inflammatory response in the lung but also contribute to the subsequent systemic inflammatory response . the systemic inflammatory response is characterized by mobilization of inflammatory cells from the bone marrow into the circulatory system , followed by their activation , as well as the production of acute phase proteins by the liver , and an increase in circulating inflammatory mediators . in this review , the primary role of am is to keep the air spaces clear by removal of all foreign materials via phagocytosis . experiments from our laboratory have shown that ams exposed to atmospheric particulate matter are able to phagocytose these particles in vivo and in vitro [ 13 , 14 ] . nonbiological particles lack specific opsonins preventing them from classic opsonin - dependent phagocytosis . despite the absence of specific opsonins , am can phagocytose unopsonized environmental particles . the class a scavenger receptor ( sr - a ) and the macrophage receptor with collagenous structure ( marco ) are considered to be the two major receptors for unopsonized particle phagocytosis by am [ 17 , 18 ] , and a deficiency in scavenger receptor function results in reduced uptake of environmental particles by am . toll - like receptors ( tlrs ) are sensors that directly recognize molecules from microbes . they are essential for initiation of the innate immune response , when interacting with pm , and also play a role in sustaining and regulating the adaptive immune response to pm . ambient pm contains small amounts of microbial materials such as lipopolysaccharide ( lps)/endotoxin [ 19 , 20 ] , beta - glucan , bacteria , and fungal spores that are thought to be the mechanism by which tlrs engage in processing pm . among the tlrs identified in humans , tlr4 and tlr2 toll - like receptor 4 initiates a signaling cascade in response to lps present in the outer membrane of gram - negative bacteria , while tlr2 initiates signals in response to zymosan ( beta - glucans ) and peptidoglycan of gram - positive bacteria . furthermore , microorganisms attached to pm can be opsonized by specific opsonins ( such as immunoglobulin fc receptors and complement receptor 3 ) that allow am to phagocytose the particles via an opsonin - dependent pathway . particulate matter is classified according to aerodynamic diameter into pm10 ( coarse particles , median aerodynamic diameter 2.510 m pm2.5 ( fine particles , median aerodynamic diameter < 2.5 m ) , and ultrafine particles ( ufp ) median aerodynamic diameter < 0.1 m ) . pm10 particles are derived predominantly from abraded soil , road dust , construction debris , and oil combustion products with bioaerosols such as fungi , bacteria , endotoxins , and pollen , while pm2.5 and ufp are primarily derived from direct emissions from combustion processes such as vehicle use of fossil fuel products , wood burning , and coal burning . although a considerable amount of data implicate pm10 and pm2.5 in adverse health effects [ 2426 ] , much less is known about the risks of ufp . in addition , several studies have shown that pm2.5 and ufp have the strongest association with adverse cardiovascular adverse effects [ 27 , 28 ] , which is a direct consequence of the systemic response induced by these particles [ 29 , 30 ] . alveolar macrophages exposed to smaller pm that have the ability to penetrate deep into the lungs significantly contribute to the systemic inflammatory response . upon contact with particulate pollutants , the capability of inducing apoptosis and inflammation varies with different particle size and concentration . in vitro studies have shown that macrophages do recognize the size and shape of their target pathogens ; therefore , their response against various particle sizes may be different . it is generally thought that the larger surface area of pm2.5 and ufp per unit concentration of pm allows more opportunity for cellular interaction and a downstream biological response . on the contrary , in vitro studies comparing the effects of the coarse and fine fraction of pm10 showed stronger proinflammatory effects for the coarse particles [ 33 , 34 ] . in addition to pm size and concentration , particle composition has also been reported to impact pm toxicity . the toxicity of pm may stem from their metal content , adhered organic compounds , or other biological components such as lps . . showed that coarse pm induced a greater inflammatory response than fine pm in rats and suggested that , in these larger particles , toxicity is due more to their biological components , such as endotoxin , than their metal content . in other studies , no apparent relationship could be established between pulmonary injury and the concentration of ambient particles or their elemental components such as sulfate ( s ) , zinc ( zn ) , manganese ( mn ) , iron ( fe ) , and copper ( cu ) . diesel exhaust particles ( deps ) without their organic constituents were no longer able to induce apoptosis or generate reactive oxygen radicals in murine and human macrophages in vitro . diesel exhaust particle is a major component of urban pm10 pollutants , which comprise 40% of total pm10 levels in los angeles . the water - soluble fraction of pollutant particles and individual soluble metals such as vanadium ( v ) , nickel ( ni ) , and fe did not induce apoptosis in human ams . in addition to these reports that emphasize the toxicity of organic components in pm , nonorganic components such as metals have also been implicated in the pathogenesis of particulate - induced pulmonary inflammation . vanadium ( v ) , bromine ( br ) , lead ( pb ) , and organic carbon had a strong association with pulmonary inflammation . stone particles of varying composition ( mylonite , gabbro , feldspar , basalt , and quartz ) induced different cytokine responses in rat ams . because ambient particles contain many other nonleachable and leachable components , further studies are needed to identify the toxicity of different particle components . in addition to pm , gaseous pollutants such as ozone also have inflammatory effects on the respiratory tract and am [ 4245 ] . ozone exposure induces the release of cytokines and fibronectin by am , increases am recruitment into asthmatic airways , and increases the eosinophilic airway response . nitrogen dioxide ( no2 ) is a precursor to photochemical smog , and its major effect on health as an outdoor pollutant is likely through the formation of ozone . recent epidemiologic studies conducted worldwide have provided valuable insight into the associations between sulfur dioxide ( so2 ) , no2 , and carbon monoxide ( co ) exposure and increases in cardiopulmonary mortality , such as respiratory and cardiovascular hospital admissions , emergency admissions caused by stroke ( no2 ) , and myocardial infarction ( no2 and co ) [ 4951 ] . in these studies , no2 inhibited ams from playing an immunosuppressive role . alveolar macrophage phagocytosis was significantly suppressed following coexposure of fine carbon particles and so2 . because air pollution contains both , the contribution of these gaseous pollutants in modulating the response of the am to pm is complex , poorly understood , and still an area of active investigation . alveolar macrophages are one of the most potent producers of proinflammatory mediators in the airways and lung . studies from our laboratory have shown that human ams exposed to urban pm ( ehc-93 ) phagocytosed these particles in vitro ( figure 1 ) [ 13 , 54 ] , and , ex vivo , they produced tumor necrosis factor alpha ( tnf- ) in a dose - dependent fashion following pm exposure . in addition , these ams produce an array of proinflammatory mediators including acute response mediators such as interleukin ( il)-1 and il-6 as well as secondary mediators , such as il-8 , and granulocyte macrophage colony - stimulating factor ( gm - csf ) [ 11 , 13 ] . interestingly , production of anti - inflammatory mediators such as il-10 was suppressed , suggesting that the am inflammatory response induced by pm is tipped toward a proinflammatory profile . the inflammatory profile of mediators produced by bronchial epithelial cells exposed to pm is distinct from those of am . alveolar macrophages are also more potent producers of the acute response mediators such as il-1 , il-6 , and tnf- than bronchial epithelial cells when exposed to the same dose of pm , suggesting that ams are the drivers of the proinflammatory response in the lung following inhalation of pm ( figure 2 ) . furthermore , instillation of supernatants from human ams incubated ex vivo with urban pm into rabbit lungs produced a systemic response similar to that produced by direct deposition of the same amount of pm directly into the lungs [ 57 , 58 ] , which implies that ams significantly contribute to the systemic inflammatory response generated following exposure to pm . depending on the microenvironment , they undergo distinct activation programs , acquiring polarized phenotypes and different functional capacities that together provide an armamentarium that protects , repairs , and sometimes damages tissues [ 60 , 61 ] . program , is induced by signals generated during th1-mediated immune responses such as interferon ( ifn)- and by exposure to components of pathogens such as bacteria [ 60 , 61 ] . the m1 polarization response is characterized by upregulation of genes relevant to inflammation and cell - mediated immunity . m2 polarization induced upon exposure to the th2 cytokines il-4 and il-13 ( referred to as alternative activation ) or immunoregulatory signals such as il-10 ( also called deactivation ) is highlighted by induced expression of receptors with scavenger functions , anti - inflammatory cytokines , and molecules implicated in tissue remodeling [ 60 , 61 ] . although pm shares many of the same ingredients and characteristics as cigarette smoke , the m1 cytokines ( il-12 and ifn- ) are increased consistently in bronchoalveolar lavage fluid ( balf ) from pm - exposed animals [ 63 , 64 ] while the m2 cytokines ( il-4 , il-10 , and il-13 ) remain at lower levels . our group previously showed that primary cultured human am , stimulated in vitro with urban pm10 , produced an array of cytokines without significantly increased levels of il-10 compared to nonstimulated am . these reports suggest that pm skews the ams to an m1 profile rather than an m2 profile . data from an influenza virus pneumonia model suggest that macrophage pro- and anti - inflammatory phenotypes are under tight control of nearby airway epithelial cells . epithelial - macrophage crosstalk seems to be an important mechanism in keeping the balance between efficient host defense and excessive inflammation and injury during infection . these responses ( m1/m2 switching ) of macrophages following pm exposure still need further investigation to assess what factors ( such as size or composition ) determine switching . figure 3 shows how pm exposure could potentially inhibit macrophage switching and thus promote a proinflammatory state . in addition to being key players in the innate immune response against microorganisms and in the initiation of adaptive immune responses , they are crucial for the clearance and processing of microorganisms , dead cells , and environmental debris in the lung tissue via phagocytosis . in contrast to cells such as neutrophils , ams are long - lived and in general are resistant to apoptotic stimuli . following activation of am , by exposure to pm , for example , they either remain in the lung airways or tissues or are removed via the lymphatic system to regional lymph nodes . several studies have demonstrated that exposure to ambient pm and diesel exhaust particles induced apoptosis in macrophages [ 37 , 71 ] . phagocytosis of apoptotic cells ( efferocytosis ) by ams is involved in the regulation of the inflammatory response and maintenance of lung homeostasis by removing dead cells before the onset of necrosis . alveolar macrophages are primarily responsible for removing and processing dead cells and debris in the airways , thereby reducing their inflammatory potential . our group recently showed that 3-hydoxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase inhibitors ( statins ) enhance the phagocytic activity of ams and promote the clearance of pm from lung tissues . promoting macrophage phagocytosis and efferocytosis could accelerate processing and clearance of pm particles from lung tissues and thereby reduce lung inflammation . autophagy consists of the fusion of autophagosomes with lysosomes , forming autolysosomes and resulting in the breakdown of encapsulated materials to components that are then available for homeostasis . intracellular nanoparticles may undergo autophagic sequestration , and autophagy dysfunction may play an important role in nanoparticle toxicity . identified an autophagic defect in the ams of smokers and concluded that the decrease in the process of autophagy leads to impaired protein aggregate clearance , dysfunctional mitochondria , and defective delivery of bacteria to lysosomes . exposure to ambient pm could decrease autophagy of am in a similar manner to smoking , but further studies are necessary to confirm this . they sense , scavenge , and phagocytose pm and in the process they produce and release early response cytokines . these cytokines stimulate neighboring airway and alveolar epithelial cells as well as tissue - resident macrophages in an auto- and paracrine manner to produce a variety of chemokines necessary to recruit other cells , such as polymorphonuclear leukocytes , to assist in processing and ultimately clearing foreign material . human airway and alveolar epithelial cells are also capable of pm endocytosis and in the process they produce mediators such as gm - csf , il-1 , il-8 , and leukemia inhibitory factor ( lif ) in a dose - dependent manner at both the mrna and protein level when exposed to ambient particles . coculture experiments of bronchial epithelial cells and am showed synergistic production of certain mediators such as il-1 , il-6 , and gm - csf . the increased il-1 production [ 54 , 76 ] is mediated by the nucleotide - binding domain and leucine - rich repeat protein 3 ( nlrp3 ) inflammasome that spreads the local inflammatory response by interacting with resident dendritic cells residing within or near the epithelium , initiating , and maintaining an adaptive immune response . these studies demonstrate the importance of the interaction of ams with other lung cells in producing lung inflammation and possibly contributing to the systemic inflammatory response following pm inhalation . after phagocytosis and internalization of pm , the organic components are digested by the endosome into peptide fragments that combine with the mhc class ii complex for presentation to cd4 + t cells , which are pivotal steps in cell - mediated and adaptive immunity . expression of mhc in ams moderately increased in response to pm exposure in healthy human subjects . pretreatment of pm with heat to degrade the organic component abolished the mhc class ii overexpression , which suggests that the organic component of pm is responsible for mhc class ii upregulation . in addition to antigen - specific mhc and t - cell receptor interaction , t cells require a costimulatory signal to be fully activated . these molecules are expressed on the cell membrane of antigen presenting cells and are upregulated by pm exposure . together , these studies illustrate the crucial role that ams play in initiating the adaptive immune response to pm exposure . however , the role of this adaptive response in the local lung and systemic inflammatory responses induced by pm exposure has not been well studied to date . earlier studies implicate ams as key effector cells responsible for generating the systemic inflammatory response associated with exposure to air pollution [ 13 , 41 , 56 , 81 , 82 ] . they produce a broad range of mediators , particularly il-6 , il-1 , mip-1 , and the hematopoietic growth factor gm - csf , when exposed to urban pm . the importance of the am producing the mediators that elicit the systemic response is supported by studies showing a correlation between the amount of particles phagocytosed by am in the lung and the magnitude of bone marrow stimulation following pm exposure ( figure 4 ) . humans exposed to an acute episode of air pollution , where the predominant pollutant was pm , showed increased levels of circulating cytokines such as il-1 and il-6 and signs of bone marrow stimulation reflected by an increase in circulating band cells counts . these cytokines are similar to those produced by am exposed to pm both ex vivo and in vivo [ 13 , 84 ] , suggesting that these mediators produced in the lung enter the circulation and contribute to the systemic response associated with exposure to pm . recent studies from our laboratory showed that mediators such as il-6 , produced in the lung from pm exposure directly , translocate into the circulation and , because the ams are the most prolific producer of these mediators following pm exposure , it is reasonable to postulate that ams are crucially important effector cells in generating the systemic inflammatory response induced by air pollution exposure . several studies from our laboratory have shown that exposure to air pollution stimulates the bone marrow in humans and in animal models [ 11 , 14 , 5658 , 85 ] and promotes the release of both polymorphonuclear leukocytes and monocytes from the marrow . the cytokine production by macrophages in the lung is of particular importance in inducing this systemic inflammatory response ; for example , gm - csf is a hematopoietic growth factor that stimulates granulocyte and monocyte differentiation and releases from the bone marrow , but it also activates circulating leukocytes and prolongs their survival in the circulation . furthermore , il-1 is one of the acute response cytokines that induces cytokine production by many cells , stimulates hematopoiesis , activates endothelial cells , is pyrogenic , and induces the acute - phase response . in addition , stimulation of liver hepatocytes by il-6 produces acute phase proteins including c - reactive protein ( crp ) , fibrinogen , and antiproteases . moreover , il-6 also stimulates hematopoiesis , specifically the production of platelets , and has a broad stimulating effect on b and t cells , as well as markedly accelerating the transit time of granulocytes through the bone marrow , releasing them into the circulation , and promoting their sequestration in microvascular beds . collectively , gm - csf , il-1 , and il-6 have the ability to elicit a systemic inflammatory response characterized by an increase in circulating leukocytes and platelets by directly stimulating the bone marrow . this bone marrow response elicited by pm exposure is thought to play a critically important role in the downstream adverse systemic health effects associated with exposure to air pollution , particularly the adverse effects on the heart and blood vessels . numerous studies have shown an association between air pollution and increased cardiovascular morbidity and mortality [ 24 , 8891 ] . the adverse cardiovascular health effects include hospital admissions and death from conditions such as acute myocardial infarction ( acute coronary syndrome ) , arrhythmias , and congestive heart failure . these hospital admissions were shown to occur within hours of a spike in air pollution exposure . elevated concentration of pm2.5 increased acute myocardial infarction within a few hours , which was confirmed by subsequent studies . proposed that an increase in blood coagulability induced by deposition of particles in the lung is associated with an increase in cardiovascular deaths in susceptible individuals . the systemic response induced by pm10 is characterized by activation of the acute - phase response , an increase in coagulation , the release of inflammatory mediators into the circulation leading to activation of the endothelium , and stimulation of the bone marrow causing the release of leukocytes and platelets . these events may contribute to destabilization of atherosclerotic plaques , making them vulnerable for rupture and thrombosis and accounting for the increase in cardiovascular events associated with episodes of air pollution . mediators such as gm - csf , il-1 , and il-6 produced by lung macrophages when exposed to ambient pm have the ability to elicit a systemic inflammatory response characterized by an increase in circulating leukocytes , platelets , and proinflammatory and prothrombotic proteins . these mediators also have the ability to activate circulating leukocytes and the endothelium of the vascular bed to promote leukocyte - endothelial adhesion and migration , contributing to atherosclerotic plaque activation and instability . our group showed that 4 weeks of exposure to ambient particles in watanabe hereditarily hyperlipidemic rabbits induced a systemic inflammatory response that included stimulation of the marrow and caused progression of atherosclerosis in both the aorta and coronary arteries , with phenotypic changes in atherosclerotic plaques characteristic of plaque vulnerability . these observations have recently been confirmed by others using ambient pm2.5 and by our group , using deps in a mouse model . to determine the role of ams in these effects , studies by our group examined the relationship between the fraction of macrophages in the lung that had phagocytosed particles and circulating mediators strongly associated with cardiovascular disease in humans such as il-6 and found a positive association [ 84 , 98 ] . in addition , our group showed an association between the extent of progression of atherosclerosis and features of plaque vulnerability and the fraction of ams that had phagocytosed pm ( figure 5 ) . collectively , these studies strongly implicate the am in eliciting the systemic inflammatory responses induced by exposure to pm as well as the downstream adverse vascular effects of air pollution . the studies mentioned earlier describe the effect of pm on ams ; however , it is understandable that there is marked interindividual variability in the response to pm . exploration of genetic predispositions and epigenetic changes associated with ambient air pollution shows promising results . polymorphisms in genes coding for glutathione - s - transferases ( gsts ) , which are enzymes responsible for the metabolism of reactive oxygen species , are correlated with the risk of lung diseases such as asthma when individuals are exposed to ambient air pollution [ 100 , 101 ] . showed that single - nucleotide polymorphisms in tlr2 and tlr4 genes significantly modified the effect of pm2.5 on the incidence of asthma . ambient air pollution can induce epigenetic changes such as dna methylation [ 103107 ] and there is further promise for genome - wide association studies ( gwas ) . little is known about the effect of gene - environment interactions on am and more research in this field is required . increasing evidence over the past 10 years has demonstrated that am plays a key role in local lung and systemic inflammatory responses induced by exposure to ambient pm . in the lung , am contributes to the magnitude and the nature of the inflammatory response by interacting with other lung cells such as bronchial epithelial cells and dendritic cells in an effort to process and clear the pm from the lung . these macrophages also produce the mediators that are associated with the systemic inflammatory response induced by pm exposure , and recent studies support the concept that these systemic mediators translocate from the lung tissues into the circulation . the adverse systemic health effects of exposure to pm , particularly the adverse cardiovascular effects , are strongly associated with the amount of pm phagocytosed by ams , underscoring the crucial role that the ams play in these adverse systemic responses of pm exposure . therefore , it is reasonable to suppose that attenuating the local and systemic inflammatory responses of ams , induced by pm exposure , would be of benefit . recent studies by our group have shown that statins reduce both local lung and systemic inflammatory responses induced by exposure to ambient pm in a rabbit model . figure 6 shows a potential mechanism by which statins could alter key pathways to achieve these local and systemic anti - inflammatory effects . downregulating the inflammatory responses further research surrounding gene - environment interactions in ams will contribute to understanding of interindividual variability and may assist in designing tailor - made therapies for air - pollution - related lung and heart diseases .

large population cohort studies have indicated an association between exposure to particulate matter and cardiopulmonary morbidity and mortality . the inhalation of toxic environmental particles and gases impacts the innate and adaptive defense systems of the lung . lung macrophages play a critically important role in the recognition and processing of any inhaled foreign material such as pathogens or particulate matter . alveolar macrophages and lung epithelial cells are the predominant cells that process and remove inhaled particulate matter from the lung . cooperatively , they produce proinflammatory mediators when exposed to atmospheric particles . these mediators produce integrated local ( lung , controlled predominantly by epithelial cells ) and systemic ( bone marrow and vascular system , controlled predominantly by macrophages ) inflammatory responses . the systemic response results in an increase in the release of leukocytes from the bone marrow and an increased production of acute phase proteins from the liver , with both factors impacting blood vessels and leading to destabilization of existing atherosclerotic plaques . this review focuses on lung macrophages and their role in orchestrating the inflammatory responses induced by exposure to air pollutants .

1. Introduction 2. Response of Lung Macrophages to Inhaled Air Pollutants 3. Macrophages and the Systemic Response Induced by PM 4. Conclusions

there are numerous sources of suspended particulate matter ( pm ) including industrial sources , automobile traffic , natural disasters , such as forest fires and volcanic eruptions , and local sources generated either in the home or workplace [ 13 ] . air pollutants can be classified by their source , chemical composition , size , mode of release ( gaseous or particulate ) , and space ( indoor or outdoor ) . in addition , data from large population cohorts have indicated an association between exposure to pm and cardiovascular morbidity and mortality [ 710 ] . mechanistically , this is thought to be due to the systemic inflammatory response induced by exposure to pm air pollution . this concept is supported by studies showing a positive association between long - term pm exposure and hematological markers of inflammation and diseases such as diabetes mellitus . inhalation of air pollution particles induces a local response in the lung that is initiated by alveolar macrophages ( ams ) and airway epithelial cells . the macrophages are several times more potent in producing proinflammatory mediators that contribute to the local inflammatory response in the lung but also contribute to the subsequent systemic inflammatory response . the systemic inflammatory response is characterized by mobilization of inflammatory cells from the bone marrow into the circulatory system , followed by their activation , as well as the production of acute phase proteins by the liver , and an increase in circulating inflammatory mediators . in this review , the primary role of am is to keep the air spaces clear by removal of all foreign materials via phagocytosis . experiments from our laboratory have shown that ams exposed to atmospheric particulate matter are able to phagocytose these particles in vivo and in vitro [ 13 , 14 ] . despite the absence of specific opsonins , am can phagocytose unopsonized environmental particles . the class a scavenger receptor ( sr - a ) and the macrophage receptor with collagenous structure ( marco ) are considered to be the two major receptors for unopsonized particle phagocytosis by am [ 17 , 18 ] , and a deficiency in scavenger receptor function results in reduced uptake of environmental particles by am . they are essential for initiation of the innate immune response , when interacting with pm , and also play a role in sustaining and regulating the adaptive immune response to pm . among the tlrs identified in humans , tlr4 and tlr2 toll - like receptor 4 initiates a signaling cascade in response to lps present in the outer membrane of gram - negative bacteria , while tlr2 initiates signals in response to zymosan ( beta - glucans ) and peptidoglycan of gram - positive bacteria . furthermore , microorganisms attached to pm can be opsonized by specific opsonins ( such as immunoglobulin fc receptors and complement receptor 3 ) that allow am to phagocytose the particles via an opsonin - dependent pathway . pm10 particles are derived predominantly from abraded soil , road dust , construction debris , and oil combustion products with bioaerosols such as fungi , bacteria , endotoxins , and pollen , while pm2.5 and ufp are primarily derived from direct emissions from combustion processes such as vehicle use of fossil fuel products , wood burning , and coal burning . in addition , several studies have shown that pm2.5 and ufp have the strongest association with adverse cardiovascular adverse effects [ 27 , 28 ] , which is a direct consequence of the systemic response induced by these particles [ 29 , 30 ] . alveolar macrophages exposed to smaller pm that have the ability to penetrate deep into the lungs significantly contribute to the systemic inflammatory response . in vitro studies have shown that macrophages do recognize the size and shape of their target pathogens ; therefore , their response against various particle sizes may be different . on the contrary , in vitro studies comparing the effects of the coarse and fine fraction of pm10 showed stronger proinflammatory effects for the coarse particles [ 33 , 34 ] . the toxicity of pm may stem from their metal content , adhered organic compounds , or other biological components such as lps . showed that coarse pm induced a greater inflammatory response than fine pm in rats and suggested that , in these larger particles , toxicity is due more to their biological components , such as endotoxin , than their metal content . in other studies , no apparent relationship could be established between pulmonary injury and the concentration of ambient particles or their elemental components such as sulfate ( s ) , zinc ( zn ) , manganese ( mn ) , iron ( fe ) , and copper ( cu ) . the water - soluble fraction of pollutant particles and individual soluble metals such as vanadium ( v ) , nickel ( ni ) , and fe did not induce apoptosis in human ams . in addition to these reports that emphasize the toxicity of organic components in pm , nonorganic components such as metals have also been implicated in the pathogenesis of particulate - induced pulmonary inflammation . ozone exposure induces the release of cytokines and fibronectin by am , increases am recruitment into asthmatic airways , and increases the eosinophilic airway response . recent epidemiologic studies conducted worldwide have provided valuable insight into the associations between sulfur dioxide ( so2 ) , no2 , and carbon monoxide ( co ) exposure and increases in cardiopulmonary mortality , such as respiratory and cardiovascular hospital admissions , emergency admissions caused by stroke ( no2 ) , and myocardial infarction ( no2 and co ) [ 4951 ] . alveolar macrophage phagocytosis was significantly suppressed following coexposure of fine carbon particles and so2 . because air pollution contains both , the contribution of these gaseous pollutants in modulating the response of the am to pm is complex , poorly understood , and still an area of active investigation . alveolar macrophages are one of the most potent producers of proinflammatory mediators in the airways and lung . studies from our laboratory have shown that human ams exposed to urban pm ( ehc-93 ) phagocytosed these particles in vitro ( figure 1 ) [ 13 , 54 ] , and , ex vivo , they produced tumor necrosis factor alpha ( tnf- ) in a dose - dependent fashion following pm exposure . in addition , these ams produce an array of proinflammatory mediators including acute response mediators such as interleukin ( il)-1 and il-6 as well as secondary mediators , such as il-8 , and granulocyte macrophage colony - stimulating factor ( gm - csf ) [ 11 , 13 ] . interestingly , production of anti - inflammatory mediators such as il-10 was suppressed , suggesting that the am inflammatory response induced by pm is tipped toward a proinflammatory profile . the inflammatory profile of mediators produced by bronchial epithelial cells exposed to pm is distinct from those of am . alveolar macrophages are also more potent producers of the acute response mediators such as il-1 , il-6 , and tnf- than bronchial epithelial cells when exposed to the same dose of pm , suggesting that ams are the drivers of the proinflammatory response in the lung following inhalation of pm ( figure 2 ) . furthermore , instillation of supernatants from human ams incubated ex vivo with urban pm into rabbit lungs produced a systemic response similar to that produced by direct deposition of the same amount of pm directly into the lungs [ 57 , 58 ] , which implies that ams significantly contribute to the systemic inflammatory response generated following exposure to pm . depending on the microenvironment , they undergo distinct activation programs , acquiring polarized phenotypes and different functional capacities that together provide an armamentarium that protects , repairs , and sometimes damages tissues [ 60 , 61 ] . program , is induced by signals generated during th1-mediated immune responses such as interferon ( ifn)- and by exposure to components of pathogens such as bacteria [ 60 , 61 ] . m2 polarization induced upon exposure to the th2 cytokines il-4 and il-13 ( referred to as alternative activation ) or immunoregulatory signals such as il-10 ( also called deactivation ) is highlighted by induced expression of receptors with scavenger functions , anti - inflammatory cytokines , and molecules implicated in tissue remodeling [ 60 , 61 ] . although pm shares many of the same ingredients and characteristics as cigarette smoke , the m1 cytokines ( il-12 and ifn- ) are increased consistently in bronchoalveolar lavage fluid ( balf ) from pm - exposed animals [ 63 , 64 ] while the m2 cytokines ( il-4 , il-10 , and il-13 ) remain at lower levels . data from an influenza virus pneumonia model suggest that macrophage pro- and anti - inflammatory phenotypes are under tight control of nearby airway epithelial cells . these responses ( m1/m2 switching ) of macrophages following pm exposure still need further investigation to assess what factors ( such as size or composition ) determine switching . in addition to being key players in the innate immune response against microorganisms and in the initiation of adaptive immune responses , they are crucial for the clearance and processing of microorganisms , dead cells , and environmental debris in the lung tissue via phagocytosis . in contrast to cells such as neutrophils , ams are long - lived and in general are resistant to apoptotic stimuli . following activation of am , by exposure to pm , for example , they either remain in the lung airways or tissues or are removed via the lymphatic system to regional lymph nodes . several studies have demonstrated that exposure to ambient pm and diesel exhaust particles induced apoptosis in macrophages [ 37 , 71 ] . phagocytosis of apoptotic cells ( efferocytosis ) by ams is involved in the regulation of the inflammatory response and maintenance of lung homeostasis by removing dead cells before the onset of necrosis . alveolar macrophages are primarily responsible for removing and processing dead cells and debris in the airways , thereby reducing their inflammatory potential . autophagy consists of the fusion of autophagosomes with lysosomes , forming autolysosomes and resulting in the breakdown of encapsulated materials to components that are then available for homeostasis . intracellular nanoparticles may undergo autophagic sequestration , and autophagy dysfunction may play an important role in nanoparticle toxicity . identified an autophagic defect in the ams of smokers and concluded that the decrease in the process of autophagy leads to impaired protein aggregate clearance , dysfunctional mitochondria , and defective delivery of bacteria to lysosomes . they sense , scavenge , and phagocytose pm and in the process they produce and release early response cytokines . these cytokines stimulate neighboring airway and alveolar epithelial cells as well as tissue - resident macrophages in an auto- and paracrine manner to produce a variety of chemokines necessary to recruit other cells , such as polymorphonuclear leukocytes , to assist in processing and ultimately clearing foreign material . human airway and alveolar epithelial cells are also capable of pm endocytosis and in the process they produce mediators such as gm - csf , il-1 , il-8 , and leukemia inhibitory factor ( lif ) in a dose - dependent manner at both the mrna and protein level when exposed to ambient particles . coculture experiments of bronchial epithelial cells and am showed synergistic production of certain mediators such as il-1 , il-6 , and gm - csf . these studies demonstrate the importance of the interaction of ams with other lung cells in producing lung inflammation and possibly contributing to the systemic inflammatory response following pm inhalation . after phagocytosis and internalization of pm , the organic components are digested by the endosome into peptide fragments that combine with the mhc class ii complex for presentation to cd4 + t cells , which are pivotal steps in cell - mediated and adaptive immunity . however , the role of this adaptive response in the local lung and systemic inflammatory responses induced by pm exposure has not been well studied to date . earlier studies implicate ams as key effector cells responsible for generating the systemic inflammatory response associated with exposure to air pollution [ 13 , 41 , 56 , 81 , 82 ] . they produce a broad range of mediators , particularly il-6 , il-1 , mip-1 , and the hematopoietic growth factor gm - csf , when exposed to urban pm . the importance of the am producing the mediators that elicit the systemic response is supported by studies showing a correlation between the amount of particles phagocytosed by am in the lung and the magnitude of bone marrow stimulation following pm exposure ( figure 4 ) . humans exposed to an acute episode of air pollution , where the predominant pollutant was pm , showed increased levels of circulating cytokines such as il-1 and il-6 and signs of bone marrow stimulation reflected by an increase in circulating band cells counts . these cytokines are similar to those produced by am exposed to pm both ex vivo and in vivo [ 13 , 84 ] , suggesting that these mediators produced in the lung enter the circulation and contribute to the systemic response associated with exposure to pm . recent studies from our laboratory showed that mediators such as il-6 , produced in the lung from pm exposure directly , translocate into the circulation and , because the ams are the most prolific producer of these mediators following pm exposure , it is reasonable to postulate that ams are crucially important effector cells in generating the systemic inflammatory response induced by air pollution exposure . several studies from our laboratory have shown that exposure to air pollution stimulates the bone marrow in humans and in animal models [ 11 , 14 , 5658 , 85 ] and promotes the release of both polymorphonuclear leukocytes and monocytes from the marrow . the cytokine production by macrophages in the lung is of particular importance in inducing this systemic inflammatory response ; for example , gm - csf is a hematopoietic growth factor that stimulates granulocyte and monocyte differentiation and releases from the bone marrow , but it also activates circulating leukocytes and prolongs their survival in the circulation . furthermore , il-1 is one of the acute response cytokines that induces cytokine production by many cells , stimulates hematopoiesis , activates endothelial cells , is pyrogenic , and induces the acute - phase response . in addition , stimulation of liver hepatocytes by il-6 produces acute phase proteins including c - reactive protein ( crp ) , fibrinogen , and antiproteases . moreover , il-6 also stimulates hematopoiesis , specifically the production of platelets , and has a broad stimulating effect on b and t cells , as well as markedly accelerating the transit time of granulocytes through the bone marrow , releasing them into the circulation , and promoting their sequestration in microvascular beds . collectively , gm - csf , il-1 , and il-6 have the ability to elicit a systemic inflammatory response characterized by an increase in circulating leukocytes and platelets by directly stimulating the bone marrow . this bone marrow response elicited by pm exposure is thought to play a critically important role in the downstream adverse systemic health effects associated with exposure to air pollution , particularly the adverse effects on the heart and blood vessels . numerous studies have shown an association between air pollution and increased cardiovascular morbidity and mortality [ 24 , 8891 ] . the adverse cardiovascular health effects include hospital admissions and death from conditions such as acute myocardial infarction ( acute coronary syndrome ) , arrhythmias , and congestive heart failure . proposed that an increase in blood coagulability induced by deposition of particles in the lung is associated with an increase in cardiovascular deaths in susceptible individuals . the systemic response induced by pm10 is characterized by activation of the acute - phase response , an increase in coagulation , the release of inflammatory mediators into the circulation leading to activation of the endothelium , and stimulation of the bone marrow causing the release of leukocytes and platelets . these events may contribute to destabilization of atherosclerotic plaques , making them vulnerable for rupture and thrombosis and accounting for the increase in cardiovascular events associated with episodes of air pollution . mediators such as gm - csf , il-1 , and il-6 produced by lung macrophages when exposed to ambient pm have the ability to elicit a systemic inflammatory response characterized by an increase in circulating leukocytes , platelets , and proinflammatory and prothrombotic proteins . these mediators also have the ability to activate circulating leukocytes and the endothelium of the vascular bed to promote leukocyte - endothelial adhesion and migration , contributing to atherosclerotic plaque activation and instability . our group showed that 4 weeks of exposure to ambient particles in watanabe hereditarily hyperlipidemic rabbits induced a systemic inflammatory response that included stimulation of the marrow and caused progression of atherosclerosis in both the aorta and coronary arteries , with phenotypic changes in atherosclerotic plaques characteristic of plaque vulnerability . to determine the role of ams in these effects , studies by our group examined the relationship between the fraction of macrophages in the lung that had phagocytosed particles and circulating mediators strongly associated with cardiovascular disease in humans such as il-6 and found a positive association [ 84 , 98 ] . in addition , our group showed an association between the extent of progression of atherosclerosis and features of plaque vulnerability and the fraction of ams that had phagocytosed pm ( figure 5 ) . collectively , these studies strongly implicate the am in eliciting the systemic inflammatory responses induced by exposure to pm as well as the downstream adverse vascular effects of air pollution . polymorphisms in genes coding for glutathione - s - transferases ( gsts ) , which are enzymes responsible for the metabolism of reactive oxygen species , are correlated with the risk of lung diseases such as asthma when individuals are exposed to ambient air pollution [ 100 , 101 ] . ambient air pollution can induce epigenetic changes such as dna methylation [ 103107 ] and there is further promise for genome - wide association studies ( gwas ) . increasing evidence over the past 10 years has demonstrated that am plays a key role in local lung and systemic inflammatory responses induced by exposure to ambient pm . in the lung , am contributes to the magnitude and the nature of the inflammatory response by interacting with other lung cells such as bronchial epithelial cells and dendritic cells in an effort to process and clear the pm from the lung . these macrophages also produce the mediators that are associated with the systemic inflammatory response induced by pm exposure , and recent studies support the concept that these systemic mediators translocate from the lung tissues into the circulation . the adverse systemic health effects of exposure to pm , particularly the adverse cardiovascular effects , are strongly associated with the amount of pm phagocytosed by ams , underscoring the crucial role that the ams play in these adverse systemic responses of pm exposure . therefore , it is reasonable to suppose that attenuating the local and systemic inflammatory responses of ams , induced by pm exposure , would be of benefit . recent studies by our group have shown that statins reduce both local lung and systemic inflammatory responses induced by exposure to ambient pm in a rabbit model . figure 6 shows a potential mechanism by which statins could alter key pathways to achieve these local and systemic anti - inflammatory effects . downregulating the inflammatory responses further research surrounding gene - environment interactions in ams will contribute to understanding of interindividual variability and may assist in designing tailor - made therapies for air - pollution - related lung and heart diseases .

in current stem cell biology and regenerative medicine , two of the greatest challenges [ 1 , 2 ] are to control the differentiation of stem cells and to ensure the purity of isolated cells . the process of stem cell differentiation is at present monitored by biological assays , namely , immunocytochemistry [ 3 , 4 ] . there is a clear need for a truly noninvasive technique which can monitor the degree of differentiation rapidly . such a technique will most likely involve a form of optical imaging or spectroscopy but must not involve the addition of any kind of biomarker . biomarkers are used to sort embryonic stem cells , in conjunction with fluorescent [ 5 , 6 ] or magnetic labels . these techniques are lengthy and time - consuming , but careful monitoring of stem cell differentiation is essential : in clinical applications , a population of fully differentiated cells is often implanted , but teratomas can result if any stem cells remain undifferentiated . there are a number of issue with the use of biomarkers for the characterization and sorting of stem cells and their derivatives . firstly , only a limited number of biomarkers exists each one being cell - specific . many cell types lack biomarkers , for example , cardiomyocytes , gastrointestinal stem cells , and corneal stem cells . secondly , the use of biomarkers raises issues with both biological researchers and clinicians , who would strongly prefer a label - free technique . finally , these biomarkers can not easily be translated ; for example , embryonic stem cell biomarkers are not always applicable to adult stem cells . fluorescent biomarkers [ 5 , 6 ] have been employed in cell sorting and characterization , but fluorescent techniques have a number of drawbacks . firstly , photobleaching means that signal levels drop over time ; so long - term studies of differentiation are prohibited . secondly , this process of photobleaching produces free radical singlet oxygen species which will damage live cells . finally , the use of biomarkers causes modification to cells ' surface chemistry , and stem cells are highly sensitive to small changes in their surface chemistry . magnetic beads can not easily be visualised in microscopy ; they must all be removed from the cells ; a large mass could cause large mechanical stresses to the cells , which can affect the cells ' behaviour . there is thus a requirement from the stem cell community for a rapid , easy , sensitive , nondestructive , noninvasive , label - free technique which can be applied both on the single cell level and to monitoring or sorting large populations of cells . this review will concentrate on label - free optical spectroscopy techniques , which are noninvasive and have sufficiently high resolution to be applied at the single cell level . white light imaging either phase contrast or differential interference contrast ( dic)can reveal the approximate level of differentiation in situ , to those who are expert in stem cell culture . however , it is only really suitable for monolayers of cells . as white light imaging is usually only qualitative , it would benefit by being replaced by a more advanced optical technique capable of a quantitative measurement on individual cells . such a technique should therefore be capable of high speed characterization , to enable large numbers of cells to be studied in monolayer cultures , embryos , and scaffolds . infrared light is absorbed by the wide variety of chemical bonds within molecules , which all have well - defined vibrational frequencies . hence , an absorption spectrum of a cell should give a characteristic snapshot of the chemistry , and an undifferentiated cell 's spectrum could differ enough from that of a differentiated cell enough to characterize them . simple infrared spectrometers use a broadband light source containing a wide range of wavelengths , which is typically passed through a cuvette of solution , through a dispersing spectrometer onto a single detector . this technique is slow , as the spectrum is built up from around 1000 sequential data points . in order to collect a full spectrum without losing the vast majority of the signal , fourier transform infrared ( ftir ) spectroscopy uses both interferometry and a fourier transform of the signal : from the time to frequency domain . a typical ftir setup is illustrated in figure 1(a ) , which requires a mirror to scan one half of the interferometer arm over a distance of a few millimetres . synchrotron sources have promised vastly improved spectral acquisition times up to 1000 times faster than benchtop ftir but it is not clear whether this is applicable to live cells , as heating by absorption may prevent any increase in speed . although an n.a . of 1.4 is achievable with objective lenses using visible light , infrared light has very low transmission through standard glass objectives ; so a parabolic mirror ( known as a cassegrain objective ) is normally used to focus light . the bonds in molecules are typically excited with infrared light of wavelengths between 2.8 and 16 m , which corresponds to a lateral resolution of 4.2 to 24 m , which is small enough to be applied to individual isolated cells or to average over groups of cells , but will not usually be cell specific when applied to an embryo or group of cells tightly bound together . ftir microscopy has been achieved on fixed adherent mesenchymal stem cells ( mscs ) with a diameter of around 50 m , but only for high - frequency ( short wavelength ) vibrations . these vibrational frequencies are described by spectroscopists as inverse wavelengths in units of cm [ wavenumbers ] . the lowest frequency vibrations occur in cells around 600 cm ( = 16.7 m ) and the highest frequencies relate to the ch stretch ( 28003000 cm , = 3.33.6 m ) and oh stretch ( ~3500 cm , = 2.8 m ) . one of the major issues with infrared radiation is its extremely low penetration depth in water , which limits the depth which can be probed in solution to the range 10100 m . to combat absorption of infrared light through a thick cuvette , attenuated total reflection- ( atr- ) ftir probes only the first micrometre above the substrate . an array of spectra can be acquired rapidly enough to perform imaging within a few minutes on live cells . the second optical technique suitable for stem cell characterization is raman spectroscopy . in the most widespread form stokes scattering visible or near - infrared light loses energy ( frequency ) by exciting molecules into their excited state , as depicted in figure 2 . this means that some of the laser light is red - shifted after interacting with the sample . a typical setup is illustrated in figure 1(b ) : after filtering out the laser , the remaining red - shifted light is passed through a spectrometer onto a cooled ccd camera . a full raman spectrum is normally acquired in 1 to 10 seconds , so is typically slower than ftir . as silicon ccds have a response which dies away rapidly at a wavelength of around 1000 nm , the longest laser wavelength which can be used is 785 nm ( algaas diode ) which is the preferred wavelength for raman spectroscopy in biology . the other popular laser wavelength for biological samples is 633 nm ( hene laser ) which has lower power than 785 nm lasers , and heating in cells and tissue is the lowest using near - ir illumination ( 7001100 nm ) . heating has been measured directly in cells using 100 mw of 1064 nm radiation in an optical trap , a temperature rise of < 1c was observed . prolonged exposure to 300 mw illumination at constant power ( continuous wave , cw ) caused photodamage a light - induced reduction in cell viability attributed to two - photon absorption . visible light ( < 700 nm ) is believed to cause photodamage at far lower thresholds than this , due to increased absorption by proteins , but shorter wavelengths produce more raman signal . the reduction in wavelengths for raman spectroscopy , compared to ftir , equates to a greatly improved lateral resolution of 350 nm and axial ( depth ) resolution of 1150 nm . this is sufficient to allow spectroscopy on individual cells , and even at the subcellular level . furthermore , raman microspectroscopy otherwise known as raman mapping or raman microscopy can be performed . maps can be produced containing the total signal under a given peak , or more subtle differences between spectra can be exploited with principal component analysis ( pca ) or cluster analysis . this reduces the large number of peaks in the raman spectrum to a smaller number of independent variables , and cluster analysis produces colour - coded maps with regions of similar chemistry deduced from the set of spectra . due to the long acquisition time required , only raman microscopy of fixed cells had been performed until recently , as live cells move far more quickly than the set of spectra could be recorded . however , recently raman microscopy has been performed on live cells with 100 mw power at 647 nm and acquisition time of 0.5 second , albeit with only 32 32 pixels . an alternative approach is to illuminate a line rather than a spot then a series of spectra can be recorded across a single ccd , each relating to a pixel along the line . this technique has been applied to live cells with 5 seconds per line ( 3 minutes per image ) with 532 nm at 3.5 mw/m intensity , again with a small number of imaging pixels . it remains to be seen whether photodamage was occurring in both of these live cell imaging publications . pca can be used to extract the most significant variations between groups of spectra acquired on large numbers of cells . thus , determining an unknown cell type from two possibilities notably , stem cell and differentiated cell can be accomplished using large numbers of spectra from known cell types . the most important differences are highlighted , rather than any uncorrelated and unimportant variations , to improve the sensitivity of the technique . two improved variants of pca are discussed in relation to ftir , and both show marked improvements in their ability to distinguish cell types . before analysis can be performed , spectra require processing to remove unwanted autofluorescence from tissue normally by baseline subtraction , as well as the removal of cosmic rays , and substrate and media contribution to the spectrum . all this processing and analysis of spectra requires significant computing power , when applied to large datasets . raman spectra consist of a large number of peaks at well - defined frequencies , as demonstrated in figures 3 and 4 . the relative intensities of the peaks change between cells , but the frequencies themselves do not shift . peaks are usually around 510 cm in width , except for the single peak at 1002 cm . this frequency relates to the in - plane vibration of the aromatic ring of the phenylalanine molecule which is highly symmetric and is narrower than the other peaks because of the lack of vibration out of the plane and lack of variation in other atoms attached to the benzene ring . frequencies in ftir can be slightly shifted by a few cm and tend to be broader . both techniques excite these vibrations to different extents ; so the relative peak intensities will be different in raman and ftir spectra . raman scattering is a weak process typically only 1 in ~10 incident photons gives rise to a raman - shifted photon . this is largely due to the excitation of the bond vibration far above its resonant frequency . in order to increase this efficiency , in coherent anti - stokes raman scattering ( cars ) [ 2528 ] the vibration is excited with two laser frequencies the difference ( or beat ) frequency between them is matched to the vibrational frequency of interest . this means that cars images are acquired in seconds , whereas a similar quality raman map would require days to complete . the process is best excited with pulsed lasers with durations of around 6 ps , which should mean that no photodamage occurs with extensive use of powers of at least 12 mw . a cars microscope , designed for biological imaging , is described in detail elsewehere , which has a lateral resolution of 350 nm and an axial resolution of 1100 nm . live cell imaging is slightly slower than that of fixed cells , but high - quality images are acquired within 1 minute . picosecond cars excitation pulses have an estimated width of around 3 cm , so are ideal for biological molecules , but this means that only one vibration ( spectral peak ) may be excited during an image . images can be acquired sequentially at different wavenumbers , by retuning one laser , but this is not ideal given the motion occurring during live cell imaging . multiplex cars [ 3034 ] uses normal ( narrowband ) pulses for one laser , p , and broadband pulses for s . the broadband supercontinuum excitation pulse is several hundred nanometres wide and is excited in a photonic crystal fibre by a femtosecond laser . a full spectrum is acquired in around 100 milliseconds on live yeast , but an estimate of photodamage suggests that 1 second per pixel would be more appropriate for eukaryotic cells . further improvements to excitation sources could see this fall to the millisecond range enabling high - quality , noninvasive full spectral mapping of live cells in minutes . a different approach to increase the speed of raman microscopy , termed stimulated raman scattering , has recently been published . in a similar way to how stimulated emission depopulates the excited state in lasers , the excited state in a simple raman excitation ( pumped with p ) can be rapidly depopulated by a second laser ( at s ) modulated in the mhz range . in this way , the signal at s is increased slightly ( stimulated raman gain ) and the pump power at p is decreased slightly ( stimulated raman loss , srl ) . monitoring either signal , filtered by a lock - in amplifier , produces images which are background - free and directly proportional to the concentration . the standard cars signal has a quadratic dependence on concentration and can have a large unwanted background . heterodyne cars is another technique which is able to circumvent both of these problems encountered in standard cars imaging . srl is a linear optical technique and is suitable for extension into optical coherence tomography deep into tissue , using low numerical aperture lenses . ftir spectroscopy was used to study murine embryonic stem cells by ami et al . . after 47 days of differentiation , changes to the absorption spectrum of fixed cells were noticed : features in the amide i band ( 16001700 cm ) were enhanced , and those in the nucleic acid region ( 8501050 cm ) diminish . this means that the overall levels of dna and rna decrease , and the alpha helix content of proteins increases over time . furthermore , new dna / rna hybrid bands at 899 cm and 954 cm start to occur around day 47 , suggesting that mrna translation is occurring at this time . german et al . employed high - intensity synchrotron radiation to probe 10 m thick cryosections of bovine cornea . they used pca to clearly distinguish the three cells types of interest : stem cells , transit - amplifying cells , and terminal differentiated cells . no biomarkers of corneal stem cells exist ; so spectroscopic techniques offer the only viable method of cell characterization here . from the same group , walsh et al . again used synchrotron ftir , this time on paraffin - embedded human intestinal crypts , which were dewaxed . the position of cells along the crypt denotes the change from stem cell location to transit - amplifying region to differentiated location . pca was used to compare spectral features and was able to separate cell types from three positions along the crypt , which is shown in figure 3 . this method of characterization was compared with tissue stained with two different immunophenotypical markers : rabbit polyclonal anti - cd133 and -catenin antibodies . the authors state that the dominant ftir absorption peak at 1080 cm , relating to the symmetric ( po2 ) stretch , is a more robust marker than the two biomarkers . as gastrointestinal stem cells lack specific biomarkers , they went on to compare ftir data against a number of chemical differences which are discussed at length in a further publication . salasznyk et al . used ftir to study osteoblasts derived from human mesenchymal stem cells after 28 days of cell culture . the spectrally derived mineral - to - matrix ratio was calculated as the ratio of the integrated areas of the phosphate absorbance ( 9001200 cm ) and protein amide i band ( 15851720 cm ) . they observed a significant decrease in the mineral - to - matrix ratio in the extracellular matrix produced by focal adhesion kinase- ( fak- ) knockdown cells when compared to untreated ( control ) cells . krafft et al . also used ftir to study human mesenchymal stem cells differentiating into osteoblasts . their samples were fixed in methanol then dried , and they were able to distinguish cells stimulated in osteogenic medium for 7 days , from nonstimulated cells . ftir microscopy on isolated adherent cells ( of size ~50 m ) showed that some of the nonstimulated cells had high levels of glycogen accumulation , and some stimulated cells had a high expression of calcium phosphate . stimulated cells had reduced levels of amide i ( at 1631 cm ) , meaning that lower concentrations of beta - sheet proteins were present . this compares well with ami et al . , who measured a higher alpha helix proportion during differentiation . bentley et al . measured ftir spectra of human corneal stem cells and their derivatives : transit amplifying cells . they made 10 m thick cryosections of cornea , which were left to dry and observed differences in synchrotron ftir spectra which were attributed to nucleic acids . they were able to distinguish between both cell types with spectra acquired from two different regions in the tissue , using pca , albeit with an overlap of 16% between the two populations . in summary , ftir is able to distinguish between stem cells and their derivatives . using synchrotron sources , the speed of data acquisition and the quality of comparison have improved greatly . even microscopy has been performed , requiring a spectral acquisition at each imaging pixel . to be able to distinguish between individual cells , rather than populations , requires subconfluent adherent cells due to the spatial resolution ( governed by the wavelength of infrared radiation ) being of the order of the cell size . the technique has until now been limited to dried samples due to the high absorption coefficient of water ; so the technique has not been used to noninvasively monitor stem cell differentiation in vitro as the required drying is clearly destructive . however , atr - ftir can be used on live cell cultures ; hence it could potentially be employed as a real - time noninvasive technique for monitoring stem cell differentiation on adherent cells ( though no results have yet been published ) . such a technique would be highly preferable to the use of biomarkers in clinical as well as research applications . one issue with atr - ftir is that it only probes the first 1 - 2 m above a substrate ; so the nucleus would only give a small contribution to the overall signal . given that nucleic acids play a large part in the reported changes in spectral signatures during differentiation , and the nucleus may remain above this penetration depth , it remains to be seen whether atr - ftir can indeed be used as a noninvasive biomarker - free analytical technique for live cell studies . notingher et al . used raman spectroscopy to investigate live murine embryonic stem cells . 100 mw of 785 nm laser light was focussed onto a spot of size 10 m 5 m 25 m . the group grew cells on a gelatine - coated quartz substrate , as plastic contains a number of vibrational bonds which are also present in cells . glass gives a strong fluorescence background ; so quartz or magnesium fluoride is preferred as a substrate . however , stem cells do not adhere well to glass and crystals ; so a thin coating to the substrate is required , such as the gelatine used in this study . great care must also be taken to ensure that the stem cells do not differentiate spontaneously on a given substrate or coating . over 16 days of differentiation , they observed a decrease in the rna peak ( at 813 cm , opo stretch ) by 75% and a drop of 50% in the dna peak ( at 788 cm , cytosine ring vibration ) . they also extracted the first principal component spectrum , reproduced in figure 4 , which reveals the spectrum responsible for most of the differences between spectra of stem cells and differentiated cells . note the high coincidence of many peaks of this principal component spectrum , with the spectrum of rna . this confirms that a reduction in rna levels dominates the chemical changes during differentiation . chan et al . acquired raman spectra on live human embryonic stem cells ( hescs ) as they differentiated into cardiomyocytes and were able to distinguish between hescs and hesc - derived cardiomyocytes with an accuracy of 66% . they found that the rna peak ( 811 cm ) and dna peaks ( e.g. , 785 cm , 1090 cm ) were all reduced in intensity during differentiation . raman spectra of fixed mesenchymal stem cells grown on gelatine - coated quartz , and used a diffraction - limited laser spot , instead of averaging over a large area . this meant that spectra could be acquired separately from within the nucleus and within the cytoplasm the spectrum from the substrate has been subtracted , but no baseline subtraction was performed to highlight the requirement for automated subtraction in quantitative spectroscopic analysis . small variations in laser power and focus position are thought to be responsible for the offset spectra at low wavenumbers . the spectra in figure 5 as expected clearly demonstrate that the nucleus contains far more dna and rna than does the cytoplasm ; and that the cytoplasm contains far more proteins and lipids than the nucleus . these cells were fixed rather than live , but a study of fixation methods on raman spectra indicates that the effect of aldehyde cross - linking fixation on spectra is minimal . [ 40 , 43 ] , implies that it is the nucleus size ( or nucleic acid density therein ) which shrinks during differentiation . hence , monitoring the nucleus size in a quantitative manner using imaging processing techniques would seem to be a good potential approach to monitoring the state of differentiation . mineralization was monitored at two frequencies : 960 cm ( p o stretch ) and 1070 cm ( po4 ) . ca5(po4)3(oh)and was by far the strongest signal ; this peak height rose linearly from zero to the dominant peak in the spectrum over the 21 days of differentiation . used raman spectroscopy to compare tissue - engineered bone derived from mesenchymal stem cells , with femoral bone . they found a very good similarity in phosphate ( 960 cm ) and carbonate ( 595 cm ) levels , with only minor spectral differences such as a larger amount of protein . liu also observed a major peak at 960 cm due to hydroxyapatite , in mineral extracted from odontoblast nodules which were formed by the differentiation of dental pulp stem cells . azrad et al . used raman spectroscopy to characterize the production of mineral content from mesenchymal stem cells , under the influence of two osteogenic agents : quality elk velvet antler ( qeva ) extract , and dexamethasone . they measured no mineralization from the control group , some mineralization from the dexamethasone - fed cells , but most mineralization from cells supplemented with the elk velvet antler . peaks indicated phosphate derivatives , which for qeva were mostly related to hydroxyapatite ( at 960 cm ) and its precursors ( amorphous calcium phosphate , ca9(po4)6h2o , at 952 cm and octacalcium phosphate , ca8(po4)65h2o , at 957 cm ) . for dexamethasone - fed cells , the dominant peak was of octacalcium phosphate , and lower amounts of hydroxyapatite and amorphous calcium phosphate were measured . gentleman et al . compared mineralized nodules in vitro from 3 sources : embryonic stem cells , neonatal calvarial osteoblasts , and adult bone - marrow - derived mesenchymal stem cells . after 28 days in osteogenic medium , sets of raman spectra were acquired of the mineralized nodules , and pca was performed on the spectra to extract their chemical components . the osteoblasts and mesenchymal stem cells both produced nodules which were chemically similar to native bone : a combination in descending order of concentration , of ( a ) carbonate - substituted hydroxyapatite , ( b ) crystalline nonsubstituted hydroxyapatite , and ( c ) amorphous phosphate species . however , embryonic stem cells produce nodules which were dominated only by the first mineral component , which was similar to synthetic carbonated hydroxyapatite . nanoindentation tests showed that these nodules derived from embryonic stem cells were more than an order of magnitude less stiff than those derived from osteoblasts and mesenchymal stem cells . these raman studies all show a similar ability to distinguish stem cells from their derivatives , that is , a similar sensitivity , as the ftir technique . the major advantage of ftir over raman is its speed ; hence mapping signals ( derived from a full spectral acquisition at each imaging pixel ) into images is easier with ftir . the major advantage of raman over ftir is that has been used on live cells , so is truly noninvasive . cars microscopy has been performed on live murine embryonic stem cells by konorov et al . , but the laser setup only permitted pixel dwell times of 300 milliseconds compared to microseconds in standard cars microscopy [ 2528 ] . the groups were unable to distinguish any individual cells or features when imaging at the dna and rna frequencies . however , cars spectroscopy showed a large reduction in the rna peak intensity ( at 811 cm ) in differentiated cells . figure 6 shows our cars microscopy image , acquired on mcf-7 human breast cancer cells which have a similarly large nucleus to cytoplasm ratio as stem cells . two sequentially acquired images are overlaid : in green , dna / rna is mapped at the phosphate backbone opo stretch frequency ( 1095 cm ) , and in red , lipids and the cytoskeleton are mapped at the ch2 deformation frequency ( 1448 cm ) . the opo stretch frequency is also present in figure 4(a)the principal component spectrum which describes most of the changes during differentiation and in figure 4(b)the rna spectrum . this method can be easily extended to monitoring the nucleus size in live stem cells , with a reduced frame rate of at least 1 image per minute ( we find that cars imaging in live cells is slower than in dried , fixed cells ) . cars microscopy is limited to monitoring one vibrational mode at a time , rather than comparing the full spectral signature . however , the rna and dna peaks have been shown to drop considerably , and rna is the dominant change to spectra . so it is possible that cars imaging will be able to measure the stage of differentiation purely by monitoring the size of each nucleus . white light imaging is less exact at measuring the nucleus size than either fluorescence or cars microscopy and could not be extended from monolayers ; so cars is preferred as a noninvasive technique to monitor nuclear size . we expect that cars should also be able to map mineralization from mesenchymal stem cells , by mapping the peak at 960 cm . the clear advantage of cars is its high speed compared to the other spectroscopic techniques . one of the most promising techniques over the coming years should be multiplex cars , which acquires a full vibrational spectrum at higher speed than raman , and is also applicable to the single cell level . this is bound to be more sensitive than monitoring just one peak in standard cars . if the spectral acquisition speed is improved to the millisecond scale , the technology could be applied to both flow cytometry and microscopy . the major noninvasive optical spectroscopy techniques suitable for analysis of stem cell differentiation have been outlined : namely , ftir , raman , and cars . ftir spectroscopy has only been performed on fixed or dried stem cells , but atr - ftir can be used in order to investigate live cells in future . raman spectroscopy has demonstrated the ability to distinguish between live stem cells and differentiated cells : both murine and human embryonic stem cells display a large reduction in peak intensities of both rna and dna . when mesenchymal stem cells differentiate into osteoblasts , they display a clear peak ( or peaks ) relating solely to mineral composition . it is well known that the nucleus to cytoplasm ( volume ) ratio in embryonic stem cells is elevated [ 5154 ] . hence , the reduction in rna and dna levels during differentiation is not entirely surprising . the assumption is therefore that the nucleus volume shrinks by around 50% during differentiation rather than the concentration of nucleic acids reducing . cars microscopy is well suited to monitoring the nucleus size or mineralization content of each individual cell ( around 960 cm ) and can be applied to large numbers of cells in cultures and to engineered tissue scaffolds . monitoring the cars signal of the rna or dna peak could also be applied to high - speed cell flow cytometry . white light imaging ( normally dic or phase contrast ) could offer a low - cost solution for monitoring the nucleus / cytoplasm ratio of cell monolayers , in conjunction with automated image recognition cytometry . cars only monitors one ( or possibly two ) spectral peaks ; so it will be less sensitive than raman and ftir which both acquire a full vibrational spectrum . multiplex cars can acquire a full spectrum and promises to replace raman spectroscopy in time , due to its improved speed . each spectroscopic technique has its own benefits and drawbacks ; so it is more suited to characterization of stem cells in different ways and on different platforms . raman and ftir spectroscopy are both most suited to monitoring cultures averaged over a large number cells . ftir does not have the required resolution to address single cells in confluent monolayer cultures , or a sufficient penetration depth for flow cytometry . raman spectroscopy is too slow to characterize enough individual cells to be a worthwhile clinical technique but could be used extensively in biomedical research . both ftir and raman techniques are more sensitive than normal cars which relies on excitation of just one spectral peak . however , cars may be sufficiently sensitive to apply to characterization of differentiation in microscopy and flow cytometry . raman and cars have been integrated into one instrument , to combine the benefits of both techniques . in future , multiplex cars promises to be the technique of choice for all platforms , due to its combined attributes of speed , full spectral analysis , and applicability to individual live cells .

there is a requirement for a noninvasive technique to monitor stem cell differentiation . several candidates based on optical spectroscopy are discussed in this review : fourier transform infrared ( ftir ) spectroscopy , raman spectroscopy , and coherent anti - stokes raman scattering ( cars ) microscopy . these techniques are briefly described , and the ability of each to distinguish undifferentiated from differentiated cells is discussed . ftir spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives . raman spectroscopy shows a clear reduction in dna and rna concentrations during embryonic stem cell differentiation ( agreeing with the well - known reduction in the nucleus to cytoplasm ratio ) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells . cars microscopy can map these dna , rna , and mineral concentrations at high speed , and mutliplex cars spectroscopy / microscopy is highlighted as the technique with most promise for future applications .

1. Introduction 2. Results and Discussion 3. Conclusions

in current stem cell biology and regenerative medicine , two of the greatest challenges [ 1 , 2 ] are to control the differentiation of stem cells and to ensure the purity of isolated cells . the process of stem cell differentiation is at present monitored by biological assays , namely , immunocytochemistry [ 3 , 4 ] . there is a clear need for a truly noninvasive technique which can monitor the degree of differentiation rapidly . biomarkers are used to sort embryonic stem cells , in conjunction with fluorescent [ 5 , 6 ] or magnetic labels . these techniques are lengthy and time - consuming , but careful monitoring of stem cell differentiation is essential : in clinical applications , a population of fully differentiated cells is often implanted , but teratomas can result if any stem cells remain undifferentiated . there are a number of issue with the use of biomarkers for the characterization and sorting of stem cells and their derivatives . many cell types lack biomarkers , for example , cardiomyocytes , gastrointestinal stem cells , and corneal stem cells . finally , these biomarkers can not easily be translated ; for example , embryonic stem cell biomarkers are not always applicable to adult stem cells . finally , the use of biomarkers causes modification to cells ' surface chemistry , and stem cells are highly sensitive to small changes in their surface chemistry . there is thus a requirement from the stem cell community for a rapid , easy , sensitive , nondestructive , noninvasive , label - free technique which can be applied both on the single cell level and to monitoring or sorting large populations of cells . this review will concentrate on label - free optical spectroscopy techniques , which are noninvasive and have sufficiently high resolution to be applied at the single cell level . white light imaging either phase contrast or differential interference contrast ( dic)can reveal the approximate level of differentiation in situ , to those who are expert in stem cell culture . such a technique should therefore be capable of high speed characterization , to enable large numbers of cells to be studied in monolayer cultures , embryos , and scaffolds . infrared light is absorbed by the wide variety of chemical bonds within molecules , which all have well - defined vibrational frequencies . hence , an absorption spectrum of a cell should give a characteristic snapshot of the chemistry , and an undifferentiated cell 's spectrum could differ enough from that of a differentiated cell enough to characterize them . this technique is slow , as the spectrum is built up from around 1000 sequential data points . in order to collect a full spectrum without losing the vast majority of the signal , fourier transform infrared ( ftir ) spectroscopy uses both interferometry and a fourier transform of the signal : from the time to frequency domain . ftir microscopy has been achieved on fixed adherent mesenchymal stem cells ( mscs ) with a diameter of around 50 m , but only for high - frequency ( short wavelength ) vibrations . the lowest frequency vibrations occur in cells around 600 cm ( = 16.7 m ) and the highest frequencies relate to the ch stretch ( 28003000 cm , = 3.33.6 m ) and oh stretch ( ~3500 cm , = 2.8 m ) . the second optical technique suitable for stem cell characterization is raman spectroscopy . in the most widespread form stokes scattering visible or near - infrared light loses energy ( frequency ) by exciting molecules into their excited state , as depicted in figure 2 . as silicon ccds have a response which dies away rapidly at a wavelength of around 1000 nm , the longest laser wavelength which can be used is 785 nm ( algaas diode ) which is the preferred wavelength for raman spectroscopy in biology . the other popular laser wavelength for biological samples is 633 nm ( hene laser ) which has lower power than 785 nm lasers , and heating in cells and tissue is the lowest using near - ir illumination ( 7001100 nm ) . prolonged exposure to 300 mw illumination at constant power ( continuous wave , cw ) caused photodamage a light - induced reduction in cell viability attributed to two - photon absorption . the reduction in wavelengths for raman spectroscopy , compared to ftir , equates to a greatly improved lateral resolution of 350 nm and axial ( depth ) resolution of 1150 nm . this is sufficient to allow spectroscopy on individual cells , and even at the subcellular level . furthermore , raman microspectroscopy otherwise known as raman mapping or raman microscopy can be performed . this reduces the large number of peaks in the raman spectrum to a smaller number of independent variables , and cluster analysis produces colour - coded maps with regions of similar chemistry deduced from the set of spectra . thus , determining an unknown cell type from two possibilities notably , stem cell and differentiated cell can be accomplished using large numbers of spectra from known cell types . the most important differences are highlighted , rather than any uncorrelated and unimportant variations , to improve the sensitivity of the technique . two improved variants of pca are discussed in relation to ftir , and both show marked improvements in their ability to distinguish cell types . before analysis can be performed , spectra require processing to remove unwanted autofluorescence from tissue normally by baseline subtraction , as well as the removal of cosmic rays , and substrate and media contribution to the spectrum . raman spectra consist of a large number of peaks at well - defined frequencies , as demonstrated in figures 3 and 4 . raman scattering is a weak process typically only 1 in ~10 incident photons gives rise to a raman - shifted photon . in order to increase this efficiency , in coherent anti - stokes raman scattering ( cars ) [ 2528 ] the vibration is excited with two laser frequencies the difference ( or beat ) frequency between them is matched to the vibrational frequency of interest . multiplex cars [ 3034 ] uses normal ( narrowband ) pulses for one laser , p , and broadband pulses for s . a different approach to increase the speed of raman microscopy , termed stimulated raman scattering , has recently been published . in a similar way to how stimulated emission depopulates the excited state in lasers , the excited state in a simple raman excitation ( pumped with p ) can be rapidly depopulated by a second laser ( at s ) modulated in the mhz range . in this way , the signal at s is increased slightly ( stimulated raman gain ) and the pump power at p is decreased slightly ( stimulated raman loss , srl ) . srl is a linear optical technique and is suitable for extension into optical coherence tomography deep into tissue , using low numerical aperture lenses . ftir spectroscopy was used to study murine embryonic stem cells by ami et al . after 47 days of differentiation , changes to the absorption spectrum of fixed cells were noticed : features in the amide i band ( 16001700 cm ) were enhanced , and those in the nucleic acid region ( 8501050 cm ) diminish . this means that the overall levels of dna and rna decrease , and the alpha helix content of proteins increases over time . they used pca to clearly distinguish the three cells types of interest : stem cells , transit - amplifying cells , and terminal differentiated cells . no biomarkers of corneal stem cells exist ; so spectroscopic techniques offer the only viable method of cell characterization here . the position of cells along the crypt denotes the change from stem cell location to transit - amplifying region to differentiated location . this method of characterization was compared with tissue stained with two different immunophenotypical markers : rabbit polyclonal anti - cd133 and -catenin antibodies . the authors state that the dominant ftir absorption peak at 1080 cm , relating to the symmetric ( po2 ) stretch , is a more robust marker than the two biomarkers . as gastrointestinal stem cells lack specific biomarkers , they went on to compare ftir data against a number of chemical differences which are discussed at length in a further publication . used ftir to study osteoblasts derived from human mesenchymal stem cells after 28 days of cell culture . the spectrally derived mineral - to - matrix ratio was calculated as the ratio of the integrated areas of the phosphate absorbance ( 9001200 cm ) and protein amide i band ( 15851720 cm ) . they observed a significant decrease in the mineral - to - matrix ratio in the extracellular matrix produced by focal adhesion kinase- ( fak- ) knockdown cells when compared to untreated ( control ) cells . also used ftir to study human mesenchymal stem cells differentiating into osteoblasts . their samples were fixed in methanol then dried , and they were able to distinguish cells stimulated in osteogenic medium for 7 days , from nonstimulated cells . ftir microscopy on isolated adherent cells ( of size ~50 m ) showed that some of the nonstimulated cells had high levels of glycogen accumulation , and some stimulated cells had a high expression of calcium phosphate . , who measured a higher alpha helix proportion during differentiation . measured ftir spectra of human corneal stem cells and their derivatives : transit amplifying cells . they were able to distinguish between both cell types with spectra acquired from two different regions in the tissue , using pca , albeit with an overlap of 16% between the two populations . in summary , ftir is able to distinguish between stem cells and their derivatives . using synchrotron sources , the speed of data acquisition and the quality of comparison have improved greatly . to be able to distinguish between individual cells , rather than populations , requires subconfluent adherent cells due to the spatial resolution ( governed by the wavelength of infrared radiation ) being of the order of the cell size . the technique has until now been limited to dried samples due to the high absorption coefficient of water ; so the technique has not been used to noninvasively monitor stem cell differentiation in vitro as the required drying is clearly destructive . however , atr - ftir can be used on live cell cultures ; hence it could potentially be employed as a real - time noninvasive technique for monitoring stem cell differentiation on adherent cells ( though no results have yet been published ) . one issue with atr - ftir is that it only probes the first 1 - 2 m above a substrate ; so the nucleus would only give a small contribution to the overall signal . given that nucleic acids play a large part in the reported changes in spectral signatures during differentiation , and the nucleus may remain above this penetration depth , it remains to be seen whether atr - ftir can indeed be used as a noninvasive biomarker - free analytical technique for live cell studies . used raman spectroscopy to investigate live murine embryonic stem cells . however , stem cells do not adhere well to glass and crystals ; so a thin coating to the substrate is required , such as the gelatine used in this study . great care must also be taken to ensure that the stem cells do not differentiate spontaneously on a given substrate or coating . over 16 days of differentiation , they observed a decrease in the rna peak ( at 813 cm , opo stretch ) by 75% and a drop of 50% in the dna peak ( at 788 cm , cytosine ring vibration ) . they also extracted the first principal component spectrum , reproduced in figure 4 , which reveals the spectrum responsible for most of the differences between spectra of stem cells and differentiated cells . note the high coincidence of many peaks of this principal component spectrum , with the spectrum of rna . this confirms that a reduction in rna levels dominates the chemical changes during differentiation . acquired raman spectra on live human embryonic stem cells ( hescs ) as they differentiated into cardiomyocytes and were able to distinguish between hescs and hesc - derived cardiomyocytes with an accuracy of 66% . they found that the rna peak ( 811 cm ) and dna peaks ( e.g. , 785 cm , 1090 cm ) were all reduced in intensity during differentiation . raman spectra of fixed mesenchymal stem cells grown on gelatine - coated quartz , and used a diffraction - limited laser spot , instead of averaging over a large area . this meant that spectra could be acquired separately from within the nucleus and within the cytoplasm the spectrum from the substrate has been subtracted , but no baseline subtraction was performed to highlight the requirement for automated subtraction in quantitative spectroscopic analysis . the spectra in figure 5 as expected clearly demonstrate that the nucleus contains far more dna and rna than does the cytoplasm ; and that the cytoplasm contains far more proteins and lipids than the nucleus . [ 40 , 43 ] , implies that it is the nucleus size ( or nucleic acid density therein ) which shrinks during differentiation . hence , monitoring the nucleus size in a quantitative manner using imaging processing techniques would seem to be a good potential approach to monitoring the state of differentiation . ca5(po4)3(oh)and was by far the strongest signal ; this peak height rose linearly from zero to the dominant peak in the spectrum over the 21 days of differentiation . used raman spectroscopy to compare tissue - engineered bone derived from mesenchymal stem cells , with femoral bone . liu also observed a major peak at 960 cm due to hydroxyapatite , in mineral extracted from odontoblast nodules which were formed by the differentiation of dental pulp stem cells . used raman spectroscopy to characterize the production of mineral content from mesenchymal stem cells , under the influence of two osteogenic agents : quality elk velvet antler ( qeva ) extract , and dexamethasone . they measured no mineralization from the control group , some mineralization from the dexamethasone - fed cells , but most mineralization from cells supplemented with the elk velvet antler . for dexamethasone - fed cells , the dominant peak was of octacalcium phosphate , and lower amounts of hydroxyapatite and amorphous calcium phosphate were measured . compared mineralized nodules in vitro from 3 sources : embryonic stem cells , neonatal calvarial osteoblasts , and adult bone - marrow - derived mesenchymal stem cells . after 28 days in osteogenic medium , sets of raman spectra were acquired of the mineralized nodules , and pca was performed on the spectra to extract their chemical components . the osteoblasts and mesenchymal stem cells both produced nodules which were chemically similar to native bone : a combination in descending order of concentration , of ( a ) carbonate - substituted hydroxyapatite , ( b ) crystalline nonsubstituted hydroxyapatite , and ( c ) amorphous phosphate species . however , embryonic stem cells produce nodules which were dominated only by the first mineral component , which was similar to synthetic carbonated hydroxyapatite . nanoindentation tests showed that these nodules derived from embryonic stem cells were more than an order of magnitude less stiff than those derived from osteoblasts and mesenchymal stem cells . these raman studies all show a similar ability to distinguish stem cells from their derivatives , that is , a similar sensitivity , as the ftir technique . cars microscopy has been performed on live murine embryonic stem cells by konorov et al . , but the laser setup only permitted pixel dwell times of 300 milliseconds compared to microseconds in standard cars microscopy [ 2528 ] . the groups were unable to distinguish any individual cells or features when imaging at the dna and rna frequencies . however , cars spectroscopy showed a large reduction in the rna peak intensity ( at 811 cm ) in differentiated cells . figure 6 shows our cars microscopy image , acquired on mcf-7 human breast cancer cells which have a similarly large nucleus to cytoplasm ratio as stem cells . two sequentially acquired images are overlaid : in green , dna / rna is mapped at the phosphate backbone opo stretch frequency ( 1095 cm ) , and in red , lipids and the cytoskeleton are mapped at the ch2 deformation frequency ( 1448 cm ) . the opo stretch frequency is also present in figure 4(a)the principal component spectrum which describes most of the changes during differentiation and in figure 4(b)the rna spectrum . this method can be easily extended to monitoring the nucleus size in live stem cells , with a reduced frame rate of at least 1 image per minute ( we find that cars imaging in live cells is slower than in dried , fixed cells ) . cars microscopy is limited to monitoring one vibrational mode at a time , rather than comparing the full spectral signature . however , the rna and dna peaks have been shown to drop considerably , and rna is the dominant change to spectra . so it is possible that cars imaging will be able to measure the stage of differentiation purely by monitoring the size of each nucleus . white light imaging is less exact at measuring the nucleus size than either fluorescence or cars microscopy and could not be extended from monolayers ; so cars is preferred as a noninvasive technique to monitor nuclear size . we expect that cars should also be able to map mineralization from mesenchymal stem cells , by mapping the peak at 960 cm . the clear advantage of cars is its high speed compared to the other spectroscopic techniques . the major noninvasive optical spectroscopy techniques suitable for analysis of stem cell differentiation have been outlined : namely , ftir , raman , and cars . ftir spectroscopy has only been performed on fixed or dried stem cells , but atr - ftir can be used in order to investigate live cells in future . raman spectroscopy has demonstrated the ability to distinguish between live stem cells and differentiated cells : both murine and human embryonic stem cells display a large reduction in peak intensities of both rna and dna . when mesenchymal stem cells differentiate into osteoblasts , they display a clear peak ( or peaks ) relating solely to mineral composition . it is well known that the nucleus to cytoplasm ( volume ) ratio in embryonic stem cells is elevated [ 5154 ] . hence , the reduction in rna and dna levels during differentiation is not entirely surprising . the assumption is therefore that the nucleus volume shrinks by around 50% during differentiation rather than the concentration of nucleic acids reducing . cars microscopy is well suited to monitoring the nucleus size or mineralization content of each individual cell ( around 960 cm ) and can be applied to large numbers of cells in cultures and to engineered tissue scaffolds . white light imaging ( normally dic or phase contrast ) could offer a low - cost solution for monitoring the nucleus / cytoplasm ratio of cell monolayers , in conjunction with automated image recognition cytometry . multiplex cars can acquire a full spectrum and promises to replace raman spectroscopy in time , due to its improved speed . each spectroscopic technique has its own benefits and drawbacks ; so it is more suited to characterization of stem cells in different ways and on different platforms . raman and ftir spectroscopy are both most suited to monitoring cultures averaged over a large number cells . raman spectroscopy is too slow to characterize enough individual cells to be a worthwhile clinical technique but could be used extensively in biomedical research . both ftir and raman techniques are more sensitive than normal cars which relies on excitation of just one spectral peak . in future , multiplex cars promises to be the technique of choice for all platforms , due to its combined attributes of speed , full spectral analysis , and applicability to individual live cells .

extraoral lesions have been most commonly described on the penis ( table 1 ) , scrotum ( table 2 ) , and vulva ( table 3 ) . tumors in genital locations have recently been referred to as vegas ( verruciform genital - associated ) xanthomas . this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors.table 1verruciform xanthomas of the peniscaseao ( year)durationracelocationmorphologycolorsize ref.1825 yearsir prepucev , dry growthpinkishna216nawpenisps forming v plaquee y1020 mm3223 monthsnaprepuceind plaquer - orange2.5 1.75 cm4235 yearsnaglans penisfirm , v , ibpink1.5 cm5 25nananananana6282 weeksbleft penile shaftpn , kery - brown15 5 mm729nawcoronal sulcusvna2.4 1.7 0.7 cm8382.5 monthswpenile shaft perineumpn , pe8 5 mm9396 monthswglans penisv , caul , softe with y1.5 1.5 0.6 cm104012 yearscl glans peniscaul , v , kery - brown4 3.2 cm1141nawglans penisnana2 cm57whole penile shaftcaul , foc ulcy7 3 cm12431 yearnaventral shaftwl ppale brown0.8 cm1345naiinner prepuceflat , wl , pnana14521 yearwglans penisni , v plaquew1.5 0.5 cm15571.5 yearswglans penisirreg surf , not wlr10 mm16565 yearsnanav , pna0.3 cm1761najinner prepuceind , v plaquey - redna67coronal sulcus and glans penisind , eroded tumor 18622 yearswsulcusp , well - def , wle - brownish1.5 2 cm19642 yearsprepucev lesionnana2064nawnear coronal sulcusni , v plaquer1.5 2 cm2171nawforeskinfirm , swollennana22722 yearswglans penisg & vr to yna2373nawinferior foreskinppink to brown6 5 2 mm2477nanaglans penissessile , wlna1 0.5 cm257710 monthswglans penisv noduley15 12 mm26857 monthswdistal foreskinv plaqueskin color1 1.5 cm27nanaicoronal sulcusflat , wl , pnana28nananaglans penisnanana29 nanananananana ao age of onset , b black , c chinese , caul cauliflower - like , def defined , e erythematous , f filiform , foc ulc focally ulcerated , ib irregular boundaries , irreg irregular , i indian , ind indurated , g & v globules and small - dotted vessels , ker keratotic , m months , na not available , ni non - indurated , p(s ) papule(s ) , pn pedunculated , r red , ref reference , sev several , surf surface , v verrucous , w white , wks weeks , wl wart - like , y yellow , y year size is reported as given in the case reports . this is measured either in diameter , length width , or length width height kukreja et al . discuss a case of a 25-year - old male who was circumcised for a penile lesion . the lesion was initially misdiagnosed as a squamous cell carcinoma , but due to the histological features of the lesion , their department determined it was actually a verruciform xanthoma the patient s original lesion was treated with circumcision ; however , its removal from the coronary sulcus was suspected to be incomplete because of severe adhesion present between the prepuce and the glans penis . a yellowish - red papule was noted at the coronary sulcus 4 months following surgery . the patient decided not to have surgery because the lesion was reported to be benign . six years later , the patient reported with an eroded tumor covering the entire coronary sulcus and much of the glans penis . the patient received a partial penectomy pellice et al . reported a verruciform xanthoma of the penis . no other information was obtained table 2verruciform xanthomas of the scrotumcaseao ( year)durationracelocationmorphologycolorsize ref.11920 yearsnallob , mlpinkish32 mm219nanananana15 11 mm335nannacyst , crateriformna0.8 cm440wrped , vna9 5 mm5407 yearsnanaped , flat , g surf , caulpink1 cm64020 yearsnana3 nod , pebbly surfy r7 , 9 , and 12 mm7443 yearsnanaped , spherical , g surf , caulpink3 cm84923 yearsjlelastic soft , ped , g surf r w y10 mm950few monthsblv , fil ppink4 2 mm10502 yearsbnasessile plaquena1.2 cm1153>1 yearnanawl , polypoidna0.5 cm12594 yearsnanamult ppink11.5 cm13593 yearsnanapolyp v nod , pebbly surfy r6 mm14593 weeknalfil pr5 4 4 mm15634 yearsjrped , v , occ bleedingr - pink2.5 2.5 cm1663nananav nod whitish4 mm17643.5 yearsnanapolypoidna1.8 cm18661 yearjna2 ker papy4 mm19682 yearsnarml nodse20 mm206812 yearsjrped , v tumorpink10 mm21703 yearsnalsl ped nodse30 mm22715 yearsjrped , elastic , soft nod , ml , erosive surfr7 10 mm23722 yearsnalnodspink6 mm24741 yearjlwell - defpinkish13 mm257510 monthsjrsoft , ped , grany3 cm26751 yearpolyp v nod , pebbly surf y r25 mm2878nanawl , ppinkna29803 yearswlpr8 5 mmcr30826 monthswlped , soft , mlpinkish15 mm31835 yearsnanaped , soft , mlpink25 mm32135 v , wl[2 , 26 , 34 , 46 , 56 ] ao age onset , b black , c case , caul cauliflower - like , cr current report , fil filiform , g granular , j japanese , l location , m months , ml mulberry - like , mult multiple , na not available , nod(s ) nodule(s ) , occ occasional , p papules , ped pedunculated , poly polypoid , r red , ref reference , sl strawberry - like , surf surface , v verrucous , w with , wk weeks , wl wart like , y yellow , y year(s ) size is reported as given in the case reports . this is measured in either in diameter , length width , or length width height three red papules about 1 mm in diameter were scattered in the surrounding area of the lesion . the patient noticed a nodule on the nonpsoriatic part of his scrotum while receiving systemic and topical puva therapy . the nodule appeared when the cumulative uva dosage reached 27.6 j / cm this patient had four 23-mm diameter red papules adjacent to his 7 mm verrucous nodule the patient also had a pulsating subcutaneous induration covered by lightly purplish skin under his scrotal nodule joshi and ovhal documented a case of a single scrotal lesion that was diagnosed as a verruciform xanthoma in their report . . stated that one patient in their study had 2 scrotal lesions appearing at different times in the same year . no other information about the lesions was given fukuda h and saito r performed a review of the japanese dermatological literature in 2005 and found 100 scrotal cases that were not cited in this review . 36.3% of the lesions were on the left of the scrotum , 31.4% were on the right of the scrotum , 6.9% were bilateral , and 25.4% did not specify furue et al . conducted a study using a sample from a scrotal verruciform xanthoma from a 69-year - old man . no other information about the patient was reported helm et al . reported an additional scrotal case with no other information table 3verruciform xanthomas of the female genitaliacaseao ( year)durationracelocmorphologycolorsize a.c.ref.1 15 days17 yearsnar ing foldwd , v plaqueenachild2 11 yearnal lab minv plaquey7 5 cmnr356 yearscvulvav lesione , y6 6 cmchild4 8 nacolr lab majthick v lesionbrownnachild51217 yearsbvulvamult v , inv lesionsnananr615 childhoodnal groin , ext gensoft , exudatingpinknachild716912 mnal lab majgran , vw - tan1.5 cmna816 lifelongnal inguinal areav lesion w small nody - tan6 3 cmchild9 30nanal lab minpolyr2.5 cmnr38wl5.0 cm10 2220 yearswgenital mucosamult v indr0.32.5 cmnr1127 since childhoodwl vulva and perver hyperkerrnachild1243 nawclitorishyperker , caul , v , wl lesiongrayish white13 mmls13448 monthsnananana13 10 mmna14 441 monthnal lab majscaly plaquepink34 mmnr1546several yearsjl lab majsoft , ped nod w new gran lesionbrown w y35 24 22 mmfep16482 yearsjvulvav surf , wdo rnasl , lc1751nanalab minv lesion , indy o2 mmvpd2884nanal vulvav lesionnananr29 nananananananana ac associated condition , ao age of onset , b black , c chinese , caul cauliflower - like , child congenital hemidysplasia with ichthyosiform nevus and limb defects , col columbian , demac demarcated , dur duration , ens epidermal nevus syndrome , ext gen external genitalia , fep fibroepithelial polyp , gran granular , ind indurated , ing inguinal , inv inverted , j japanese , ker keratotic , l left , loc location , lab maj labia majora , lab min labia minora , lc lymphangioma circumscriptum , lmm leiomyomatosis of uterine corpus , lp lichen planus , ls lichen sclerosus , m months , mult multiple , na not available , nod nodule , nr none reported , o orange , p papule , ped pedunculated , per perineum , poly polypoid , r red , rd radiodermatitis , ref reference , sl severe lymphedema , v verrucous , vpd vulvar paget disease , w with , wd well - demarcated , wl wart - like , y years the age of onset was not given size is reported as given in the case reports . this is measured in either in diameter , length width , or length width height this young girl developed multiple skin lesions 15 days after birth involving the right inguinal fold , gluteal fold , and leg . she also had lesions on her finger and toe nails and interdigital spaces this young girl had multiple small papules around the anus and on her left thigh . she was treated with topical imiquimod cream 5% , and the lesions almost entirely cleared 4 months after initiating treatment this columbian girl was born with skin lesions . by the age of 19 months she had non - verrucous hypopigmented bands and streaks on her right arm . at at age 8 she had linear patches and plaques on the right side of her body from her neck to inguinal areas , along with hyperkeratotic and discolored fingernails and a linear band on her left middle finger extending to her nail the vulvar lesion was initially removed by co2 laser ablasion , but it recurred 8 years later and was successfully treated with wide local excision . this woman had lesions on her plantar creases , lateral feet , dorsal surface of toes , palmar creases , dorsal aspect of third and fourth right fingers , a scaly plaque on her posterior right ear , multiple genital lesions , and a large plaque on the left inguinal fold this woman had disseminated verruciform xanthoma . she had a 20-year history of multiple 0.51 cm discrete hyperkeratotic plaques on the dorsal left food , medial calf , and medial and lateral left knee . she also developed lesions on the plantar left food , buccal mucosa , hard palate , left axilla , and left labia majora this woman had laser ablation of her lesion , but the lesion recurred 16 months following treatment . she then had surgical removal , but the removal was incomplete , and the verruciform xanthoma recurred 2 years later daimaru et al . described a case of verruciform xanthoma of the vulva . no other information was obtained verruciform xanthomas of the penis ao age of onset , b black , c chinese , caul cauliflower - like , def defined , e erythematous , f filiform , foc ulc focally ulcerated , ib irregular boundaries , irreg irregular , i indian , ind indurated , g & v globules and small - dotted vessels , ker keratotic , m months , na not available , ni non - indurated , p(s ) papule(s ) , pn pedunculated , r red , ref reference , sev several , surf surface , v verrucous , w white , wks weeks , wl wart - like , y yellow , y year size is reported as given in the case reports . this is measured either in diameter , length width , or length width height kukreja et al . discuss a case of a 25-year - old male who was circumcised for a penile lesion . the lesion was initially misdiagnosed as a squamous cell carcinoma , but due to the histological features of the lesion , their department determined it was actually a verruciform xanthoma the patient s original lesion was treated with circumcision ; however , its removal from the coronary sulcus was suspected to be incomplete because of severe adhesion present between the prepuce and the glans penis . a yellowish - red papule was noted at the coronary sulcus 4 months following surgery . the patient decided not to have surgery because the lesion was reported to be benign . six years later , the patient reported with an eroded tumor covering the entire coronary sulcus and much of the glans penis . the patient received a partial penectomy pellice et al . reported a verruciform xanthoma of the penis . no other information was obtained verruciform xanthomas of the scrotum ao age onset , b black , c case , caul cauliflower - like , cr current report , fil filiform , g granular , j japanese , l location , m months , ml mulberry - like , mult multiple , na not available , nod(s ) nodule(s ) , occ occasional , p papules , ped pedunculated , poly polypoid , r red , ref reference , sl strawberry - like , surf surface , v verrucous , w with , wk weeks , wl wart like , y yellow , y year(s ) size is reported as given in the case reports . this is measured in either in diameter , length width , or length width height three red papules about 1 mm in diameter were scattered in the surrounding area of the lesion . the patient noticed a nodule on the nonpsoriatic part of his scrotum while receiving systemic and topical puva therapy . the nodule appeared when the cumulative uva dosage reached 27.6 j / cm this patient had four 23-mm diameter red papules adjacent to his 7 mm verrucous nodule the patient also had a pulsating subcutaneous induration covered by lightly purplish skin under his scrotal nodule joshi and ovhal documented a case of a single scrotal lesion that was diagnosed as a verruciform xanthoma in their report . stated that one patient in their study had 2 scrotal lesions appearing at different times in the same year . no other information about the lesions was given fukuda h and saito r performed a review of the japanese dermatological literature in 2005 and found 100 scrotal cases that were not cited in this review . 36.3% of the lesions were on the left of the scrotum , 31.4% were on the right of the scrotum , 6.9% were bilateral , and 25.4% did not specify furue et al . conducted a study using a sample from a scrotal verruciform xanthoma from a 69-year - old man . no other information about the patient was reported helm et al . reported an additional scrotal case with no other information verruciform xanthomas of the female genitalia ac associated condition , ao age of onset , b black , c chinese , caul cauliflower - like , child congenital hemidysplasia with ichthyosiform nevus and limb defects , col columbian , demac demarcated , dur duration , ens epidermal nevus syndrome , ext gen external genitalia , fep fibroepithelial polyp , gran granular , ind indurated , ing inguinal , inv inverted , j japanese , ker keratotic , l left , loc location , lab maj labia majora , lab min labia minora , lc lymphangioma circumscriptum , lmm leiomyomatosis of uterine corpus , lp lichen planus , ls lichen sclerosus , m months , mult multiple , na not available , nod nodule , nr none reported , o orange , p papule , ped pedunculated , per perineum , poly polypoid , r red , rd radiodermatitis , ref reference , sl severe lymphedema , v verrucous , vpd vulvar paget disease , w with , wd well - demarcated , wl wart - like , y years the age of onset was not given size is reported as given in the case reports . this is measured in either in diameter , length width , or length width height this young girl developed multiple skin lesions 15 days after birth involving the right inguinal fold , gluteal fold , and leg . she also had lesions on her finger and toe nails and interdigital spaces this young girl had multiple small papules around the anus and on her left thigh . she was treated with topical imiquimod cream 5% , and the lesions almost entirely cleared 4 months after initiating treatment this columbian girl was born with skin lesions . by the age of 19 months she had non - verrucous hypopigmented bands and streaks on her right arm . at 3 years old the lesion extended to her hand and fingers . at age 8 she had linear patches and plaques on the right side of her body from her neck to inguinal areas , along with hyperkeratotic and discolored fingernails and a linear band on her left middle finger extending to her nail the vulvar lesion was initially removed by co2 laser ablasion , but it recurred 8 years later and was successfully treated with wide local excision . this woman had lesions on her plantar creases , lateral feet , dorsal surface of toes , palmar creases , dorsal aspect of third and fourth right fingers , a scaly plaque on her posterior right ear , multiple genital lesions , and a large plaque on the left inguinal fold this woman had disseminated verruciform xanthoma . she had a 20-year history of multiple 0.51 cm discrete hyperkeratotic plaques on the dorsal left food , medial calf , and medial and lateral left knee . she also developed lesions on the plantar left food , buccal mucosa , hard palate , left axilla , and left labia majora this woman had laser ablation of her lesion , but the lesion recurred 16 months following treatment . she then had surgical removal , but the removal was incomplete , and the verruciform xanthoma recurred 2 years later daimaru et al . described a case of verruciform xanthoma of the vulva . no other information was obtained verruciform xanthoma is a wart - like lesion that most commonly occurs in the oral mucosa . it was first described as a xanthoma - like nevus by sachs in 1903 . in 1971 , shafer coined the term verruciform xanthoma while describing 15 cases in the oral cavity . these lesions were reported as asymptomatic , pale or hyperkeratotic , with a pebbly surface and verrucous appearance . the most common extraoral verruciform xanthomas are reported in the genital region ; however , other reported extra - oral locations include the anal region , ear , forearm , foot , hand , leg , nose , and sacrum [ 6571 , 74 ] . in 2003 , philipsen et al . did a profile of 282 oral verruciform xanthomas they found a slight male predominance below the age of 50 , with a male : female ratio of 1.6:1 , and a slight female predominance above the age of 50 , with a male : female ratio of 0.8:1 ; 73.4% of the oral verruciform xanthomas were on the masticatory mucosa . the majority of cases were reported in caucasians ( 139 patients ) and japanese ( 109 patients ) , but there were also reports of african americans , asians , and south americans . clinically , verruciform xanthomas have a similar appearance to bowenoid papulosis , condyloma acuminatum , erythroplasia of queyrat , granular cell tumor , giant molluscum contagiosum , seborrheic keratosis , squamous cell carcinoma , verruca vulgaris , verrucous carcinoma , and vulvar intraepithelial neoplasia ( fig . 1 ) [ 11 , 27 , 40 , 75 , 76 ] . 1distant ( a ) and closer ( b ) views showing a pedunculated papule protruding from the patient s left side of the scrotum of an 83-year - old heterosexual monogamous man who had no history of sexually transmitted diseases and no reported hiv risk factors . cutaneous examination revealed a flesh - colored 8 5 mm wart - like elongated papule localized to the left side of his scrotum . a snip excision was performed for biopsy and removal of the lesion distant ( a ) and closer ( b ) views showing a pedunculated papule protruding from the patient s left side of the scrotum of an 83-year - old heterosexual monogamous man who had no history of sexually transmitted diseases and no reported hiv risk factors . cutaneous examination revealed a flesh - colored 8 5 mm wart - like elongated papule localized to the left side of his scrotum . a snip excision was performed for biopsy and removal of the lesion the histologic differential diagnosis of verruciform xanthoma includes condyloma accuminatum , granular cell epulis , granular cell tumor , verruca vulgaris , and verrucous carcinoma . these features include acanthotic epidermis with parakeratosis that extends deep into the epithelium , uniformly elongated rete ridges , neutrophilic infiltrate in the dermis , and foamy histiocytes throughout the dermal papillae ( fig . 2 ) . the foam cells stain periodic acid schiff - positive and express cd68 antigen , indicating the presence of glycogen granules in monocyte - macrophage lineage cells [ 12 , 13 , 17 , 52 , 55].fig . 2microscopic examination of the lesion from the 83-year - old man was performed . low magnification ( a ) shows a pedunculated tumor with acanthosis , papillomatosis , and elongation of the rete ridges . intermediate magnification ( b , c ) reveals parakeratosis and neutrophilic inflammation in the dermis . correlation of the clinical features and the pathologic changes establish a diagnosis of verruciform xanthoma . the lesion was completely removed at the time of biopsy , and the patient applied mupirocin 2% ointment to the site . ( hematoxylin and eosin : a = 2 , b = 10 , c = 20 , d = 40 ) microscopic examination of the lesion from the 83-year - old man was performed . low magnification ( a ) shows a pedunculated tumor with acanthosis , papillomatosis , and elongation of the rete ridges . intermediate magnification correlation of the clinical features and the pathologic changes establish a diagnosis of verruciform xanthoma . the lesion was completely removed at the time of biopsy , and the patient applied mupirocin 2% ointment to the site . ( hematoxylin and eosin : a = 2 , b = 10 , c = 20 , d = 40 ) the pathogenesis of verruciform xanthoma is unknown . it has been hypothesized that the lesions are associated with human papilloma virus , but multiple studies have found this association to be unlikely [ 9 , 18 , 82 , 83 ] . he states that the inflammatory response damages keratinocytes , which release lipids that are then engulfed by macrophages , leading to the accumulation of foam cells . found that damaged keratinocytes release cytokines that attract neutrophils and stimulate rapid growth of the epidermis , supporting zegarelli et al.s hypothesis . other investigators have speculated that verruciform xanthomas may be due to an immunologic reaction [ 28 , 84 , 86 ] . oliveira et al . proposed that verruciform xanthomas are formed by an autoimmune reaction inducing apoptosis of epithelial cells , similarly to lichen planus . this is supported by multiple cases of verruciform xanthomas reported in association with lichen planus [ 43 , 85 ] . however , there is not sufficient evidence to conclude a clear mechanism of pathogenesis associated with verruciform xanthomas . the first verruciform xanthoma of the penis was reported in 1981 by kraemer et al . . to date , there are 31 cases of penile verruciform xanthomas in the literature that have been described in 29 men . the age of onset of the lesions ranged from 8 to 85 years , with a mean of 54.5 years . the duration of the penile verruciform xanthomas prior to establishing the diagnosis ranged from 2 weeks to 25 years , with a mean duration of 3.7 years . the locations ( of the 27 lesions for which the site was specified ) include the coronal sulcus ( 18.5% , n = 5 ) , glans ( 37.0% , n = 10 ) , prepuce ( 29.6% , n = 8) , and shaft ( 14.8% , n = 4 ) . the colors varied ; including brown , erythematous , pink , and yellow . verruciform xanthomas of the penis have been reported following necrotizing fasciitis of the anogenital region , radical removal of initial verruciform xanthoma with grafting of the foreskin , and transurethral prostate resection . the first scrotal verruciform xanthoma was described in 1984 by al - nafussi et al . . fukuda and saito carried out a review of the japanese literature and found that 81% of verruciform xanthomas in the pubic area were located on the scrotum . including the 102 cases reviewed by fukuda and saito , kono suggested that the verruciform xanthomas may be related to irritation of the scrotum by the japanese custom of sitting on the floor . this is an interesting hypothesis since there is a significant number of scrotal verruciform xanthomas reported in the japanese literature [ 34 , 35 , 42 ] . these findings support zegarreli et al.s proposal that verruciform xanthoma formation may be linked to epithelial degeneration due to irritation . the age of onset was given for 26 of the scrotal lesions , and ranged from ages 19 to 83 years , with a mean age of 59.5 years . the duration of the tumors , prior to establishing the diagnosis , varied from 3 weeks to 20 years . scrotal lesions have been associated with arteriovenous haemangioma , epidermolytic acanthoma [ 25 , 34 ] , graft versus host disease following bone marrow transplant for acute lymphoblastic leukemia , human papillomavirus , and psoriasis in a patient undergoing psoralen and ultraviolet a ( puva ) therapy . in addition to the scrotal and penile tumors , verruciform xanthoma of the genitalia has been reported on the inguinal folds . the man had a 7 5 cm well - demarcated plaque on his left inguinal fold that extended over the thigh and onto the scrotum . he also had a 1 2 cm plaque in the right inguinal fold . the first reported extraoral lesions were two cases of verruciform xanthoma of the vulva , described by santa cruz and martin in 1979 . a total of 28 additional vulvar verruciform xanthomas have since been reported in 27 women . vulvar verruciform xanthomas have been described in a variety of patients , including african american , chinese , caucasian , columbian , and japanese women . the age of onset of the female genital lesions ranges from shortly after birth [ in association with congenital hemidysplasia with ichthyosiform erythroderma and limb defects ( child syndrome ) ] to age 85 years , with a mean age of 43.2 years . a total of 27 locations have been reported , which included the clitoris ( 11.1% , n = 3 ) , external genitalia and groin ( 3.7% , n = 1 ) , fourchette ( 7.4% , n = 2 ) , genital mucosa ( 3.7% , n = 1 ) , inguinal fold ( 7.4% , n = 2 ) , labia majora ( 25.9% , n = 7 ) , labia minora ( 18.5% , n = 5 ) , and vulva not otherwise specified ( 22.2% , n = 6 ) . the color of the lesion was most commonly yellowish orange . vulvar lesions have been found in association with child syndrome [ 44 , 47 , 49 , 52 , 59 , 60 ] , fibroepithelial polyp , leiomyomatosis of uterus , lichen planus , lichen sclerosus [ 43 , 54 ] , lymphangioma circumscriptum with severe lymphedema , radiodermatitis , and vulvar paget s disease . the verruciform xanthomas are cured by complete excision ; however , cases of recurrence have been described [ 13 , 43 , 76 ] . laser ablation resulted in the recurrence in two cases of vulvar lesions [ 43 , 76 ] . however , joo et al . successfully removed a scrotal xanthoma with shave debulking and fractionated co2 laser ablation . guo et al . successfully treated a large ( 7 5 mm ) lesion of the labia minora with imiquiod cream 5% . verruciform xanthoma is a wart - like lesion that most commonly occurs in the oral mucosa . it was first described as a xanthoma - like nevus by sachs in 1903 . in 1971 , shafer coined the term verruciform xanthoma while describing 15 cases in the oral cavity . these lesions were reported as asymptomatic , pale or hyperkeratotic , with a pebbly surface and verrucous appearance . the most common extraoral verruciform xanthomas are reported in the genital region ; however , other reported extra - oral locations include the anal region , ear , forearm , foot , hand , leg , nose , and sacrum [ 6571 , 74 ] . in 2003 , philipsen et al . did a profile of 282 oral verruciform xanthomas they found a slight male predominance below the age of 50 , with a male : female ratio of 1.6:1 , and a slight female predominance above the age of 50 , with a male : female ratio of 0.8:1 ; 73.4% of the oral verruciform xanthomas were on the masticatory mucosa . the majority of cases were reported in caucasians ( 139 patients ) and japanese ( 109 patients ) , but there were also reports of african americans , asians , and south americans . clinically , verruciform xanthomas have a similar appearance to bowenoid papulosis , condyloma acuminatum , erythroplasia of queyrat , granular cell tumor , giant molluscum contagiosum , seborrheic keratosis , squamous cell carcinoma , verruca vulgaris , verrucous carcinoma , and vulvar intraepithelial neoplasia ( fig . 1 ) [ 11 , 27 , 40 , 75 , 76 ] . 1distant ( a ) and closer ( b ) views showing a pedunculated papule protruding from the patient s left side of the scrotum of an 83-year - old heterosexual monogamous man who had no history of sexually transmitted diseases and no reported hiv risk factors . cutaneous examination revealed a flesh - colored 8 5 mm wart - like elongated papule localized to the left side of his scrotum . a snip excision was performed for biopsy and removal of the lesion distant ( a ) and closer ( b ) views showing a pedunculated papule protruding from the patient s left side of the scrotum of an 83-year - old heterosexual monogamous man who had no history of sexually transmitted diseases and no reported hiv risk factors . cutaneous examination revealed a flesh - colored 8 5 mm wart - like elongated papule localized to the left side of his scrotum . a snip excision was performed for biopsy and removal of the lesion the histologic differential diagnosis of verruciform xanthoma includes condyloma accuminatum , granular cell epulis , granular cell tumor , verruca vulgaris , and verrucous carcinoma . these features include acanthotic epidermis with parakeratosis that extends deep into the epithelium , uniformly elongated rete ridges , neutrophilic infiltrate in the dermis , and foamy histiocytes throughout the dermal papillae ( fig . 2 ) . the foam cells stain periodic acid schiff - positive and express cd68 antigen , indicating the presence of glycogen granules in monocyte - macrophage lineage cells [ 12 , 13 , 17 , 52 , 55].fig . 2microscopic examination of the lesion from the 83-year - old man was performed . low magnification ( a ) shows a pedunculated tumor with acanthosis , papillomatosis , and elongation of the rete ridges . intermediate magnification ( b , c ) reveals parakeratosis and neutrophilic inflammation in the dermis . correlation of the clinical features and the pathologic changes establish a diagnosis of verruciform xanthoma . the lesion was completely removed at the time of biopsy , and the patient applied mupirocin 2% ointment to the site . ( hematoxylin and eosin : a = 2 , b = 10 , c = 20 , d = 40 ) microscopic examination of the lesion from the 83-year - old man was performed . low magnification ( a ) shows a pedunculated tumor with acanthosis , papillomatosis , and elongation of the rete ridges . intermediate magnification ( b , c ) reveals parakeratosis and neutrophilic inflammation in the dermis . high magnification ( d ) reveals numerous foamy histiocytes in the widened dermal papillae . correlation of the clinical features and the pathologic changes establish a diagnosis of verruciform xanthoma . the lesion was completely removed at the time of biopsy , and the patient applied mupirocin 2% ointment to the site . ( hematoxylin and eosin : a = 2 , b = 10 , c = 20 , d = 40 ) it has been hypothesized that the lesions are associated with human papilloma virus , but multiple studies have found this association to be unlikely [ 9 , 18 , 82 , 83 ] . he states that the inflammatory response damages keratinocytes , which release lipids that are then engulfed by macrophages , leading to the accumulation of foam cells . found that damaged keratinocytes release cytokines that attract neutrophils and stimulate rapid growth of the epidermis , supporting zegarelli et al.s hypothesis . other investigators have speculated that verruciform xanthomas may be due to an immunologic reaction [ 28 , 84 , 86 ] . oliveira et al . proposed that verruciform xanthomas are formed by an autoimmune reaction inducing apoptosis of epithelial cells , similarly to lichen planus . this is supported by multiple cases of verruciform xanthomas reported in association with lichen planus [ 43 , 85 ] . however , there is not sufficient evidence to conclude a clear mechanism of pathogenesis associated with verruciform xanthomas . the first verruciform xanthoma of the penis was reported in 1981 by kraemer et al . . to date , there are 31 cases of penile verruciform xanthomas in the literature that have been described in 29 men . the age of onset of the lesions ranged from 8 to 85 years , with a mean of 54.5 years . the duration of the penile verruciform xanthomas prior to establishing the diagnosis ranged from 2 weeks to 25 years , with a mean duration of 3.7 years . the locations ( of the 27 lesions for which the site was specified ) include the coronal sulcus ( 18.5% , n = 5 ) , glans ( 37.0% , n = 10 ) , prepuce ( 29.6% , n = 8) , and shaft ( 14.8% , n = 4 ) . the colors varied ; including brown , erythematous , pink , and yellow . verruciform xanthomas of the penis have been reported following necrotizing fasciitis of the anogenital region , radical removal of initial verruciform xanthoma with grafting of the foreskin , and transurethral prostate resection . the first scrotal verruciform xanthoma was described in 1984 by al - nafussi et al . . fukuda and saito carried out a review of the japanese literature and found that 81% of verruciform xanthomas in the pubic area were located on the scrotum . including the 102 cases reviewed by fukuda and saito , kono suggested that the verruciform xanthomas may be related to irritation of the scrotum by the japanese custom of sitting on the floor . this is an interesting hypothesis since there is a significant number of scrotal verruciform xanthomas reported in the japanese literature [ 34 , 35 , 42 ] . these findings support zegarreli et al.s proposal that verruciform xanthoma formation may be linked to epithelial degeneration due to irritation . the age of onset was given for 26 of the scrotal lesions , and ranged from ages 19 to 83 years , with a mean age of 59.5 years . the duration of the tumors , prior to establishing the diagnosis , varied from 3 weeks to 20 years . scrotal lesions have been associated with arteriovenous haemangioma , epidermolytic acanthoma [ 25 , 34 ] , graft versus host disease following bone marrow transplant for acute lymphoblastic leukemia , human papillomavirus , and psoriasis in a patient undergoing psoralen and ultraviolet a ( puva ) therapy . in addition to the scrotal and penile tumors , verruciform xanthoma of the genitalia has been reported on the inguinal folds . the man had a 7 5 cm well - demarcated plaque on his left inguinal fold that extended over the thigh and onto the scrotum . he also had a 1 2 cm plaque in the right inguinal fold . the first reported extraoral lesions were two cases of verruciform xanthoma of the vulva , described by santa cruz and martin in 1979 . a total of 28 additional vulvar verruciform xanthomas have since been reported in 27 women . vulvar verruciform xanthomas have been described in a variety of patients , including african american , chinese , caucasian , columbian , and japanese women . the age of onset of the female genital lesions ranges from shortly after birth [ in association with congenital hemidysplasia with ichthyosiform erythroderma and limb defects ( child syndrome ) ] to age 85 years , with a mean age of 43.2 years . a total of 27 locations have been reported , which included the clitoris ( 11.1% , n = 3 ) , external genitalia and groin ( 3.7% , n = 1 ) , fourchette ( 7.4% , n = 2 ) , genital mucosa ( 3.7% , n = 1 ) , inguinal fold ( 7.4% , n = 2 ) , labia majora ( 25.9% , n = 7 ) , labia minora ( 18.5% , n = 5 ) , and vulva not otherwise specified ( 22.2% , n = 6 ) . vulvar lesions have been found in association with child syndrome [ 44 , 47 , 49 , 52 , 59 , 60 ] , fibroepithelial polyp , leiomyomatosis of uterus , lichen planus , lichen sclerosus [ 43 , 54 ] , lymphangioma circumscriptum with severe lymphedema , radiodermatitis , and vulvar paget s disease . the verruciform xanthomas are cured by complete excision ; however , cases of recurrence have been described [ 13 , 43 , 76 ] . laser ablation resulted in the recurrence in two cases of vulvar lesions [ 43 , 76 ] . however , joo et al . successfully removed a scrotal xanthoma with shave debulking and fractionated co2 laser ablation . guo et al . successfully treated a large ( 7 5 mm ) lesion of the labia minora with imiquiod cream 5% . the verruciform xanthomas are cured by complete excision ; however , cases of recurrence have been described [ 13 , 43 , 76 ] . laser ablation resulted in the recurrence in two cases of vulvar lesions [ 43 , 76 ] . however , joo et al . successfully removed a scrotal xanthoma with shave debulking and fractionated co2 laser ablation . guo et al . successfully treated a large ( 7 5 mm ) lesion of the labia minora with imiquiod cream 5% . verruciform xanthomas are benign , asymptomatic wart - like lesions most commonly found in the oral cavity . although they appear similarly to skin lesions caused by tumors or viral infections , they can be distinguished based on histologic evaluation . the defining pathologic features include hyperkeratosis with parakeratosis , acanthosis , elongated rete ridges , neutrophilic inflammation in the dermis , and foam cells in the dermal papillae . there have been 194 cases of verruciform genital - associated ( vegas ) xanthomas reported in the literature . patients may seek treatment because of concern of a sexually transmitted disease or the lesion may be discovered as an incidental finding during complete cutaneous skin examination . verruciform xanthomas of the genitalia share similar histologic characteristics with verruciform xanthomas of the oral cavity . the lesions have been reported in association with a variety of cutaneous diseases and systemic conditions . verruciform xanthomas are usually asymptomatic and may be present for many years before being treated . fractionated co2 laser therapy and imiquimoid cream have also been used to successfully remove the lesions . this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors . the datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

introductionverruciform xanthoma is a wart - like benign lesion . the classic histologic appearance consists of foamy histiocytes within elongated dermal papillae and epithelial acanthosis . the lesion most commonly occurs in the oral cavity , but has been reported in extra - oral sites such as the penis , scrotum , and vulva . the clinical and histologic characteristics of verruciform genital - associated ( vegas ) xanthomas of the penis , scrotum , and vulva are reviewed.methodspubmed was used to search the following term : verruciform xanthoma . the relevant papers were obtained and reviewed.resultsthere have been 193 cases of genital - associated verruciform xanthomas . there were 164 in men and 29 in women . similar to verruciform xanthomas of the oral mucosa , they presented as asymptomatic lesions , demonstrated foam cells in the dermal papillae , and were typically managed successfully with surgical excision.conclusionverruciform xanthoma is a benign lesion characterized by a wart - like growth that is most commonly seen in the oral mucosa . verruciform xanthomas of the genital region have been coined vegas xanthomas . vegas xanthomas have been reported in association with a variety of diseases , as well as in healthy individuals . biopsy is required for diagnosis , and complete surgical excision is typically curative .

Introduction History Differential Diagnosis Pathogenesis Verruciform Xanthomas of the Penis (Table Verruciform Xanthomas of the Scrotum (Table Verruciform Xanthomas of the Inguinal Fold Verruciform Xanthomas of the Female Genitalia (Table Treatment Conclusion Disclosures Compliance with Ethics Guidelines Data Availability Open Access

extraoral lesions have been most commonly described on the penis ( table 1 ) , scrotum ( table 2 ) , and vulva ( table 3 ) . this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors.table 1verruciform xanthomas of the peniscaseao ( year)durationracelocationmorphologycolorsize ref.1825 yearsir prepucev , dry growthpinkishna216nawpenisps forming v plaquee y1020 mm3223 monthsnaprepuceind plaquer - orange2.5 1.75 cm4235 yearsnaglans penisfirm , v , ibpink1.5 cm5 25nananananana6282 weeksbleft penile shaftpn , kery - brown15 5 mm729nawcoronal sulcusvna2.4 1.7 0.7 cm8382.5 monthswpenile shaft perineumpn , pe8 5 mm9396 monthswglans penisv , caul , softe with y1.5 1.5 0.6 cm104012 yearscl glans peniscaul , v , kery - brown4 3.2 cm1141nawglans penisnana2 cm57whole penile shaftcaul , foc ulcy7 3 cm12431 yearnaventral shaftwl ppale brown0.8 cm1345naiinner prepuceflat , wl , pnana14521 yearwglans penisni , v plaquew1.5 0.5 cm15571.5 yearswglans penisirreg surf , not wlr10 mm16565 yearsnanav , pna0.3 cm1761najinner prepuceind , v plaquey - redna67coronal sulcus and glans penisind , eroded tumor 18622 yearswsulcusp , well - def , wle - brownish1.5 2 cm19642 yearsprepucev lesionnana2064nawnear coronal sulcusni , v plaquer1.5 2 cm2171nawforeskinfirm , swollennana22722 yearswglans penisg & vr to yna2373nawinferior foreskinppink to brown6 5 2 mm2477nanaglans penissessile , wlna1 0.5 cm257710 monthswglans penisv noduley15 12 mm26857 monthswdistal foreskinv plaqueskin color1 1.5 cm27nanaicoronal sulcusflat , wl , pnana28nananaglans penisnanana29 nanananananana ao age of onset , b black , c chinese , caul cauliflower - like , def defined , e erythematous , f filiform , foc ulc focally ulcerated , ib irregular boundaries , irreg irregular , i indian , ind indurated , g & v globules and small - dotted vessels , ker keratotic , m months , na not available , ni non - indurated , p(s ) papule(s ) , pn pedunculated , r red , ref reference , sev several , surf surface , v verrucous , w white , wks weeks , wl wart - like , y yellow , y year size is reported as given in the case reports . the lesion was initially misdiagnosed as a squamous cell carcinoma , but due to the histological features of the lesion , their department determined it was actually a verruciform xanthoma the patient s original lesion was treated with circumcision ; however , its removal from the coronary sulcus was suspected to be incomplete because of severe adhesion present between the prepuce and the glans penis . no other information was obtained table 2verruciform xanthomas of the scrotumcaseao ( year)durationracelocationmorphologycolorsize ref.11920 yearsnallob , mlpinkish32 mm219nanananana15 11 mm335nannacyst , crateriformna0.8 cm440wrped , vna9 5 mm5407 yearsnanaped , flat , g surf , caulpink1 cm64020 yearsnana3 nod , pebbly surfy r7 , 9 , and 12 mm7443 yearsnanaped , spherical , g surf , caulpink3 cm84923 yearsjlelastic soft , ped , g surf r w y10 mm950few monthsblv , fil ppink4 2 mm10502 yearsbnasessile plaquena1.2 cm1153>1 yearnanawl , polypoidna0.5 cm12594 yearsnanamult ppink11.5 cm13593 yearsnanapolyp v nod , pebbly surfy r6 mm14593 weeknalfil pr5 4 4 mm15634 yearsjrped , v , occ bleedingr - pink2.5 2.5 cm1663nananav nod whitish4 mm17643.5 yearsnanapolypoidna1.8 cm18661 yearjna2 ker papy4 mm19682 yearsnarml nodse20 mm206812 yearsjrped , v tumorpink10 mm21703 yearsnalsl ped nodse30 mm22715 yearsjrped , elastic , soft nod , ml , erosive surfr7 10 mm23722 yearsnalnodspink6 mm24741 yearjlwell - defpinkish13 mm257510 monthsjrsoft , ped , grany3 cm26751 yearpolyp v nod , pebbly surf y r25 mm2878nanawl , ppinkna29803 yearswlpr8 5 mmcr30826 monthswlped , soft , mlpinkish15 mm31835 yearsnanaped , soft , mlpink25 mm32135 v , wl[2 , 26 , 34 , 46 , 56 ] ao age onset , b black , c case , caul cauliflower - like , cr current report , fil filiform , g granular , j japanese , l location , m months , ml mulberry - like , mult multiple , na not available , nod(s ) nodule(s ) , occ occasional , p papules , ped pedunculated , poly polypoid , r red , ref reference , sl strawberry - like , surf surface , v verrucous , w with , wk weeks , wl wart like , y yellow , y year(s ) size is reported as given in the case reports . reported an additional scrotal case with no other information table 3verruciform xanthomas of the female genitaliacaseao ( year)durationracelocmorphologycolorsize a.c.ref.1 15 days17 yearsnar ing foldwd , v plaqueenachild2 11 yearnal lab minv plaquey7 5 cmnr356 yearscvulvav lesione , y6 6 cmchild4 8 nacolr lab majthick v lesionbrownnachild51217 yearsbvulvamult v , inv lesionsnananr615 childhoodnal groin , ext gensoft , exudatingpinknachild716912 mnal lab majgran , vw - tan1.5 cmna816 lifelongnal inguinal areav lesion w small nody - tan6 3 cmchild9 30nanal lab minpolyr2.5 cmnr38wl5.0 cm10 2220 yearswgenital mucosamult v indr0.32.5 cmnr1127 since childhoodwl vulva and perver hyperkerrnachild1243 nawclitorishyperker , caul , v , wl lesiongrayish white13 mmls13448 monthsnananana13 10 mmna14 441 monthnal lab majscaly plaquepink34 mmnr1546several yearsjl lab majsoft , ped nod w new gran lesionbrown w y35 24 22 mmfep16482 yearsjvulvav surf , wdo rnasl , lc1751nanalab minv lesion , indy o2 mmvpd2884nanal vulvav lesionnananr29 nananananananana ac associated condition , ao age of onset , b black , c chinese , caul cauliflower - like , child congenital hemidysplasia with ichthyosiform nevus and limb defects , col columbian , demac demarcated , dur duration , ens epidermal nevus syndrome , ext gen external genitalia , fep fibroepithelial polyp , gran granular , ind indurated , ing inguinal , inv inverted , j japanese , ker keratotic , l left , loc location , lab maj labia majora , lab min labia minora , lc lymphangioma circumscriptum , lmm leiomyomatosis of uterine corpus , lp lichen planus , ls lichen sclerosus , m months , mult multiple , na not available , nod nodule , nr none reported , o orange , p papule , ped pedunculated , per perineum , poly polypoid , r red , rd radiodermatitis , ref reference , sl severe lymphedema , v verrucous , vpd vulvar paget disease , w with , wd well - demarcated , wl wart - like , y years the age of onset was not given size is reported as given in the case reports . no other information was obtained verruciform xanthomas of the penis ao age of onset , b black , c chinese , caul cauliflower - like , def defined , e erythematous , f filiform , foc ulc focally ulcerated , ib irregular boundaries , irreg irregular , i indian , ind indurated , g & v globules and small - dotted vessels , ker keratotic , m months , na not available , ni non - indurated , p(s ) papule(s ) , pn pedunculated , r red , ref reference , sev several , surf surface , v verrucous , w white , wks weeks , wl wart - like , y yellow , y year size is reported as given in the case reports . the lesion was initially misdiagnosed as a squamous cell carcinoma , but due to the histological features of the lesion , their department determined it was actually a verruciform xanthoma the patient s original lesion was treated with circumcision ; however , its removal from the coronary sulcus was suspected to be incomplete because of severe adhesion present between the prepuce and the glans penis . no other information was obtained verruciform xanthomas of the scrotum ao age onset , b black , c case , caul cauliflower - like , cr current report , fil filiform , g granular , j japanese , l location , m months , ml mulberry - like , mult multiple , na not available , nod(s ) nodule(s ) , occ occasional , p papules , ped pedunculated , poly polypoid , r red , ref reference , sl strawberry - like , surf surface , v verrucous , w with , wk weeks , wl wart like , y yellow , y year(s ) size is reported as given in the case reports . reported an additional scrotal case with no other information verruciform xanthomas of the female genitalia ac associated condition , ao age of onset , b black , c chinese , caul cauliflower - like , child congenital hemidysplasia with ichthyosiform nevus and limb defects , col columbian , demac demarcated , dur duration , ens epidermal nevus syndrome , ext gen external genitalia , fep fibroepithelial polyp , gran granular , ind indurated , ing inguinal , inv inverted , j japanese , ker keratotic , l left , loc location , lab maj labia majora , lab min labia minora , lc lymphangioma circumscriptum , lmm leiomyomatosis of uterine corpus , lp lichen planus , ls lichen sclerosus , m months , mult multiple , na not available , nod nodule , nr none reported , o orange , p papule , ped pedunculated , per perineum , poly polypoid , r red , rd radiodermatitis , ref reference , sl severe lymphedema , v verrucous , vpd vulvar paget disease , w with , wd well - demarcated , wl wart - like , y years the age of onset was not given size is reported as given in the case reports . no other information was obtained verruciform xanthoma is a wart - like lesion that most commonly occurs in the oral mucosa . the most common extraoral verruciform xanthomas are reported in the genital region ; however , other reported extra - oral locations include the anal region , ear , forearm , foot , hand , leg , nose , and sacrum [ 6571 , 74 ] . did a profile of 282 oral verruciform xanthomas they found a slight male predominance below the age of 50 , with a male : female ratio of 1.6:1 , and a slight female predominance above the age of 50 , with a male : female ratio of 0.8:1 ; 73.4% of the oral verruciform xanthomas were on the masticatory mucosa . a snip excision was performed for biopsy and removal of the lesion the histologic differential diagnosis of verruciform xanthoma includes condyloma accuminatum , granular cell epulis , granular cell tumor , verruca vulgaris , and verrucous carcinoma . these features include acanthotic epidermis with parakeratosis that extends deep into the epithelium , uniformly elongated rete ridges , neutrophilic infiltrate in the dermis , and foamy histiocytes throughout the dermal papillae ( fig . correlation of the clinical features and the pathologic changes establish a diagnosis of verruciform xanthoma . this is supported by multiple cases of verruciform xanthomas reported in association with lichen planus [ 43 , 85 ] . the first verruciform xanthoma of the penis was reported in 1981 by kraemer et al . verruciform xanthomas of the penis have been reported following necrotizing fasciitis of the anogenital region , radical removal of initial verruciform xanthoma with grafting of the foreskin , and transurethral prostate resection . vulvar verruciform xanthomas have been described in a variety of patients , including african american , chinese , caucasian , columbian , and japanese women . verruciform xanthoma is a wart - like lesion that most commonly occurs in the oral mucosa . the most common extraoral verruciform xanthomas are reported in the genital region ; however , other reported extra - oral locations include the anal region , ear , forearm , foot , hand , leg , nose , and sacrum [ 6571 , 74 ] . did a profile of 282 oral verruciform xanthomas they found a slight male predominance below the age of 50 , with a male : female ratio of 1.6:1 , and a slight female predominance above the age of 50 , with a male : female ratio of 0.8:1 ; 73.4% of the oral verruciform xanthomas were on the masticatory mucosa . a snip excision was performed for biopsy and removal of the lesion the histologic differential diagnosis of verruciform xanthoma includes condyloma accuminatum , granular cell epulis , granular cell tumor , verruca vulgaris , and verrucous carcinoma . these features include acanthotic epidermis with parakeratosis that extends deep into the epithelium , uniformly elongated rete ridges , neutrophilic infiltrate in the dermis , and foamy histiocytes throughout the dermal papillae ( fig . this is supported by multiple cases of verruciform xanthomas reported in association with lichen planus [ 43 , 85 ] . verruciform xanthomas of the penis have been reported following necrotizing fasciitis of the anogenital region , radical removal of initial verruciform xanthoma with grafting of the foreskin , and transurethral prostate resection . the first reported extraoral lesions were two cases of verruciform xanthoma of the vulva , described by santa cruz and martin in 1979 . vulvar verruciform xanthomas have been described in a variety of patients , including african american , chinese , caucasian , columbian , and japanese women . verruciform xanthomas are benign , asymptomatic wart - like lesions most commonly found in the oral cavity . the defining pathologic features include hyperkeratosis with parakeratosis , acanthosis , elongated rete ridges , neutrophilic inflammation in the dermis , and foam cells in the dermal papillae . there have been 194 cases of verruciform genital - associated ( vegas ) xanthomas reported in the literature . verruciform xanthomas of the genitalia share similar histologic characteristics with verruciform xanthomas of the oral cavity . the lesions have been reported in association with a variety of cutaneous diseases and systemic conditions .

jarid2 ( jumonji , at - rich interactive domain 2 ) is the founding member of the jumonji family of proteins that can demethylate histones , although jarid2 itself is unable to do so ( klose et al . , 2006 ; landeira and fisher , 2011 ) . jarid2 was discovered in 1995 as a regulator of neural development in a gene trap screen in mice ( takeuchi et al . , 1995 ) , and jarid2 deficiency was later shown to result in a range of phenotypes with variable severity and onset with defects in heart and neural tube formation and hypoplasia of liver , spleen , and blood tissues ( jung et al . , 2005 ; landeira and fisher , 2011 ; takeuchi et al . , 2006 ) . in humans , mutations in jarid2 have been linked to congenital defects including nonsyndromic cleft lip , spina bifida , and congenital heart abnormalities ( scapoli et al . , 2010 ; 2004 ) , and haploinsufficiency is linked to intellectual disability and brain dysfunction including schizophrenia ( bary et al . , 2013 ; celestino - soper et al . , 2012 ; , jarid2 is known to inhibit myogenic differentiation in mouse and human cells ( walters et al . , 2014 ) , is important for the scheduled proliferation of epidermal stem and progenitor cells ( mejetta et al . , 2011 ) , and is thought to input into cell - cycle control by regulating cyclin d1 ( nakajima et al . , 2011 ; toyoda et al . , , jarid2 is particularly abundant and has been implicated as a hub component of transcriptional networks that underpin pluripotency ( assou et al . , 2009 ; kim et al . , 2008 ; loh et al . , 2006 ; sun et al . , 2008 ; zhou et al . , 2007 ) . escs lacking jarid2 remain viable and pluripotent but are unable to efficiently differentiate in culture and show alterations in growth kinetics and in colony morphology ( landeira et al . , 2010 ; li et al . , 2010 ; pasini et al . , 2010 ; shen et al . , 2009 ) . jarid2 is a critical component of the polycomb repressor complex 2 ( prc2 ) , implicated in guiding the deposition of h3k27me3 across the genome ( landeira et al . , 2010 ; li et al . , 2010 ; , 2010 ; peng et al . , 2009 ; shen et al . , 2009 ) . in escs , jarid2 binds to a similar set of genomic sites as core prc2 components , such as embryonic ectoderm development ( eed ) , suppressor of zeste 12 ( suz12 ) , and enhancer of zeste 2 ( ezh2 ) ( landeira et al . , 2010 ; , 2010 ; peng et al . , 2009 ; shen et al . , 2009 ) . in drosophila , jarid2 also binds to sites that broadly overlap with other core prc2 members , albeit at slightly different levels and with some exceptions ( herz et al . , 2012 ) . although these results support a role for jarid2 in prc2 recruitment , in concert with co - factors such as aebp2 , pcl1 , 2 , 3 , and esprc2p48 ( hunkapiller et al . , 2012 ; kalb et al . , 2014 ; kim et al . , 2008 ; walker et al . , 2010 ; zhang et al . , 2011 ) , the consequence of jarid2 loss for h3k27me3 levels is unclear , as both increased ( peng et al . , 2009 ; shen et al . , 2009 ) and decreased h3k27methylation have been reported ( landeira et al . , 2010 ; li et al . , 2010 ; in addition , recent studies of x - inactivation have shown that , while jarid2 is necessary for efficient prc2 recruitment , jarid2 binds to xist rna - associated chromatin domains independently of prc2 ( da rocha et al . , 2014 ) . jarid2 also binds to other non - coding rnas in escs ( kaneko et al . , 2014 ) and has been shown to bind nucleosomes directly ( son et al . these findings suggest that jarid2 may have multiple functions in pluripotent escs and could act as an intermediate in the chromatin events that occur as pluripotent cells move toward differentiation ( da rocha et al . , 2014 ) . consistent with this view , jarid2-null escs grow and self - renew robustly in culture but are severely compromised in their capacity to generate mesoderm , endoderm , or ectoderm lineages in vitro ( landeira et al . , 2010 ; jarid2-null mouse embryos develop normally until e10.5 or beyond ( jung et al . , 2005 ; takeuchi et al . , 2006 ) , suggesting that jarid2-null escs may be capable of differentiation but fail to do so in standard in vitro cultures . here , we examined the properties of jarid2-null escs derived from different sources , as well as the factors that were consistently de - regulated in these cells . our results show that jarid2-null escs express constitutively high levels of nanog proteins , indicative of a naive pluripotent state , together with dramatically reduced planar cell polarity ( pcp ) and wnt signaling components . in particular , expression of prickle1 , fzd2 , wnt9a , e - cadherin , and -catenin activity were all reduced in jarid2-null escs , while wnt antagonists such as sfrp1 were upregulated . remarkably , this inverse correlation between pcp / wnt signaling and nanog expression was replicated in wild - type escs depleted of prickle1/fzd2/wnt9a , or engineered to overexpress nanog . co - culture of jarid2-null escs with wild - type partners allowed the mutant cells to regain variable nanog expression and -catenin activity and restored their capability for differentiation . surprisingly , we showed that disruption of pcp / wnt signaling early in mouse embryogenesis , by introducing jarid2-null escs ( but not eed - null escs ) , resulted in multiple icm formation . this emphasizes the importance of the jarid2-nanog - pcp / wnt core circuit for normal development and revealed a non - canonical ( prc2-independent ) role for jarid2 in coordinating this process . pluripotency factor expression by escs has been generally assumed to be unaffected by deletion of prc2 ( azuara et al . , 2006 ; boyer et al . , 2006 ; lee et al . , 2006 ; consistent with this , jarid2-null escs ( landeira et al . , 2010 ) expressed substantial levels of each of the core pluripotency factors oct4 , sox2 , and nanog as indicated by western blotting analysis ( figure 1a , clones e4 and e8 ) . surprisingly , however , we detected an inverse correlation between jarid2 gene dose and nanog expression [ jm8(wild - type ) < a08 ( ) < e4 ( ) and e8 ( ) , figure 1a ] . at the single - cell level , immuno - labeling for nanog followed by fluorescence microscopy ( figure 1b ) or flow cytometry analysis ( figure 1c ) revealed that the increased expression of nanog by jarid2-null escs was due to a reduced proportion of nanog - low cells . specifically , jarid2-null escs appeared homogenously nanog - high ( figure 1b , lower panel shows nanog labeling of virtually all oct4 cells ) , whereas nanog - high and -low cells were easily discerned among parental wild - type jm8 escs ( upper panel ) . this difference was confirmed by flow cytometry ( compare green and gray traces , figure 1c ) where mutant e8 cells showed a reduced proportion of nanog - low cells . to verify that altered nanog expression was due to jarid2 depletion rather than simply clonal variation between different esc lines , we examined the properties jarid2 mutant cells that had been independently generated by others ( shen et al . , 2009 ) . these jarid2-null ( ) escs also expressed constitutively high levels of nanog protein detected by western blotting ( figure 1d ) , fluorescence - activated cell sorting ( facs ) analysis ( figure 1e , 82% nanog - high ) , and immunofluorescence microscopy ( figure 1f ) ( lower - right panel ) , as compared to matched wild - type controls ( jarid2 fl / fl ) . in addition , we noticed that , among cells plated at low density , jarid2-null escs failed to properly self - organize into coherent colonies appearing as either spread ( such as e8 ) or disorganized overlapping layers of nanog - high cells ( white arrows ) . to quantify such defects , we assessed the nuclear shape and dimensions of four to eight cell colonies that arose after plating e8 and jm8 escs at limiting dilution ( figure s1a ) . this unbiased comparison confirmed a profound difference in the nuclear height / width ratios of cells occupying central and peripheral positions within jarid2-null colonies ( figures s1b and s1c ) . taken together , these data suggest that jarid2 has a role in regulating both nanog levels and cell - cell interactions in escs . prior studies of escs lacking prc1 ( ring1b ) or prc2 ( eed ) components have shown similar levels of nanog and oct4 as matched controls ( eskeland et al . , 2010 ; leeb et al . , 2010 ) suggesting that the effect of jarid2 depletion on nanog expression might not be mediated by prc2 . furthermore , as jarid2 does not directly bind to the nanog promoter in mouse escs ( pasini et al . , 2010 ) , we investigated whether other repressors might be responsible for regulating nanog expression differentially between jarid2-null and wild - type escs . chromatin immunoprecipitation ( chip ) analysis revealed that , although both cell lines expressed similar levels of tcf3 ( figure s1d ) , wild - type escs showed a significant enrichment for tcf3 bound to a consensus site in the nanog promoter , as compared to jarid2-null samples ( figure s1e ) . this result was consistent with tcf3 mediating the repression of nanog in escs ( cole et al . , 2008 ) and suggests that jarid2 may modulate tcf3 activity . tcf3 is a critical component of canonical wnt signaling pathways that is implicated in regulating the balance between stem cell self - renewal and differentiation . mouse escs maintained in media supplemented with inhibitors of glycogen synthase kinase 3 and fgf - mapk ( 2i ) have elevated nanog levels ( wray et al . , 2010 ; ying et al . , 2008 ) and , like jarid2-null escs , are less prone to differentiation . to discover genes downstream of jarid2 , we examined publically available gene - expression microarray data for jarid2 escs ( shen et al . , 2009 ) and found that among the 413 genes misregulated relative to controls ( fold change > 2 , p value < 0.05 ) many have roles in cell adhesion , the cytoskeleton , and extracellular matrix or are implicated in development ( figure s2a ) . we examined published chip sequencing ( chip - seq ) data for jarid2 binding in undifferentiated escs ( pasini et al . , 2010 ) to uncover which genes might be directly regulated by jarid2 . among the 1,146 genic promoters enriched for jarid2 binding , we noticed a strong preference for genes critical in wnt signaling ( 29 genes ) and wnt - related pathways ( figure s2b ) . to explore this further , gene - expression profiling was performed to formally determine whether genes involved in wnt signaling pathways were substantially deregulated in escs lacking jarid2 . for this , esc clones that lacked jarid2 ( e8 and jarid2 cells ) were compared with wild - type escs ( jm8 and jarid2 fl / fl ) , and mrna expression of wnt signaling components was quantified by qpcr using commercially available pcr plates . among the candidates showing significant de - regulation in escs lacking jarid2 ( p < 0.05 ) were prickle1 , fzd2 , wisp1 , wnt9a , and fosl1 ( downregulated , figure 2a , left ) and aes , dkk1 , sfrp1 , and wnt11 ( upregulated , figure 2a , right ) . these candidates included genes with binding sites for jarid2 within their promoters ( prickle , fzd2 , fosl1 , wnt9a , wnt11 , dkk1 , and sfrp1 , figure s2c ) as reported previously ( peng et al . , 2009 ) . careful pairwise comparison of gene - expression levels relative to matched parental controls verified a significant up- or downregulation of these genes in jarid2-null escs ( figure 2b , where black bars show wild - type and white bars indicate jarid2 nulls ) . differences between the two sources of jarid2-deficient escs were apparent only for dkk1 , where expression appears particularly sensitive to cell density ( data not shown ) . fzd2 and prickle1 are both well - established core components of the non - canonical wnt , or pcp pathway in mammals , wnt11 is a ligand associated with pcp ( gray et al . , 2011 ) , and dkk1 and sfrp1 are secreted wnt inhibitors involved in pcp signaling in vertebrates ( caneparo et al . 2008 ) ( as depicted schematically in figure 2c ) . to check whether jarid2-null escs have defects in planar cell polarity , we examined the distribution of prickle1 and vangl1 proteins ( green ) in wild - type and jarid2-null escs using immunofluorescence with previously characterized antisera ( figure 2d ) . both proteins were abundant within the cytoplasm and nucleus of wild - type escs ( upper panels ) , whereas in jarid2 nulls ( e8 ) prickle1 and vangl1 levels were reduced in nuclei and there was a paucity of both proteins in the cytoplasm . this difference was confirmed using quantitative analysis of labeling intensity ( figure s2d ) that showed a significant decrease ( 5- to 10-fold ) in prickle1 and vangl1 in the cytoplasm of jarid2-null escs . e - cadherin labeling was also low in jarid2-null escs and appeared patchy ( figure 2d , green lower right ) as compared with contiguous labeling of cell boundaries in colonies of wild - type escs ( figure 2d , green upper right ) . these data highlight a deficiency of pcp proteins in jarid2-null escs . to extend this analysis , we generated three additional jarid2-null esc lines , using crispr / cas9 to engineer mutations into the third exon of jarid2 ( figures s2e clones were selected that showed frameshift mutation to both endogenous jarid2 alleles ( figure s2 g ) , and western blots confirmed the absence of detectable jarid2 proteins ( figure 2e ) . rt - pcr analysis showed that prickle1 , fzd2 , and wnt9a expression was significantly reduced in each mutant clone relative to parental escs ( figure 2f ) , and flow cytometry analysis ( figure 2 g ) indicated a characteristic switch to constitutive nanog - high expression ( green versus gray trace ) as illustrated for a jarid2#3 mutant cells . this extends previous observations made with established jarid2-null lines , indicating that deregulated pcp and nanog expression was likely to be a direct consequence of jarid2 removal rather secondary effects or adaption to culture . using a similar crispr / cas9 approach , we also generated esc lines lacking prickle1 , fzd2 , and wnt9a ( figures s2e six clones were identified with frameshift mutations in one or both alleles of each of these genes , and each mutant cell line showed a switch to constitutive nanog - high expression and defects in colony formation ( spread or layered this is exemplified for pcp#5 ( figures 2h2j ) , a clone with frameshift mutations in both endogenous prickle1 and fzd2 alleles and a single wnt9a allele ( figure s2 g ) . this cell line showed dramatically reduced expression of all three genes ( figure 2h ) , expressed nanog constitutively ( figure 2i , green ) ( figure 2j , green trace ) , and showed aberrant clonal morphology ( figure 2i , right , arrows ) . taken together , these data showed a hitherto - unrecognized role for jarid2 in regulating non - canonical wnt signaling and nanog expression in undifferentiated escs . although jarid2 binds to the promoters of prickle1 , fzd2 , wnt9a , wnt11 , dkk1 , and sfrp1 ( pasini et al . , 2010 ) ( figure s2c ) , chip analysis revealed similar h3k27me3 levels at these targets in jarid2-wild - type ( jm8 ) , heterozygous ( a08 ) , and null esc lines ( e4 , e8 ) ( figure s3a ) . this observation , together with the fact that prickle1 , fzd2 , and wnt9a expression apparently requires jarid2 , suggests that jarid2 may regulate pcp / wnt signaling in escs in a way that is independent of its canonical role as a component of prc2 . a comparison of the replication timing of a panel of genes involved in pcp / wnt signaling in wild - type ( black bars ) and jarid2-null escs ( white bars , figure s3b ) also showed similar temporal profiles , suggesting that the chromatin structure of these genes was not radically different in the absence of jarid2 . we examined whether canonical wnt signaling ( -catenin mediated ) was also impaired in escs lacking jarid2 . as shown in figure 3a , western blots revealed broadly similar amounts of -catenin in whole - cell extracts derived from jarid2-null ( e8 ) , heterozygous ( a08 ) , and wild - type escs ( jm8 ) . however , using antibody specific for active forms of -catenin ( figure 3b ) , we observed reduced levels of active -catenin in escs lacking jarid2 ( e8 , jarid2 ) compared to wild - type cells ( jm8 , jarid2fl / fl ) . this reduction was confirmed in functional assays using topflash ( luciferase - based ) reporters . -catenin activity was significantly reduced in both established ( e8 and jarid2 ) and newly generated ( jarid2#3 ) jarid2-null escs ( figure 3c , white bars ) compared with controls ( black bars ) , and also in prickle1/fzd2/wnt9a depleted escs ( pcp#5 , gray bar ) . since each of these mutant escs also expressed nanog constitutively , we asked whether nanog expression per se was important for reduced -catenin activity . escs that stably express elevated levels of nanog ( figure 3d , nanog white bars ) were generated as described previously ( theunissen et al . , 2011 ) . these cells showed significant reductions in -catenin activity and reduced expression of fzd2 , prickle1 , and wnt9a ( figures 3e and 3f ) , providing compelling evidence of a robust regulatory circuit between nanog , pcp and -catenin activity that is sensitive to jarid2 ( illustrated in figure 3 g ) . operationally , this balance between nanog and wnt signaling was also evident in escs expressing wild - type levels of nanog ( chambers et al . , 2007 ) , where nanog expression and -catenin activity were inversely correlated ( figure 3h ) . colonies that respond poorly to differentiation cues ( landeira et al . , 2010 ) . to better understand whether defective wnt signaling in jarid2-null escs might underlie their inability to form coherent differentiation - responsive colonies , we initiated a series of cell mixing / sorting experiments in which jarid2-null e8 cells were co - cultured with wild - type or jarid2 heterozygous partners . e8 cells express gfp ( landeira et al . , 2010 ) allowing these cells to be easily tracked in co - culture . escs were mixed in a 1:1 ratio , plated on gelatin - coated plates , and analyzed 1624 hr after mixing ( figure 4a ) . jarid2-null escs were unable to form tight colonies when plated alone or with heterozygous ( a08 ) partners ( top , green ) , but this capacity was restored when mixed with ( unlabeled ) wild - type escs ( figure 4a , lower image ) . co - culture of e8 cells with wild - type escs also resulted in preferential sorting of mutant cells to the periphery of colonies ( figure 4a , histograms right ) , similar to the peripheral sorting of nanog - high cells seen in wild - type jm8 colonies ( figure 1f ) . although co - culture of e8 and wild - type escs increased overall e - cadherin expression in mixed colonies ( figure s4a , red ) , a close examination revealed that e - cadherin expression by mutant cells remained low in cultures at 16 hr ( figure s4b ) . these data showed that jarid2-null escs have intrinsic defects in canonical and non - canonical wnt signaling , likely to contribute to aberrant cell - cell interactions . remarkably , co - culture of jarid2-null ( gfp ) escs with wild - type partners resulted in a switch from constitutive nanog - high expression ( figure 4b , e8 left ) to a profile that was indistinguishable from wild - type escs ( figure 4b , e8 right ) . exposure of jarid2-null escs to wild - type escs also restored -catenin activity in the mutant cells ( figure 4c ) and improved their capacity to differentiate ( figures 4d and 4e ) . following co - culture and leukemia - inhibitory factor ( lif ) withdrawal , jarid2-null e8-derived ( gfp ) cells expressing neural - associated markers such as nestin and mash1 ( red ) were readily identified within mixed cultures ( 47% of gfp cells expressed nestin ) . neural induction was less efficient than in control wild - type cultures ( > 80% nestin positive by day 10 , figure s4c ) , and fewer cells were recovered overall ( 5%10% ) . co - culture of e8 cells with jarid2-null escs , fibroblasts , t or b cells , or with conditioned media derived from wild - type escs did not rescue the capability of e8 cells to differentiate ( data not shown ) or enhance -catenin activity in these cells ( as shown in figure s4d ) . because -catenin activity is reportedly required for esc differentiation ( atlasi et al . , 2013 ; lyashenko et al . , 2011 ) , jarid2-null escs may be unable to execute differentiation efficiently because of low -catenin activity . in this scenario , co - culture with wild - type escs restores -catenin activity and may enable mutant escs to differentiate ( figure s4e shows a hypothetical scheme ) . to investigate whether jarid2 regulation of nanog pcp / wnt signaling is likely to be important in vivo as well as in vitro , we examined the distribution of prickle1 in the pre - implantation mouse embryo . depletion of pcp components , including prickle1 , has been reported to lead to defects in cell adhesion that affect the development of early mouse embryos ( larue et al . , 1994 ; na et al . 2009 ) , and previous studies have shown that although prickle1 is detected at e5.5 and earlier ( tao et al . , 2009 , 2012 ) , canonical wnt signaling is operational slightly later in development ( na et al . , 2007 ) . we examined mouse embryos from e3 to e4.5 for prickle1 expression using dapi as a counterstain ( blue ) to assess cell number . immuno - labeling with antibodies to nanog ( red ) , oct4 ( data not shown ) , or gata6 ( red ) was used to assess the stage and orientation of the developing inner cell mass ( icm ) ( figures 5a and 5b ) . as illustrated in figure 5a , prickle1 ( green ) was abundantly detected in 32- and 64-cell embryos and showed a prominent cytoplasmic distribution in nanog positive cells destined to form the epiblast ( region outlined in white , figure 5a ) . by e4.5 , nanog - high cells were condensed to the pole of the embryo where they expressed high levels of prickle1 distributed equivalently between nuclei and cytoplasm . the surrounding gata6-expressing cells ( red ) also expressed high levels of prickle1 ( green ) , but in this case expression was predominantly nuclear ( figure 5b , region highlighted in white ) . an exception was of a small group of two to six mural trophectoderm cells , in which prickle1 expression was high and located in nuclei and particularly within nucleoli ( summarized in the schematic diagram shown in figure 5c ) . the selective distribution of cytoplasmic prickle1 in cells of the epiblast versus primitive endoderm or trophectoderm was confirmed in optical sections of e4.5 embryos as shown in figure s5a ( green labeling and traces ) . e - cadherin ( red , figure 5d ) was prominent throughout the blastocyst at these stages . escs that lack jarid2 expressed high levels of nanog and low levels of prickle1 , vangl1 , and -catenin activity and show severely compromised differentiation ( landeira et al . , 2010 ) . as these defects were partially compensated by in vitro co - culture with wild - type escs , we asked to what extent mutant cells would contribute to the developing embryo . wild - type e3.5 blastocysts were injected with either 1015 gfpjarid2-null escs ( e8 ) or an identical number of gfp - labeled wild - type escs ( jm8 ) and then were cultured for 1620 hr before being examined . remarkably , we found that blastocysts injected with jarid2-null escs routinely initiated the formation of more than a single icm ( 16/39 , illustrated in figure 5e , lower panels ) , a feature that was not detected in any of the controls injected with wild - type escs ( 0/90 , upper panels ) . importantly , although these secondary icms appeared to be focused around small clusters of jarid2-null escs ( marked in green ) , in which higher levels of nanog might be anticipated , gata6 expression ( red ) was induced in the underlying wild - type cells ( figure 5f ) . this suggested that injection the mutant escs was sufficient to instruct neighboring wild - type blastomeres to upregulate gata6 and thus contribute to the formation of multiple icms . to distinguish whether multiple icm formation was due to defective prc2 function , or defective nanog , pcp , and wnt circuitry , we injected e3.5 blastocysts with escs lacking prickle1/fzd2/wnt9a , overexpressing nanog , or that lacked eed ( azuara et al . , 2006 ) a core component of the prc2 complex . as shown in table 1 , injection of wild - type ( e14 ) and eed - deficient ( b1.3 ) escs generated only a single icm in multiple experiments , whereas blastocysts injected with equivalent numbers of jarid2-null ( jarid2#3 ) , prickle1/fzd2/wnt9a - depleted ( pcp#5 ) , or nanog - overexpressing escs formed multiple icms in approximately 35%48% of samples . first , we show that jarid2 , a polycomb group member implicated in prc2 recruitment , chromatin structure , and gene repression , acts as a positive regulator of pcp / wnt signaling pathways in escs . absence of jarid2 or depletion of prickle1/fzd2/wnt9a results in constitutive nanog expression by esc , which , in turn , results in reduced levels of active -catenin . these observations identify an important jarid2-sensitive core circuit in escs that regulates pluripotency and differentiation . in previous studies , jarid2-null escs were shown to have a profoundly compromised ability to differentiate in culture ( landeira et al . , 2010 ; li et al . , 2010 ; pasini et al . , 2010 ; peng et al . , 2009 ; shen et al . , 2009 ) . here , we show that canonical and non - canonical wnt signaling is reduced in jarid2-null escs , and these cells are unable to establish coherent colonies with each other . introduction of wild - type escs supports the formation of colonies and increases -catenin activity within jarid2-null escs , allowing them to properly execute differentiation programs in response to appropriate cues , although the nature of the support offered by wild - type escs is at present unclear . these results are perhaps surprising in view of reports implicating jarid2 in the regulation of notch1 ( mysliwiec et al . , 2011 ) , or showing that nanog represses the expression of dkk1 ( a secreted wnt inhibitor ) in escs to enhance -catenin expression ( marucci et al . , 2014 ) . current literature contains many conflicting reports on whether -catenin is essential or dispensable for maintaining esc pluripotency ( okumura et al . , 2013 ; soncin et al . , 2009 ; ten berge et al . , 2011 ; wray et al . , 2011 this discrepancy probably reflects the fact that -catenin is pleotropic and regulates both gene transcription and cell adhesion ( reviewed in brembeck et al . , 2006 and heuberger and birchmeier , 2010 ) . in addition , the requirement for and effects of -catenin appear to be context dependent ( lyashenko et al . , 2011 ; okumura et al . , 2013 ; wray et al . , 2011 ; yi et al . , 2008 ) and are reported to differ between naive and primed escs ( kurek et al . , our data show that jarid2-null escs maintained in lif and serum have low -catenin activity despite unchanged levels of total protein . co - culture of mutant cells both restored -catenin activity and allowed them to differentiate , consistent with several reports showing that nuclear -catenin is required to execute differentiation in vitro ( atlasi et al . previous studies have shown that non - canonical wnt signaling is present in mouse blastocyst before implantation ( tao et al . , 2009 , 2012 ) . early blastomeres express cytoplasmic dishevelled ( dvl ) proteins but may not activate canonical wnt signaling as judged by sensitive bat - gal wnt reporters ( na et al . , 2007 ) . overexpression of dvl , a downstream target of prickle1 , vangl , and frizzled ( fz ) ( gmez - orte et al . , 2013 ; 2011 ) , has been shown to dramatically alter the morphology and adhesion properties of 4-cell stage mouse embryos , resulting in a lack of coherence in the resulting blastocyst ( na et al . , 2007 ) . likewise , deletion of prickle1 in mouse embryos results in local disorganization of epiblast tissue ( tao et al . , 2009 ) and early embryonic death ( e5.5e6.6 ) , while lower levels of wnt9a promotes cell proliferation ( xiang et al . , 2008 we show that pcp expression is reduced in jarid2-null escs , and their introduction into e3.5 blastocysts induces the formation of multiple icms in around half of the embryos . canonical wnt signaling is thought to begin at implantation in the mouse ( na et al . , 2007 ) , and it is tempting to speculate that the group of cells in the mural trophectoderm identified by high levels of prickle1 in nucleoli may be cells destined to mediate implantation into the uterus . whatever the explanation , our demonstration that wild - type cells can rescue the differentiation of jarid2-null escs in vitro may provide important insights for understanding the conflicting differentiation status of jarid2 cells in vivo and in vitro . in homogenous clonally derived cultures , mutant cells are unable to establish bona fide cell - cell interactions or enhance -catenin activity , whereas in heterogenous cultures ( with wild - type esc partners ) or in vivo ( with other blastocyst - derived cells ) -catenin activity and pcp / wnt signaling may be partially compensated . our discovery that jarid2 is a regulator of pcp / wnt is also important for re - evaluating some of the emerging evidence that jarid2 mutations ( or snps ) are risk factors for several human diseases . genetic studies have for example linked jarid2 with nonsyndromic cleft lip ( scapoli et al . , 2010 ) . in mice , jarid2 is highly expressed in epithelial cells and in the merging palatal shelves . in this context , as well as in congenital heart defects where jarid2 mutations have also been reported ( volcik et al . , 2004 ) , the potential for jarid2 mutations to de - regulate pcp / wnt signaling might be very informative for understanding the molecular basis of these malformations and could potentially offer different opportunities for intervention . in the case of cancer , jarid2 mutations have been linked to metastases at diagnosis in soft - tissue sarcoma ( walters et al . , 2014 ) , to non - small cell lung carcinoma ( manceau et al . , 2013 ) , t - all , and to myeloproliferative disease ( saunthararajah and maciejewski , 2012 ) . although it is possible that jarid2 has an impact on these diseases based on its canonical role in prc2-mediated chromatin modulation , it is also possible that jarid2 is more directly involved in metastatic progression through its potential impact on cell sorting , cellular adhesion , and pcp / wnt signaling . thus , in addition to influencing prc2 recruitment and h3k27 hmtase activity in escs , we have shown that jarid2 is necessary to maintain a balance between nanog expression and pcp / wnt/-catenin in escs that is essential to enable them to properly respond to differentiation cues . regulation of this core circuit is also critical for normal pre - implantation development , since it appears to enable clusters of developing blastocysts to be discriminated and form a single inner cell mass . the discovery that jarid2 regulates pcp / wnt signaling in addition to its canonical role in prc2 highlights an important intersection between cell signaling and chromatin - based regulation , relevant for understanding the interplay between pluripotency and differentiation . esc lines were grown using standard conditions on 0.1% gelatin - coated dishes in the presence of lif and 10% fetal calf serum . neural differentiation was carried out as described previously ( conti et al . , 2005 ) . analysis of wnt signaling pathway genes was performed using sybr green pcr array rt profiler ( sabioscience ) . gene - expression analysis by rt - qpcr using sybr green ( qiagen ) was performed as previously described ( landeira et al . , 2010 ) . western blots were carried out using the following antibodies : mouse antisera to total -catenin ( bd biosciences ) and active -catenin ( millipore ) , rabbit antisera to nanog ( cosmo bio ) , jarid2 ( abcam ) , and goat antisera to oct4 ( santa cruz biotechnology ) , sox2 ( santa cruz ) , tcf3 ( santa cruz ) , and lamin b ( santa cruz ) . immunofluorescence analyses of esc colonies and mouse blastocysts were carried out using the following primary antibodies : mouse antibodies against oct4 ( bd ) , e - cadherin ( bd ) , mash1 ( bd ) ; rabbit antisera against nanog ( cosmo bio ) , vangl1 ( sigma ) , and prickle1 ( gift from a.g . bassuk ) ( bassuk et al . , 2008 ) ; and goat antisera against gata6 ( r&d systems ) and nestin ( santa cruz ) . flow cytometry analysis of nanog expression was carried out as previously described ( festuccia and chambers , 2011 ) with the following modifications : 2 10e cells were stained using rabbit antisera against nanog ( 2.5 g / ml , cosmo bio ) and anti - rabbit conjugated to alexa 647 ( 6 g / ml , molecular probes ) . 10 to 15 gfp - expressing escs were injected in the inner cell mass of c57bl/6 blastocysts at e3.5 . injected embryos were cultured for 16 hr in ksom media after which they were fixed for immunofluorescence analysis . single - guide rna sequences were cloned into human codon optimized spcas9 and chimeric guide rna expressing px330 plasmid ( cong et al . , 2013 ) followed by co - transfection into escs . cells ( 10 per well ) were transfected with 1 g topflash and 0.1 g renilla luciferase plasmids in 6-well plates using lipofectamine 2000 reagent ( life technologies ) and analyzed using the dual luciferase reporter assay system ( promega ) . 2 10 escs were crosslinked using 1% formaldehyde . tcf3 chip was carried out as described previously ( cole et al . , 2008 ) . chip was carried out as described previously ( landeira et al . , 2010 ) . animals used in the study were maintained and handled according to the guidelines of the imperial college animal welfare ethical review committee and the regulations set out by the british home office .

summaryjarid2 is part of the polycomb repressor complex 2 ( prc2 ) responsible for genome - wide h3k27me3 deposition . unlike other prc2-deficient embryonic stem cells ( escs ) , however , jarid2-deficient escs show a severe differentiation block , altered colony morphology , and distinctive patterns of deregulated gene expression . here , we show that jarid2/ escs express constitutively high levels of nanog but reduced pcp signaling components wnt9a , prickle1 , and fzd2 and lowered -catenin activity . depletion of wnt9a / prickle1/fzd2 from wild - type escs or overexpression of nanog largely phenocopies these cellular defects . co - culture of jarid2/ with wild - type escs restores variable nanog expression and -catenin activity and can partially rescue the differentiation block of mutant cells . in addition , we show that escs lacking jarid2 or wnt9a / prickle1/fzd2 or overexpressing nanog induce multiple icm formation when injected into normal e3.5 blastocysts . these data describe a previously unrecognized role for jarid2 in regulating a core pluripotency and wnt / pcp signaling circuit that is important for esc differentiation and for pre - implantation development .

Introduction Results Discussion Experimental Procedures Author Contributions

, 2014 ) , is important for the scheduled proliferation of epidermal stem and progenitor cells ( mejetta et al . jarid2 is a critical component of the polycomb repressor complex 2 ( prc2 ) , implicated in guiding the deposition of h3k27me3 across the genome ( landeira et al . in escs , jarid2 binds to a similar set of genomic sites as core prc2 components , such as embryonic ectoderm development ( eed ) , suppressor of zeste 12 ( suz12 ) , and enhancer of zeste 2 ( ezh2 ) ( landeira et al . although these results support a role for jarid2 in prc2 recruitment , in concert with co - factors such as aebp2 , pcl1 , 2 , 3 , and esprc2p48 ( hunkapiller et al . our results show that jarid2-null escs express constitutively high levels of nanog proteins , indicative of a naive pluripotent state , together with dramatically reduced planar cell polarity ( pcp ) and wnt signaling components . in particular , expression of prickle1 , fzd2 , wnt9a , e - cadherin , and -catenin activity were all reduced in jarid2-null escs , while wnt antagonists such as sfrp1 were upregulated . remarkably , this inverse correlation between pcp / wnt signaling and nanog expression was replicated in wild - type escs depleted of prickle1/fzd2/wnt9a , or engineered to overexpress nanog . co - culture of jarid2-null escs with wild - type partners allowed the mutant cells to regain variable nanog expression and -catenin activity and restored their capability for differentiation . surprisingly , we showed that disruption of pcp / wnt signaling early in mouse embryogenesis , by introducing jarid2-null escs ( but not eed - null escs ) , resulted in multiple icm formation . this emphasizes the importance of the jarid2-nanog - pcp / wnt core circuit for normal development and revealed a non - canonical ( prc2-independent ) role for jarid2 in coordinating this process . , 2010 ) expressed substantial levels of each of the core pluripotency factors oct4 , sox2 , and nanog as indicated by western blotting analysis ( figure 1a , clones e4 and e8 ) . surprisingly , however , we detected an inverse correlation between jarid2 gene dose and nanog expression [ jm8(wild - type ) < a08 ( ) < e4 ( ) and e8 ( ) , figure 1a ] . specifically , jarid2-null escs appeared homogenously nanog - high ( figure 1b , lower panel shows nanog labeling of virtually all oct4 cells ) , whereas nanog - high and -low cells were easily discerned among parental wild - type jm8 escs ( upper panel ) . to verify that altered nanog expression was due to jarid2 depletion rather than simply clonal variation between different esc lines , we examined the properties jarid2 mutant cells that had been independently generated by others ( shen et al . these jarid2-null ( ) escs also expressed constitutively high levels of nanog protein detected by western blotting ( figure 1d ) , fluorescence - activated cell sorting ( facs ) analysis ( figure 1e , 82% nanog - high ) , and immunofluorescence microscopy ( figure 1f ) ( lower - right panel ) , as compared to matched wild - type controls ( jarid2 fl / fl ) . in addition , we noticed that , among cells plated at low density , jarid2-null escs failed to properly self - organize into coherent colonies appearing as either spread ( such as e8 ) or disorganized overlapping layers of nanog - high cells ( white arrows ) . prior studies of escs lacking prc1 ( ring1b ) or prc2 ( eed ) components have shown similar levels of nanog and oct4 as matched controls ( eskeland et al . , 2010 ) , we investigated whether other repressors might be responsible for regulating nanog expression differentially between jarid2-null and wild - type escs . chromatin immunoprecipitation ( chip ) analysis revealed that , although both cell lines expressed similar levels of tcf3 ( figure s1d ) , wild - type escs showed a significant enrichment for tcf3 bound to a consensus site in the nanog promoter , as compared to jarid2-null samples ( figure s1e ) . among the 1,146 genic promoters enriched for jarid2 binding , we noticed a strong preference for genes critical in wnt signaling ( 29 genes ) and wnt - related pathways ( figure s2b ) . for this , esc clones that lacked jarid2 ( e8 and jarid2 cells ) were compared with wild - type escs ( jm8 and jarid2 fl / fl ) , and mrna expression of wnt signaling components was quantified by qpcr using commercially available pcr plates . among the candidates showing significant de - regulation in escs lacking jarid2 ( p < 0.05 ) were prickle1 , fzd2 , wisp1 , wnt9a , and fosl1 ( downregulated , figure 2a , left ) and aes , dkk1 , sfrp1 , and wnt11 ( upregulated , figure 2a , right ) . , 2011 ) , and dkk1 and sfrp1 are secreted wnt inhibitors involved in pcp signaling in vertebrates ( caneparo et al . to check whether jarid2-null escs have defects in planar cell polarity , we examined the distribution of prickle1 and vangl1 proteins ( green ) in wild - type and jarid2-null escs using immunofluorescence with previously characterized antisera ( figure 2d ) . both proteins were abundant within the cytoplasm and nucleus of wild - type escs ( upper panels ) , whereas in jarid2 nulls ( e8 ) prickle1 and vangl1 levels were reduced in nuclei and there was a paucity of both proteins in the cytoplasm . e - cadherin labeling was also low in jarid2-null escs and appeared patchy ( figure 2d , green lower right ) as compared with contiguous labeling of cell boundaries in colonies of wild - type escs ( figure 2d , green upper right ) . to extend this analysis , we generated three additional jarid2-null esc lines , using crispr / cas9 to engineer mutations into the third exon of jarid2 ( figures s2e clones were selected that showed frameshift mutation to both endogenous jarid2 alleles ( figure s2 g ) , and western blots confirmed the absence of detectable jarid2 proteins ( figure 2e ) . rt - pcr analysis showed that prickle1 , fzd2 , and wnt9a expression was significantly reduced in each mutant clone relative to parental escs ( figure 2f ) , and flow cytometry analysis ( figure 2 g ) indicated a characteristic switch to constitutive nanog - high expression ( green versus gray trace ) as illustrated for a jarid2#3 mutant cells . using a similar crispr / cas9 approach , we also generated esc lines lacking prickle1 , fzd2 , and wnt9a ( figures s2e six clones were identified with frameshift mutations in one or both alleles of each of these genes , and each mutant cell line showed a switch to constitutive nanog - high expression and defects in colony formation ( spread or layered this is exemplified for pcp#5 ( figures 2h2j ) , a clone with frameshift mutations in both endogenous prickle1 and fzd2 alleles and a single wnt9a allele ( figure s2 g ) . taken together , these data showed a hitherto - unrecognized role for jarid2 in regulating non - canonical wnt signaling and nanog expression in undifferentiated escs . although jarid2 binds to the promoters of prickle1 , fzd2 , wnt9a , wnt11 , dkk1 , and sfrp1 ( pasini et al . , 2010 ) ( figure s2c ) , chip analysis revealed similar h3k27me3 levels at these targets in jarid2-wild - type ( jm8 ) , heterozygous ( a08 ) , and null esc lines ( e4 , e8 ) ( figure s3a ) . this observation , together with the fact that prickle1 , fzd2 , and wnt9a expression apparently requires jarid2 , suggests that jarid2 may regulate pcp / wnt signaling in escs in a way that is independent of its canonical role as a component of prc2 . a comparison of the replication timing of a panel of genes involved in pcp / wnt signaling in wild - type ( black bars ) and jarid2-null escs ( white bars , figure s3b ) also showed similar temporal profiles , suggesting that the chromatin structure of these genes was not radically different in the absence of jarid2 . as shown in figure 3a , western blots revealed broadly similar amounts of -catenin in whole - cell extracts derived from jarid2-null ( e8 ) , heterozygous ( a08 ) , and wild - type escs ( jm8 ) . however , using antibody specific for active forms of -catenin ( figure 3b ) , we observed reduced levels of active -catenin in escs lacking jarid2 ( e8 , jarid2 ) compared to wild - type cells ( jm8 , jarid2fl / fl ) . -catenin activity was significantly reduced in both established ( e8 and jarid2 ) and newly generated ( jarid2#3 ) jarid2-null escs ( figure 3c , white bars ) compared with controls ( black bars ) , and also in prickle1/fzd2/wnt9a depleted escs ( pcp#5 , gray bar ) . since each of these mutant escs also expressed nanog constitutively , we asked whether nanog expression per se was important for reduced -catenin activity . these cells showed significant reductions in -catenin activity and reduced expression of fzd2 , prickle1 , and wnt9a ( figures 3e and 3f ) , providing compelling evidence of a robust regulatory circuit between nanog , pcp and -catenin activity that is sensitive to jarid2 ( illustrated in figure 3 g ) . operationally , this balance between nanog and wnt signaling was also evident in escs expressing wild - type levels of nanog ( chambers et al . , 2007 ) , where nanog expression and -catenin activity were inversely correlated ( figure 3h ) . to better understand whether defective wnt signaling in jarid2-null escs might underlie their inability to form coherent differentiation - responsive colonies , we initiated a series of cell mixing / sorting experiments in which jarid2-null e8 cells were co - cultured with wild - type or jarid2 heterozygous partners . jarid2-null escs were unable to form tight colonies when plated alone or with heterozygous ( a08 ) partners ( top , green ) , but this capacity was restored when mixed with ( unlabeled ) wild - type escs ( figure 4a , lower image ) . co - culture of e8 cells with wild - type escs also resulted in preferential sorting of mutant cells to the periphery of colonies ( figure 4a , histograms right ) , similar to the peripheral sorting of nanog - high cells seen in wild - type jm8 colonies ( figure 1f ) . although co - culture of e8 and wild - type escs increased overall e - cadherin expression in mixed colonies ( figure s4a , red ) , a close examination revealed that e - cadherin expression by mutant cells remained low in cultures at 16 hr ( figure s4b ) . remarkably , co - culture of jarid2-null ( gfp ) escs with wild - type partners resulted in a switch from constitutive nanog - high expression ( figure 4b , e8 left ) to a profile that was indistinguishable from wild - type escs ( figure 4b , e8 right ) . exposure of jarid2-null escs to wild - type escs also restored -catenin activity in the mutant cells ( figure 4c ) and improved their capacity to differentiate ( figures 4d and 4e ) . neural induction was less efficient than in control wild - type cultures ( > 80% nestin positive by day 10 , figure s4c ) , and fewer cells were recovered overall ( 5%10% ) . co - culture of e8 cells with jarid2-null escs , fibroblasts , t or b cells , or with conditioned media derived from wild - type escs did not rescue the capability of e8 cells to differentiate ( data not shown ) or enhance -catenin activity in these cells ( as shown in figure s4d ) . because -catenin activity is reportedly required for esc differentiation ( atlasi et al . in this scenario , co - culture with wild - type escs restores -catenin activity and may enable mutant escs to differentiate ( figure s4e shows a hypothetical scheme ) . to investigate whether jarid2 regulation of nanog pcp / wnt signaling is likely to be important in vivo as well as in vitro , we examined the distribution of prickle1 in the pre - implantation mouse embryo . by e4.5 , nanog - high cells were condensed to the pole of the embryo where they expressed high levels of prickle1 distributed equivalently between nuclei and cytoplasm . the surrounding gata6-expressing cells ( red ) also expressed high levels of prickle1 ( green ) , but in this case expression was predominantly nuclear ( figure 5b , region highlighted in white ) . escs that lack jarid2 expressed high levels of nanog and low levels of prickle1 , vangl1 , and -catenin activity and show severely compromised differentiation ( landeira et al . as these defects were partially compensated by in vitro co - culture with wild - type escs , we asked to what extent mutant cells would contribute to the developing embryo . wild - type e3.5 blastocysts were injected with either 1015 gfpjarid2-null escs ( e8 ) or an identical number of gfp - labeled wild - type escs ( jm8 ) and then were cultured for 1620 hr before being examined . remarkably , we found that blastocysts injected with jarid2-null escs routinely initiated the formation of more than a single icm ( 16/39 , illustrated in figure 5e , lower panels ) , a feature that was not detected in any of the controls injected with wild - type escs ( 0/90 , upper panels ) . importantly , although these secondary icms appeared to be focused around small clusters of jarid2-null escs ( marked in green ) , in which higher levels of nanog might be anticipated , gata6 expression ( red ) was induced in the underlying wild - type cells ( figure 5f ) . to distinguish whether multiple icm formation was due to defective prc2 function , or defective nanog , pcp , and wnt circuitry , we injected e3.5 blastocysts with escs lacking prickle1/fzd2/wnt9a , overexpressing nanog , or that lacked eed ( azuara et al . as shown in table 1 , injection of wild - type ( e14 ) and eed - deficient ( b1.3 ) escs generated only a single icm in multiple experiments , whereas blastocysts injected with equivalent numbers of jarid2-null ( jarid2#3 ) , prickle1/fzd2/wnt9a - depleted ( pcp#5 ) , or nanog - overexpressing escs formed multiple icms in approximately 35%48% of samples . first , we show that jarid2 , a polycomb group member implicated in prc2 recruitment , chromatin structure , and gene repression , acts as a positive regulator of pcp / wnt signaling pathways in escs . absence of jarid2 or depletion of prickle1/fzd2/wnt9a results in constitutive nanog expression by esc , which , in turn , results in reduced levels of active -catenin . here , we show that canonical and non - canonical wnt signaling is reduced in jarid2-null escs , and these cells are unable to establish coherent colonies with each other . introduction of wild - type escs supports the formation of colonies and increases -catenin activity within jarid2-null escs , allowing them to properly execute differentiation programs in response to appropriate cues , although the nature of the support offered by wild - type escs is at present unclear . , our data show that jarid2-null escs maintained in lif and serum have low -catenin activity despite unchanged levels of total protein . co - culture of mutant cells both restored -catenin activity and allowed them to differentiate , consistent with several reports showing that nuclear -catenin is required to execute differentiation in vitro ( atlasi et al . overexpression of dvl , a downstream target of prickle1 , vangl , and frizzled ( fz ) ( gmez - orte et al . , 2008 we show that pcp expression is reduced in jarid2-null escs , and their introduction into e3.5 blastocysts induces the formation of multiple icms in around half of the embryos . , 2007 ) , and it is tempting to speculate that the group of cells in the mural trophectoderm identified by high levels of prickle1 in nucleoli may be cells destined to mediate implantation into the uterus . whatever the explanation , our demonstration that wild - type cells can rescue the differentiation of jarid2-null escs in vitro may provide important insights for understanding the conflicting differentiation status of jarid2 cells in vivo and in vitro . in homogenous clonally derived cultures , mutant cells are unable to establish bona fide cell - cell interactions or enhance -catenin activity , whereas in heterogenous cultures ( with wild - type esc partners ) or in vivo ( with other blastocyst - derived cells ) -catenin activity and pcp / wnt signaling may be partially compensated . thus , in addition to influencing prc2 recruitment and h3k27 hmtase activity in escs , we have shown that jarid2 is necessary to maintain a balance between nanog expression and pcp / wnt/-catenin in escs that is essential to enable them to properly respond to differentiation cues . regulation of this core circuit is also critical for normal pre - implantation development , since it appears to enable clusters of developing blastocysts to be discriminated and form a single inner cell mass .

switching -glucosidase inhibitors to miglitol reduced glucose fluctuations and circulating cardiovascular disease ( cvd ) risk factors in type 2 diabetic japanese patientsreducing glucose fluctuations may reduce the development of cvd in type 2 diabetic patients large - scale cohort studies such as diabetes epidemiology : collaborative analysis of diagnostic criteria in europe ( decode ) and funagata have shown that impaired glucose tolerance ( igt ) is strongly associated with subsequent incidence of cardiovascular disease ( cvd ) [ 13 ] . the study to prevent non - insulin - dependent diabetes mellitus ( stop - niddm ) and meta - analysis of risk improvement under acarbose ( meria7 ) trials have demonstrated that inhibition of postprandial hyperglycemia by the -glucosidase inhibitor ( -gi ) acarbose reduces pronounced cvd events in subjects with igt and type 2 diabetes [ 4 , 5 ] . these results suggest that inhibition of postprandial hyperglycemia , rather than the total rise of glucose throughout the day , in type 2 diabetic patients is important for preventing cvd development . recent studies have suggested that adhesion molecules such as e - selectin , intercellular adhesion molecule ( icam)-1 , and vascular cell adhesion molecule ( vcam)-1 , which are expressed in the vascular endothelium and induce leukocyte attachment to the blood vessels , are involved in the development of arteriosclerosis - related diabetic complications , including cvd . in addition , the chemokine monocyte chemoattractant protein ( mcp)-1 is a key mediator of the arteriosclerosis - related diabetic complications via monocyte / macrophage trafficking to the vascular endothelium in diabetic conditions . it has been reported in cell studies that hyperglycemia induces expression of icam-1 , vcam-1 , e - selectin , and mcp-1 in vascular endothelial cells [ 79 ] . previous longitudinal and cross - sectional studies including japanese populations have demonstrated that serum concentrations of soluble ( s ) se - selectin in particular , as well as sicam-1 and svcam-1 , are positively associated with arteriosclerosis - related clinical parameters and the subsequent incidence of cvd in type 2 diabetic patients [ 1013 ] . moreover , many longitudinal and cross - sectional studies have demonstrated that circulating mcp-1 concentrations are strongly and positively associated with atherosclerosis - associated clinical parameters in healthy subjects , subjects with obesity , or subjects with type 2 diabetes [ 1416 ] . our previous study demonstrated that switching -gi from acarbose or voglibose to miglitol , which has a greater effect on reducing 1 h postprandial glucose levels than other -gis , in type 2 diabetic patients reduced glucose fluctuations and messenger rna ( mrna ) levels of inflammatory cytokines such as interleukin ( il)-1 and tumor necrosis factor ( tnf)- , which are known to induce attachment of activated leukocytes to blood vessels , in peripheral leukocytes and circulating tnf- protein levels . however , whether circulating levels of soluble adhesion molecules and mcp-1 are suppressed by miglitol treatment in type 2 diabetic patients has not been determined . in this study , we examined whether switching from acarbose or voglibose to miglitol in type 2 diabetic patients reduced glucose fluctuations and circulating levels of soluble adhesion molecules such as se - selectin , sicam-1 , svcam-1 , and mcp-1 . this study was a prospective exploratory trial conducted in a hospital setting ( naka kinen clinic , ibaraki ) in japan . we first reviewed the clinical records of potential subjects and identified those that met the criteria of inclusion and exclusion . inclusion criteria were male and female patients with type 2 diabetes , hba1c values ranging from 6.9 to 8.3 % , and treatment with the highest approved doses of -gis ( 100 mg acarbose or 0.3 mg voglibose at each meal ) in combination with insulin or a sulfonylurea for at least 6 months , who visited the hospital between may 2007 and april 2008 . the number of patients compliant with the inclusion criteria was 196 type 2 diabetic patients who visited the clinic during the study period ( n = 1,136 ) . among these patients , we excluded from the study patients considered inappropriate , e.g. pregnant , possibly pregnant , or young ( patients younger than 20 years of age ) . four patients with severe nephropathy ( serum creatinine 2 mg/100 ml ) were excluded . we also excluded patients with severe clinical conditions , such as hepatic disorders , cvd , impaired pulmonary function , pancreatopathy , cancer , infectious diseases , external injury , and perioperative patients . we selected 47 patients matching the above criteria and all patients were enrolled as previously reported . the patients had been undergoing stable treatment for at least 3 months before entering the study . subjects prior -gis were switched to miglitol at a dose of 50 mg / meal , and continued for 3 months . anthropometric data were measured and blood samples collected from each patient before and 3 months after the switch to miglitol . before and 3 months after the switch , subjects were questioned regarding abdominal distension , flatulence , and abnormalities of bowel function using a questionnaire consisting of a visual analog scale ( vas ) from 1 to 10 , with 1 indicating no problems in daily life and 10 indicating an inability to perform activities of daily living . before and 3 months after the switch , each patient was asked by medical staff whether symptoms consistent with hypoglycemia , such as hand and foot trepidations and palpitations , had occurred at least once or never during each 1-month period . the prescriptions for medications other than -gis including insulin units for patients were not changed during the trial . among the subjects , four patients dropped out during the trial . overall , 43 patients completed the trial and were included in the analysis of the relationship between glucose fluctuation and inflammatory cytokine mrna levels in peripheral leukocytes , as previously reported . among the subjects who completed the trial , we reanalyzed 35 patients because serum samples were missing from eight patients . all patients in the study provided informed consent for use of their personal and health information in our analysis . the study protocol was approved by the ethics committee of the university of shizuoka , shizuoka , japan . before and 3 months after the switch to miglitol , basic parameters in the morning following an overnight fast state were measured . body heights and weights were measured using instruments ( body heights : ad-6225a ; body weight : ad6207a ; a&d co. , ltd , tokyo , japan ) . triglycerides ( tgl ) , total cholesterol ( t - cho ) , high - density lipoprotein ( hdl ) , and c - reactive protein ( crp ) were measured in blood samples with an auto - analyzer ( 7180 ; hitachi high - technologies co. , ltd , tokyo , japan ) using kits ( tgl : m / pm ; t - cho : l m / pm ; hdl cholesterol [ hdl - c ] : l m/2-pm ; crp : lt - hs ii ; wako chemicals , osaka , japan ) . fasting plasma glucose and hba1c were measured using instruments ( fasting plasma glucose : ga-1171 ; hba1c : ha8181 ; arkray , inc . , kyoto , japan ) . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . self - monitoring of blood glucose ( smbg ) was performed over 5 days within 1 month before the switch ( baseline ) and within 1 month before the end of the trial ( after the switch ) . smbg was performed just before and 1 h after each meal ( six time points per day ) using a glutest neo smbg device ( sanwa kagaku kenkyusho , nagoya , japan ) . the smbg data over 5 days within 1 month before the switch and the end of the trial were averaged . m - values were determined from the averages of the smbg values using the formula [ 10 log(blood glucose level/120 ) ] + ( blood glucose level blood glucose level)/20 . blood samples for serum protein were obtained just before and 3 months after the switch to miglitol . serum protein concentrations of mcp-1 were measured using a milliplex human cytokine / chemokine immunoassay kit ( millipore , billerica , ma , usa ) , and adhesion molecules ( se - selectin , sicam-1 and svcam-1 ) and total plasminogen activator inhibitor ( tpai)-1 were measured using a milliplex cvd panel 1 immunoassay kit ( millipore ) . serum fatty acid - binding protein ( fabp ) 4 concentrations were measured using a human adipocyte fabp enzyme - linked immunosorbent assay ( biovendor inc . , the mean intra - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 reported by the manufacturers were 6.1 , 11.2 , 7.9 , 4.5 , 11.8 , and 2.5 % , respectively . the inter - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 were 12.0 , 13.4 , 9.7 , 8.5 , 12.5 , and 3.9 % , respectively . all statistical analyses were performed using excel 2007 for windows ( microsoft corporation , redmond , wa , usa ) . this study was a prospective exploratory trial conducted in a hospital setting ( naka kinen clinic , ibaraki ) in japan . we first reviewed the clinical records of potential subjects and identified those that met the criteria of inclusion and exclusion . inclusion criteria were male and female patients with type 2 diabetes , hba1c values ranging from 6.9 to 8.3 % , and treatment with the highest approved doses of -gis ( 100 mg acarbose or 0.3 mg voglibose at each meal ) in combination with insulin or a sulfonylurea for at least 6 months , who visited the hospital between may 2007 and april 2008 . the number of patients compliant with the inclusion criteria was 196 type 2 diabetic patients who visited the clinic during the study period ( n = 1,136 ) . among these patients , we excluded from the study patients considered inappropriate , e.g. pregnant , possibly pregnant , or young ( patients younger than 20 years of age ) . four patients with severe nephropathy ( serum creatinine 2 mg/100 ml ) were excluded . we also excluded patients with severe clinical conditions , such as hepatic disorders , cvd , impaired pulmonary function , pancreatopathy , cancer , infectious diseases , external injury , and perioperative patients . we selected 47 patients matching the above criteria and all patients were enrolled as previously reported . the patients had been undergoing stable treatment for at least 3 months before entering the study . subjects prior -gis were switched to miglitol at a dose of 50 mg / meal , and continued for 3 months . anthropometric data were measured and blood samples collected from each patient before and 3 months after the switch to miglitol . before and 3 months after the switch , subjects were questioned regarding abdominal distension , flatulence , and abnormalities of bowel function using a questionnaire consisting of a visual analog scale ( vas ) from 1 to 10 , with 1 indicating no problems in daily life and 10 indicating an inability to perform activities of daily living . before and 3 months after the switch , each patient was asked by medical staff whether symptoms consistent with hypoglycemia , such as hand and foot trepidations and palpitations , had occurred at least once or never during each 1-month period . the prescriptions for medications other than -gis including insulin units for patients were not changed during the trial . among the subjects , four patients dropped out during the trial . overall , 43 patients completed the trial and were included in the analysis of the relationship between glucose fluctuation and inflammatory cytokine mrna levels in peripheral leukocytes , as previously reported . among the subjects who completed the trial , we reanalyzed 35 patients because serum samples were missing from eight patients . all patients in the study provided informed consent for use of their personal and health information in our analysis . the study protocol was approved by the ethics committee of the university of shizuoka , shizuoka , japan . before and 3 months after the switch to miglitol , basic parameters in the morning following an overnight fast state were measured . body heights and weights were measured using instruments ( body heights : ad-6225a ; body weight : ad6207a ; a&d co. , ltd , tokyo , japan ) . triglycerides ( tgl ) , total cholesterol ( t - cho ) , high - density lipoprotein ( hdl ) , and c - reactive protein ( crp ) were measured in blood samples with an auto - analyzer ( 7180 ; hitachi high - technologies co. , ltd , tokyo , japan ) using kits ( tgl : m / pm ; t - cho : l m / pm ; hdl cholesterol [ hdl - c ] : l m/2-pm ; crp : lt - hs ii ; wako chemicals , osaka , japan ) . fasting plasma glucose and hba1c were measured using instruments ( fasting plasma glucose : ga-1171 ; hba1c : ha8181 ; arkray , inc . , kyoto , japan ) . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . self - monitoring of blood glucose ( smbg ) was performed over 5 days within 1 month before the switch ( baseline ) and within 1 month before the end of the trial ( after the switch ) . smbg was performed just before and 1 h after each meal ( six time points per day ) using a glutest neo smbg device ( sanwa kagaku kenkyusho , nagoya , japan ) . the smbg data over 5 days within 1 month before the switch and the end of the trial were averaged . m - values were determined from the averages of the smbg values using the formula [ 10 log(blood glucose level/120 ) ] + ( blood glucose level blood glucose level)/20 . blood samples for serum protein were obtained just before and 3 months after the switch to miglitol . serum protein concentrations of mcp-1 were measured using a milliplex human cytokine / chemokine immunoassay kit ( millipore , billerica , ma , usa ) , and adhesion molecules ( se - selectin , sicam-1 and svcam-1 ) and total plasminogen activator inhibitor ( tpai)-1 were measured using a milliplex cvd panel 1 immunoassay kit ( millipore ) . serum fatty acid - binding protein ( fabp ) 4 concentrations were measured using a human adipocyte fabp enzyme - linked immunosorbent assay ( biovendor inc . , the mean intra - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 reported by the manufacturers were 6.1 , 11.2 , 7.9 , 4.5 , 11.8 , and 2.5 % , respectively . the inter - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 were 12.0 , 13.4 , 9.7 , 8.5 , 12.5 , and 3.9 % , respectively . all statistical analyses were performed using excel 2007 for windows ( microsoft corporation , redmond , wa , usa ) . significant differences between two groups were determined by paired student s t tests . values of p < 0.05 were considered significant . we obtained data from 35 type 2 diabetic patients whose mean hba1c values were 7.26 0.51 % at baseline . among these patients , the mean age , bmi , and duration of type 2 diabetes were 65.8 9.5 years , 21.8 2.8 kg / m , and 20.5 11.3 years , respectively.table 1baseline patient characteristicssex ( male / female)17/18age ( years)65.8 9.5bmi ( kg / m)21.8 2.8hba1c ( % ) 7.26 0.51duration of diabetes ( years)20.5 11.3diabetic complications retinopathy21 neuropathy15 nephropathy0 any one or more of these complications25hyperlipidemia22 prescription of statins18hypertension19 prescription of angiotensin receptor blockers10assigned caloric intake ( kcal)1,495 151combined drugs insulin21 intermediate - acting16 long - acting4 pre - mixed ( intermediate - acting and rapid - acting)1 sulfonylurea14prior -glucosidase inhibitor acarbose ( 100 mg three times daily)30 voglibose ( 0.3 mg three times daily)5data are expressed as mean sd , or frequency bmi body mass index baseline patient characteristics data are expressed as mean sd , or frequency table 2 shows the clinical characteristics just before and 3 months after switching from acarbose or voglibose to miglitol . switching to miglitol did not affect vas values for digestive symptoms such as abdominal distention , flatulence , and abnormalities of bowel function . the -gi switch had no effects on levels of hba1c , fasting glucose , t - cho , and crp . the results indicate that the switch from acarbose or voglibose to miglitol did not affect basic clinical parameters.table 2clinical characteristics at baseline and 3 months after switching to miglitol n baseline3 months p - valuehba1c ( % ) 357.26 0.517.27 0.610.817fasting glucose ( mg/100 ml)35130.6 29.6129.0 30.20.771triglycerides ( mg/100 ml)3573.9 35.977.8 34.40.501total cholesterol ( mg/100 ml)33179.9 28.4183.8 27.40.340crp ( mg/100 ml)350.09 0.160.08 0.180.815abdominal distention ( score 110)352.6 2.12.8 2.10.546flatulence ( score 110)354.2 2.73.1 2.00.161abnormalities of bowel function ( score 110)291.7 1.22.1 1.50.206data are expressed as mean sd , or frequencystatistical analyses were performed using two - sided , paired student s t test crp c - reactive protein clinical characteristics at baseline and 3 months after switching to miglitol data are expressed as mean sd , or frequency statistical analyses were performed using two - sided , paired student s t test crp c - reactive protein figure 1 shows blood glucose concentrations pre- and post - meals compared with periods just before and after the -gi switch . blood glucose concentrations were significantly higher just before lunch ( p = 0.018 ) , significantly lower 1 h after lunch ( p = 0.012 ) , significantly higher just before dinner ( p < 0.001 ) , and significantly lower 1 h after dinner ( p = 0.045 ) after the switch compared with before the switch . m - values were significantly reduced by the switch to miglitol ( p = 0.010 ) . glucose fluctuations were improved by the switch without changing the total rise of glucose ( hba1c).fig . 1effects on glucose fluctuations of switching from the highest approved doses of the -glucosidase inhibitors acarbose or voglibose to a medium dose of miglitol in patients with type 2 diabetes mellitus . a glucose concentrations determined by smbg . b m - value . asterisks denote significant differences compared with the value before switching to miglitol ( * p < 0.05 and * * p < 0.01 ) . smbg self - monitoring of blood glucose , sd standard deviation effects on glucose fluctuations of switching from the highest approved doses of the -glucosidase inhibitors acarbose or voglibose to a medium dose of miglitol in patients with type 2 diabetes mellitus . a glucose concentrations determined by smbg . b m - value . asterisks denote significant differences compared with the value before switching to miglitol ( * p < 0.05 and * * p < 0.01 ) . smbg self - monitoring of blood glucose , sd standard deviation serum protein concentrations of cvd risk factors are shown in fig . 2 . serum mcp-1 and se - selectin concentrations decreased at levels of 82 % ( p < 0.001 ) and 78 % ( p = 0.014 ) , respectively , and serum svcam-1 concentrations increased at levels of 107 % ( p = 0.014 ) 3 months after the switch compared with baseline . serum protein concentrations of sicam-1 , tpai-1 , and fabp4 were unchanged by the switch . these results indicate the switch from acarbose or voglibose to miglitol reduced circulating protein concentrations of cvd risk factors such as mcp-1 and se-selectin.fig . 2serum protein levels of cvd risk factors at baseline and 3 months after switching to miglitol . asterisks denote significant differences compared with the value before switching to miglitol ( * p < 0.05 and * * p < 0.01 ) . cvd cardiovascular disease , sd standard deviation , mcp monocyte chemoattractant protein , vcam vascular cell adhesion molecule , icam intercellular adhesion molecule , tpai total plasminogen activator inhibitor , fabp4 fatty acid binding protein , s soluble serum protein levels of cvd risk factors at baseline and 3 months after switching to miglitol . asterisks denote significant differences compared with the value before switching to miglitol ( * p < 0.05 and * * p < 0.01 ) . cvd cardiovascular disease , sd standard deviation , mcp monocyte chemoattractant protein , vcam vascular cell adhesion molecule , icam intercellular adhesion molecule , tpai total plasminogen activator inhibitor , fabp4 fatty acid binding protein , s soluble in large - scale cohort studies , such as decode and funagata , it has been reported that postprandial hyperglycemia , rather than hba1c , is closely associated with subsequent incidence of cvd [ 13 ] . additionally , the stop - niddm and meria7 trials have demonstrated that inhibition of postprandial hyperglycemia by the -gi acarbose greatly reduces cvd events in subjects with igt and type 2 diabetes [ 4 , 5 ] . thus , reduction of glucose fluctuations by miglitol may reduce cvd incidence in type 2 diabetic patients . in addition , we previously reported in 43 type 2 diabetic patients from the same sample that mrna levels of inflammatory cytokines , such as il-1 and tnf- , in peripheral leukocytes and circulating tnf- proteins were reduced by the switch to miglitol . in this study we reanalyzed serum samples of 35 patients from the same sample and found that serum protein concentrations of mcp-1 and se - selectin were reduced by the switch . mcp-1 induces migration of leukocytes to blood vessels and e - selectin facilitates leukocytes rolling onto the endothelium , resulting in the induction of the adhesion of leukocytes to blood vessels [ 21 , 22 ] . together , the results of this study and our previous study indicate that the switching from an -gi ( acarbose or voglibose ) to miglitol suppresses glucose fluctuations , inflammatory cytokine expression in peripheral leukocytes , and circulating protein concentrations of mcp-1 , se - selectin , and tnf- in type 2 diabetic patients in a clinical setting in japan . serum protein concentrations of sicam-1 , tpai-1 , and fabp4 were not altered and svcam-1 was slightly increased by the switch to miglitol . sicam-1 and svcam-1 participate in inducing leukocyte attachment to blood vessels after leukocyte migration and rolling of leukocytes around blood vessels . pai-1 expressed from adipose tissues promotes atherogenesis by forming blocked blood vessels by inducing blood coagulation , and fabp4 expressed from adipose tissues and macrophages enhances atherogenesis by tracking cholesterol in atheromatosis . thus , -gis , including miglitol , may inhibit cvd development by repressing the initial step of atheromatosis , i.e. inhibition of circulating mcp-1 and se - selectin proteins via inhibition of postprandial hyperglycemia and glucose fluctuations . however , the associations between glucose fluctuations and the concentrations of circulating cvd risk factors in type 2 diabetic patients , as well as in subjects with igt and healthy subjects , remain unclear . thus , there is a need to examine the associations between glucose fluctuations and the concentrations of circulating cvd risk factors in subjects with type 2 diabetes or igt and healthy subjects in cross - sectional studies . additionally , whether subjects with higher circulating concentrations of cvd risk factors accompanied by glucose fluctuations had higher subsequent incidence of cvd should be explored in cohort studies . in addition , randomized , double - blind , placebo - controlled ( rct ) trials are needed to examine whether repression of circulating cvd risk factor concentrations by miglitol , but less so by other -gis , reduces the subsequent incidence of cvd in type 2 diabetic patients . tpai-1 and fabp4 thus , switching -gis from acarbose or voglibose to miglitol may not reduce lipid abnormalities related to atherogenesis risk . it has been reported from an rct conducted in germany that drugs improving lipid metabolism ( insulin resistance ) such as metformin and pioglitazone and their combination reduced tpai-1 concentrations in type 2 diabetic patients receiving stable basal insulin therapy , although it is still unclear whether circulating fabp4 concentrations are reduced by these drugs . the combination of miglitol with these drugs for improving insulin resistance may reduce cvd development by decreasing circulating concentrations of tpai-1 , mcp-1 , and se - selectin . switching from acarbose or voglibose to miglitol for 3 months has been found to reduce hypoglycemic symptoms and blood glucose concentrations between meals . it has been shown that hypoglycemia is strongly and positively associated with subsequent cvd incidence . thus , reducing hypoglycemia using miglitol may reduce cvd risk ; however , hypoglycemic symptoms in our trials were self - reported . the self - reported hypoglycemic symptoms were limited because they may be underreported by patients to medical staff . a previous study has demonstrated that postprandial hyperglycemia within 1 h after a standard meal loading was higher , and that over 1 h was lower , in viscerally obese japanese subjects treated with miglitol compared with those treated with acarbose . in addition , it was reported that treatment with miglitol , but not with acarbose or voglibose , in japanese women who had undergone a total gastrectomy reduced reactive hypoglycemia . combining our results with those of previous studies , treatment with miglitol could be a lower risk of hypoglycemia rather than other -gis . further large - scale studies should examine whether miglitol treatment of type 2 diabetic patients reduces hypoglycemia assessed by smbg and hypoglycemic symptoms , such as hypoglycemia - induced lethargy , compared with other -gis . additionally , whether slight and severe degrees of hypoglycemia induce circulating protein concentrations of mcp-1 and se - selectin , and whether the reduction of hypoglycemia by miglitol reduces circulating protein concentrations of mcp-1 and se - selectin and cvd incidence in type 2 diabetic patients , should be examined . in addition , it should be noted that we analyzed samples from 35 of the 43 patients who completed the study because serum samples were not obtained from eight patients . our previous study using the same sample demonstrated that glucose fluctuations in 43 type 2 diabetic japanese patients were reduced by switching from acarbose or voglibose to miglitol for 3 months . in this study , we obtained the same result in 35 patients . thus , missing data from the eight patients would be less likely to affect the results of this study . it has been reported that an increase of the postprandial incremental area under the curve of blood glucose in a single oral meal test in eight type 2 diabetic patients was reduced by miglitol treatment at doses of 50 , 75 , 100 , and 200 mg . an rct of 36 type 2 diabetic patients found that postprandial blood glucose levels were reduced by ~50 % in patients treated with miglitol compared with those treated with placebo . a double - blind , crossover design in 15 type 2 diabetic patients found that treatment with miglitol ( 300 mg / day ) effectively reduced postprandial blood glucose levels over 8 weeks . in addition , a previous study reported that treatment with miglitol in 24 viscerally obese subjects reduced glucose fluctuations and circulating il-6 concentrations versus acarbose treatment . in addition , our previous study reported that the switch of -gi from acarbose or voglibose to miglitol in 43 type 2 diabetic patients reduced glucose fluctuations and expression of inflammatory cytokine genes , such as il-1 and tnf- , in peripheral leukocytes and the circulating protein concentrations of tnf- . from these studies , we considered that our sample of 35 type 2 diabetic japanese patients is comparable ; however , a large - scale rct is needed to examine whether miglitol reduces glucose fluctuations and circulating concentrations of cvd risk factors in type 2 diabetic patients compared with other -gis . recent studies have suggested that blood glucose profiles monitored by smbg are not always correlated with continuous glucose monitoring ( cgm ) , particularly given that measurement of blood glucose concentrations by smbg often omit hypoglycemic events entirely [ 32 , 33 ] . a study of ten type 2 diabetic patients hospitalized for 4 days found that glucose fluctuations , which were monitored by cgm , in a standard meal loading were reduced effectively by treatment with miglitol ( 50 mg ) compared with acarbose ( 100 mg ) . in addition , in this study we demonstrated that switching -gis from acarbose or voglibose to miglitol in type 2 diabetic japanese patients reduced glucose fluctuations , which were assessed by the averages at just before and 1 h after each meal measured over 5 days by smbg . combining our results with the results from cgm in a previous study however , it is still unclear whether glucose fluctuations were lower in type 2 diabetic patients who were treated longer with miglitol than in those who were treated longer with other -gis . although cgm during the treatment of -gis were performed under oral meal loading tests at breakfast , lunch , and dinner in patients hospitalized for 4 days in the previous study , the diet during days when smbg was performed in our trials was dependent on each patient . rct trials , in which dietary habits are well controlled , should examine whether glucose fluctuations by long - term cgm are lower in type 2 diabetic patients treated with miglitol than in those treated with acarbose or voglibose . it should be noted that our trial is a prospective exploratory trial that is not an rct , which introduces some confounding factors and bias in our trial . indeed , it has been reported that hba1c has a duration across the year that is highly detected during spring and gradually decreases by autumn in japan . one of the other possibilities is that lifestyles such as dietary and exercise habits in patients were changed during the trial . in this trial , in addition , miglitol treatment may reduce a patient s appetite because the change of -gi to miglitol treatment inhibits symptoms of hypoglycemia and reduction of blood glucose levels during a meal ; however , our results indicate that the change of -gi to miglitol reduced glucose fluctuation but not hba1c . thus , the effect is most likely a result of the effects of miglitol because changes in dietary and exercise habits may alter hba1c levels . whether miglitol treatment reduces circulating cvd risk factors including mcp-1 and se - selectin in type 2 diabetic japanese patients needs to be examined in an rct the results of this study indicate that switching from acarbose or voglibose to miglitol for 3 months suppressed glucose fluctuations and serum protein concentrations of mcp-1 and se - selectin more effectively than the prior -gi . fuchigami is an employee of sanwa kagaku kenkyusho co. , ltd , nagoya , japan .

background and objectivesin this study we examined the effects of switching -glucosidase inhibitors ( -gi ) from acarbose or voglibose to miglitol on glucose fluctuations and circulating concentrations of cardiovascular disease risk factors , such as soluble adhesion molecules ( se - selectin , sicam-1 and svcam-1 ) , a chemokine monocyte chemoattractant protein ( mcp)-1 , plasminogen activator inhibitor-1 , and fatty acid - binding protein 4 , in type 2 diabetic patients for 3 months.methodswe enrolled 47 japanese patients with type 2 diabetes , with hba1c levels with 7.26 0.5 % ( mean standard deviation ) , and who were treated with the highest approved dose of acarbose ( 100 mg / meal ) or voglibose ( 0.3 mg / meal ) in combination with insulin or sulfonylurea . patients prior -gis were switched to a medium dose of miglitol ( 50 mg / meal ) , and the new treatments were maintained for 3 months . thirty - five patients who completed the 3-month study and provided serum samples were analyzed.resultsthe switch to miglitol for 3 months did not affect hba1c , fasting glucose , triglycerides , total - cholesterol or c - reactive protein levels , or result in any adverse events . glucose fluctuations were significantly improved by the change in treatment ( m - value : 10.54 4.32 to 8.36 2.54 ) , while serum protein concentrations of mcp-1 ( 525.04 288.06428.11 163.78 pg / ml ) and se - selectin ( 18.65 9.7714.50 6.26 ng / ml ) were suppressed.conclusionour results suggest that switching from acarbose or voglibose to miglitol for 3 months suppressed glucose fluctuations and serum protein levels of mcp-1 and se - selectin in type 2 diabetic japanese patients , with fewer adverse effects .

Key Points Introduction Methods Study Population Measurements Statistical Analysis Results Discussion Conclusion Conflict of interest

switching -glucosidase inhibitors to miglitol reduced glucose fluctuations and circulating cardiovascular disease ( cvd ) risk factors in type 2 diabetic japanese patientsreducing glucose fluctuations may reduce the development of cvd in type 2 diabetic patients large - scale cohort studies such as diabetes epidemiology : collaborative analysis of diagnostic criteria in europe ( decode ) and funagata have shown that impaired glucose tolerance ( igt ) is strongly associated with subsequent incidence of cardiovascular disease ( cvd ) [ 13 ] . the study to prevent non - insulin - dependent diabetes mellitus ( stop - niddm ) and meta - analysis of risk improvement under acarbose ( meria7 ) trials have demonstrated that inhibition of postprandial hyperglycemia by the -glucosidase inhibitor ( -gi ) acarbose reduces pronounced cvd events in subjects with igt and type 2 diabetes [ 4 , 5 ] . these results suggest that inhibition of postprandial hyperglycemia , rather than the total rise of glucose throughout the day , in type 2 diabetic patients is important for preventing cvd development . recent studies have suggested that adhesion molecules such as e - selectin , intercellular adhesion molecule ( icam)-1 , and vascular cell adhesion molecule ( vcam)-1 , which are expressed in the vascular endothelium and induce leukocyte attachment to the blood vessels , are involved in the development of arteriosclerosis - related diabetic complications , including cvd . previous longitudinal and cross - sectional studies including japanese populations have demonstrated that serum concentrations of soluble ( s ) se - selectin in particular , as well as sicam-1 and svcam-1 , are positively associated with arteriosclerosis - related clinical parameters and the subsequent incidence of cvd in type 2 diabetic patients [ 1013 ] . our previous study demonstrated that switching -gi from acarbose or voglibose to miglitol , which has a greater effect on reducing 1 h postprandial glucose levels than other -gis , in type 2 diabetic patients reduced glucose fluctuations and messenger rna ( mrna ) levels of inflammatory cytokines such as interleukin ( il)-1 and tumor necrosis factor ( tnf)- , which are known to induce attachment of activated leukocytes to blood vessels , in peripheral leukocytes and circulating tnf- protein levels . however , whether circulating levels of soluble adhesion molecules and mcp-1 are suppressed by miglitol treatment in type 2 diabetic patients has not been determined . in this study , we examined whether switching from acarbose or voglibose to miglitol in type 2 diabetic patients reduced glucose fluctuations and circulating levels of soluble adhesion molecules such as se - selectin , sicam-1 , svcam-1 , and mcp-1 . inclusion criteria were male and female patients with type 2 diabetes , hba1c values ranging from 6.9 to 8.3 % , and treatment with the highest approved doses of -gis ( 100 mg acarbose or 0.3 mg voglibose at each meal ) in combination with insulin or a sulfonylurea for at least 6 months , who visited the hospital between may 2007 and april 2008 . the number of patients compliant with the inclusion criteria was 196 type 2 diabetic patients who visited the clinic during the study period ( n = 1,136 ) . subjects prior -gis were switched to miglitol at a dose of 50 mg / meal , and continued for 3 months . triglycerides ( tgl ) , total cholesterol ( t - cho ) , high - density lipoprotein ( hdl ) , and c - reactive protein ( crp ) were measured in blood samples with an auto - analyzer ( 7180 ; hitachi high - technologies co. , ltd , tokyo , japan ) using kits ( tgl : m / pm ; t - cho : l m / pm ; hdl cholesterol [ hdl - c ] : l m/2-pm ; crp : lt - hs ii ; wako chemicals , osaka , japan ) . serum protein concentrations of mcp-1 were measured using a milliplex human cytokine / chemokine immunoassay kit ( millipore , billerica , ma , usa ) , and adhesion molecules ( se - selectin , sicam-1 and svcam-1 ) and total plasminogen activator inhibitor ( tpai)-1 were measured using a milliplex cvd panel 1 immunoassay kit ( millipore ) . serum fatty acid - binding protein ( fabp ) 4 concentrations were measured using a human adipocyte fabp enzyme - linked immunosorbent assay ( biovendor inc . , the mean intra - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 reported by the manufacturers were 6.1 , 11.2 , 7.9 , 4.5 , 11.8 , and 2.5 % , respectively . the inter - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 were 12.0 , 13.4 , 9.7 , 8.5 , 12.5 , and 3.9 % , respectively . inclusion criteria were male and female patients with type 2 diabetes , hba1c values ranging from 6.9 to 8.3 % , and treatment with the highest approved doses of -gis ( 100 mg acarbose or 0.3 mg voglibose at each meal ) in combination with insulin or a sulfonylurea for at least 6 months , who visited the hospital between may 2007 and april 2008 . the number of patients compliant with the inclusion criteria was 196 type 2 diabetic patients who visited the clinic during the study period ( n = 1,136 ) . subjects prior -gis were switched to miglitol at a dose of 50 mg / meal , and continued for 3 months . triglycerides ( tgl ) , total cholesterol ( t - cho ) , high - density lipoprotein ( hdl ) , and c - reactive protein ( crp ) were measured in blood samples with an auto - analyzer ( 7180 ; hitachi high - technologies co. , ltd , tokyo , japan ) using kits ( tgl : m / pm ; t - cho : l m / pm ; hdl cholesterol [ hdl - c ] : l m/2-pm ; crp : lt - hs ii ; wako chemicals , osaka , japan ) . serum protein concentrations of mcp-1 were measured using a milliplex human cytokine / chemokine immunoassay kit ( millipore , billerica , ma , usa ) , and adhesion molecules ( se - selectin , sicam-1 and svcam-1 ) and total plasminogen activator inhibitor ( tpai)-1 were measured using a milliplex cvd panel 1 immunoassay kit ( millipore ) . serum fatty acid - binding protein ( fabp ) 4 concentrations were measured using a human adipocyte fabp enzyme - linked immunosorbent assay ( biovendor inc . , the mean intra - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 reported by the manufacturers were 6.1 , 11.2 , 7.9 , 4.5 , 11.8 , and 2.5 % , respectively . the inter - assay coefficients of variation for mcp-1 , se - selectin , sicam-1 , svcam-1 , tpai-1 , and fabp4 were 12.0 , 13.4 , 9.7 , 8.5 , 12.5 , and 3.9 % , respectively . we obtained data from 35 type 2 diabetic patients whose mean hba1c values were 7.26 0.51 % at baseline . among these patients , the mean age , bmi , and duration of type 2 diabetes were 65.8 9.5 years , 21.8 2.8 kg / m , and 20.5 11.3 years , respectively.table 1baseline patient characteristicssex ( male / female)17/18age ( years)65.8 9.5bmi ( kg / m)21.8 2.8hba1c ( % ) 7.26 0.51duration of diabetes ( years)20.5 11.3diabetic complications retinopathy21 neuropathy15 nephropathy0 any one or more of these complications25hyperlipidemia22 prescription of statins18hypertension19 prescription of angiotensin receptor blockers10assigned caloric intake ( kcal)1,495 151combined drugs insulin21 intermediate - acting16 long - acting4 pre - mixed ( intermediate - acting and rapid - acting)1 sulfonylurea14prior -glucosidase inhibitor acarbose ( 100 mg three times daily)30 voglibose ( 0.3 mg three times daily)5data are expressed as mean sd , or frequency bmi body mass index baseline patient characteristics data are expressed as mean sd , or frequency table 2 shows the clinical characteristics just before and 3 months after switching from acarbose or voglibose to miglitol . switching to miglitol did not affect vas values for digestive symptoms such as abdominal distention , flatulence , and abnormalities of bowel function . the -gi switch had no effects on levels of hba1c , fasting glucose , t - cho , and crp . the results indicate that the switch from acarbose or voglibose to miglitol did not affect basic clinical parameters.table 2clinical characteristics at baseline and 3 months after switching to miglitol n baseline3 months p - valuehba1c ( % ) 357.26 0.517.27 0.610.817fasting glucose ( mg/100 ml)35130.6 29.6129.0 30.20.771triglycerides ( mg/100 ml)3573.9 35.977.8 34.40.501total cholesterol ( mg/100 ml)33179.9 28.4183.8 27.40.340crp ( mg/100 ml)350.09 0.160.08 0.180.815abdominal distention ( score 110)352.6 2.12.8 2.10.546flatulence ( score 110)354.2 2.73.1 2.00.161abnormalities of bowel function ( score 110)291.7 1.22.1 1.50.206data are expressed as mean sd , or frequencystatistical analyses were performed using two - sided , paired student s t test crp c - reactive protein clinical characteristics at baseline and 3 months after switching to miglitol data are expressed as mean sd , or frequency statistical analyses were performed using two - sided , paired student s t test crp c - reactive protein figure 1 shows blood glucose concentrations pre- and post - meals compared with periods just before and after the -gi switch . m - values were significantly reduced by the switch to miglitol ( p = 0.010 ) . 1effects on glucose fluctuations of switching from the highest approved doses of the -glucosidase inhibitors acarbose or voglibose to a medium dose of miglitol in patients with type 2 diabetes mellitus . smbg self - monitoring of blood glucose , sd standard deviation effects on glucose fluctuations of switching from the highest approved doses of the -glucosidase inhibitors acarbose or voglibose to a medium dose of miglitol in patients with type 2 diabetes mellitus . smbg self - monitoring of blood glucose , sd standard deviation serum protein concentrations of cvd risk factors are shown in fig . serum mcp-1 and se - selectin concentrations decreased at levels of 82 % ( p < 0.001 ) and 78 % ( p = 0.014 ) , respectively , and serum svcam-1 concentrations increased at levels of 107 % ( p = 0.014 ) 3 months after the switch compared with baseline . serum protein concentrations of sicam-1 , tpai-1 , and fabp4 were unchanged by the switch . these results indicate the switch from acarbose or voglibose to miglitol reduced circulating protein concentrations of cvd risk factors such as mcp-1 and se-selectin.fig . cvd cardiovascular disease , sd standard deviation , mcp monocyte chemoattractant protein , vcam vascular cell adhesion molecule , icam intercellular adhesion molecule , tpai total plasminogen activator inhibitor , fabp4 fatty acid binding protein , s soluble serum protein levels of cvd risk factors at baseline and 3 months after switching to miglitol . cvd cardiovascular disease , sd standard deviation , mcp monocyte chemoattractant protein , vcam vascular cell adhesion molecule , icam intercellular adhesion molecule , tpai total plasminogen activator inhibitor , fabp4 fatty acid binding protein , s soluble in large - scale cohort studies , such as decode and funagata , it has been reported that postprandial hyperglycemia , rather than hba1c , is closely associated with subsequent incidence of cvd [ 13 ] . in addition , we previously reported in 43 type 2 diabetic patients from the same sample that mrna levels of inflammatory cytokines , such as il-1 and tnf- , in peripheral leukocytes and circulating tnf- proteins were reduced by the switch to miglitol . in this study we reanalyzed serum samples of 35 patients from the same sample and found that serum protein concentrations of mcp-1 and se - selectin were reduced by the switch . together , the results of this study and our previous study indicate that the switching from an -gi ( acarbose or voglibose ) to miglitol suppresses glucose fluctuations , inflammatory cytokine expression in peripheral leukocytes , and circulating protein concentrations of mcp-1 , se - selectin , and tnf- in type 2 diabetic patients in a clinical setting in japan . serum protein concentrations of sicam-1 , tpai-1 , and fabp4 were not altered and svcam-1 was slightly increased by the switch to miglitol . however , the associations between glucose fluctuations and the concentrations of circulating cvd risk factors in type 2 diabetic patients , as well as in subjects with igt and healthy subjects , remain unclear . thus , there is a need to examine the associations between glucose fluctuations and the concentrations of circulating cvd risk factors in subjects with type 2 diabetes or igt and healthy subjects in cross - sectional studies . tpai-1 and fabp4 thus , switching -gis from acarbose or voglibose to miglitol may not reduce lipid abnormalities related to atherogenesis risk . it has been reported from an rct conducted in germany that drugs improving lipid metabolism ( insulin resistance ) such as metformin and pioglitazone and their combination reduced tpai-1 concentrations in type 2 diabetic patients receiving stable basal insulin therapy , although it is still unclear whether circulating fabp4 concentrations are reduced by these drugs . the combination of miglitol with these drugs for improving insulin resistance may reduce cvd development by decreasing circulating concentrations of tpai-1 , mcp-1 , and se - selectin . switching from acarbose or voglibose to miglitol for 3 months has been found to reduce hypoglycemic symptoms and blood glucose concentrations between meals . further large - scale studies should examine whether miglitol treatment of type 2 diabetic patients reduces hypoglycemia assessed by smbg and hypoglycemic symptoms , such as hypoglycemia - induced lethargy , compared with other -gis . additionally , whether slight and severe degrees of hypoglycemia induce circulating protein concentrations of mcp-1 and se - selectin , and whether the reduction of hypoglycemia by miglitol reduces circulating protein concentrations of mcp-1 and se - selectin and cvd incidence in type 2 diabetic patients , should be examined . in addition , it should be noted that we analyzed samples from 35 of the 43 patients who completed the study because serum samples were not obtained from eight patients . our previous study using the same sample demonstrated that glucose fluctuations in 43 type 2 diabetic japanese patients were reduced by switching from acarbose or voglibose to miglitol for 3 months . a double - blind , crossover design in 15 type 2 diabetic patients found that treatment with miglitol ( 300 mg / day ) effectively reduced postprandial blood glucose levels over 8 weeks . in addition , our previous study reported that the switch of -gi from acarbose or voglibose to miglitol in 43 type 2 diabetic patients reduced glucose fluctuations and expression of inflammatory cytokine genes , such as il-1 and tnf- , in peripheral leukocytes and the circulating protein concentrations of tnf- . from these studies , we considered that our sample of 35 type 2 diabetic japanese patients is comparable ; however , a large - scale rct is needed to examine whether miglitol reduces glucose fluctuations and circulating concentrations of cvd risk factors in type 2 diabetic patients compared with other -gis . a study of ten type 2 diabetic patients hospitalized for 4 days found that glucose fluctuations , which were monitored by cgm , in a standard meal loading were reduced effectively by treatment with miglitol ( 50 mg ) compared with acarbose ( 100 mg ) . in addition , in this study we demonstrated that switching -gis from acarbose or voglibose to miglitol in type 2 diabetic japanese patients reduced glucose fluctuations , which were assessed by the averages at just before and 1 h after each meal measured over 5 days by smbg . combining our results with the results from cgm in a previous study however , it is still unclear whether glucose fluctuations were lower in type 2 diabetic patients who were treated longer with miglitol than in those who were treated longer with other -gis . rct trials , in which dietary habits are well controlled , should examine whether glucose fluctuations by long - term cgm are lower in type 2 diabetic patients treated with miglitol than in those treated with acarbose or voglibose . whether miglitol treatment reduces circulating cvd risk factors including mcp-1 and se - selectin in type 2 diabetic japanese patients needs to be examined in an rct the results of this study indicate that switching from acarbose or voglibose to miglitol for 3 months suppressed glucose fluctuations and serum protein concentrations of mcp-1 and se - selectin more effectively than the prior -gi .

cell therapy ( ct ) in the form of intramuscular ( i m ) administration of satellite cell - derived myoblasts ( scdms ) is a potential treatment for muscle pathologies . its first indication was duchenne muscular dystrophy , but later clinical applications sought treatment of selectively affected muscles , such as the cricopharyngeal muscle in oculopharyngeal muscular dystrophy , the tibialis anterior in patients with fascioscapular muscular dystrophy , the urinary sphincter in urinary incontinence , and the external anal sphincter in anal incontinence . note that satellite cell derived is specified to make a difference between myoblasts involved in postnatal muscle regeneration ( i.e. , scdms ) and somite - derived myoblasts involved in muscle embryogenesis . ( 1 ) when injected scdms fuse with the myofibers of the host tissue , graft - derived myonuclei allow gene complementation , which would be useful for the treatment of genetic myopathies . ( 2 ) to the extent that the transplanted scdms fuse with each other , they could generate new myofibers , which would be useful to recover muscles that lost myofibers due to pathological causes . ( 3 ) since some transplanted scdms become satellite cells,6 , 7 , 8 , 9 , 10 , 11 , 12 this ct could repopulate the reserve of muscle - specific stem cells in the recipient . in animals , scdms definitely produced the first13 , 14 , 15 , 16 and second outcomes,14 , 17 and there is potential evidence of the third one.6 , 14 , 18 an important issue in ct is the early post - transplantation cell survival . the fact that there is a significant death of the grafted cells was in fact reported in several types of cts , including transplantation of neurons , hepatocytes , cardiac cells , and islets . with regard to scdm transplantation , it was reported using different methods of detection in mice11 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 and pigs that the grafted scdms experience considerable mortality within the first days after their i m administration . however , the observations in mice showed significant differences between different research teams , especially in relation to the dynamics and intensity of the cell death . however , this problem was never studied in non - human primates ( nhps ) , which are the optimal model for human extrapolation in preclinical transplantation research because of the narrow phylogenetic relationship and biological similitudes . therefore , we analyzed the post - ct death of scdms in macaques ( table 1 ) , both to obtain information more extrapolable to the human and to help clarify an issue in which there are conflicting interpretations . for this , we proliferated autologous or allogeneic scdms in vitro , which were radiolabeled with [ c]thymidine before i m administration to quantify the grafted - cell death by measuring the radioactivity in the dna isolated from the cell - grafted muscles.table 1monkeys included in the study , indicating details of the protocol and quantification of acute rejectionmonkeyspeciesistype of graftorigin of cellsareporter proteinamount of cells per sitecell death pre - ct ( % ) bfollow - updead cellscct ex vivodcd8 cells at post - ct day 7e1rhesus allomonkey 22 102.47 daysxx allomonkey 23.3 104.87 days x automonkey 43.5 1087 days allons19-gal1.5 1077 days+++11rhesusyesallomonkey 2-gal1.7 10133 weeks12cynoyesallons19-gal3 1053 weeks13cynoyesallons19-gal2.7 107.93 weeks-gal , -galactosidase ; allo , allogeneic ; auto , autologous ; ct , cell transplantation ; cyno , cynomolgus ; is , immunosuppression ; , the macaque female included in the study.ans19 : identification of the cynomolgus donor of the cells.bestimated by a trypan blue exclusion test.cinjection of cells killed by freezing and thawing.dcells injected into pieces of muscle taken at euthanasia.enumber of cd8 cells per mm of muscle section as follows : , < 3 ; + , 310 ; + + , 1030 ; + + + , > 30 . monkeys included in the study , indicating details of the protocol and quantification of acute rejection -gal , -galactosidase ; allo , allogeneic ; auto , autologous ; ct , cell transplantation ; cyno , cynomolgus ; is , immunosuppression ; , the macaque female included in the study . , < 3 ; + , 310 ; + + , 1030 ; + + + , > 30 . we tested whether there were differences in the radiolabel detection when the cells were injected ex vivo on a muscle fragment ( where we could ensure that all cells were inside the muscle sample before freezing ) compared to a cell - grafted site performed in vivo and biopsied 1 hr later ( we waited 1 hr to allow the saline of the cell suspension to be absorbed ) , which was our first choice as a reference for the radiolabel loss . the radioactivity in the muscles injected with the [ c]thymidine - labeled cells was measured in the total dna isolated from the muscle samples in a liquid scintillation counter as counts per minute ( cpm ) and normalized per million of injected cells . the radioactivity measured in the muscle fragments injected ex vivo was 3,763 1,076 cpm/10 cells and in the biopsies sampled 1 hr post - ct in vivo was 3,324 1,139/10 cells , with no significant difference ( figure 1a).figure 1quantification of [ c]thymidine in validation tests(a ) comparing the radiolabel measured in the scintillation counter in cpm per 10 of grafted cells , there was no significant difference between muscle fragments injected with cells ex vivo and biopsies of the regions injected with cells in vivo and sampled 1 hr later . ( b and c ) since the presence or absence of radioactivity in the biopsies is extrapolated as survival or death of the grafted cells , we wanted to evaluate the effectiveness of the monkey organism in the present context to remove the radioactivity present in dead cells after 1 day of their administration . this was evaluated by transplanting cells killed by freezing and thawing in a site that was sampled 1 day later . comparing the results in cpm per 10 cells ( b ) , there was again no significant difference between muscle fragments injected with cells ex vivo and biopsies 1 hr post - ct in vivo . there was a significant decrease of radioactivity detected at day 1 in the site injected with the dead cells . ( c ) taking as 100% either muscle fragments injected ex vivo or biopsies 1 hr post - ct , the loss of [ c]thymidine detected in the site grafted with dead cells was very similar , respectively , at 86.6% 7.4% and 86.2% 6.4% ( indicated by arrows ) . the bars correspond to the mean with the sd except for those used for the 100% reference , which were fixed to that value . quantification of [ c]thymidine in validation tests ( a ) comparing the radiolabel measured in the scintillation counter in cpm per 10 of grafted cells , there was no significant difference between muscle fragments injected with cells ex vivo and biopsies of the regions injected with cells in vivo and sampled 1 hr later . ( b and c ) since the presence or absence of radioactivity in the biopsies is extrapolated as survival or death of the grafted cells , we wanted to evaluate the effectiveness of the monkey organism in the present context to remove the radioactivity present in dead cells after 1 day of their administration . this was evaluated by transplanting cells killed by freezing and thawing in a site that was sampled 1 day later . comparing the results in cpm per 10 cells ( b ) , there was again no significant difference between muscle fragments injected with cells ex vivo and biopsies 1 hr post - ct in vivo . there was a significant decrease of radioactivity detected at day 1 in the site injected with the dead cells . ( c ) taking as 100% either muscle fragments injected ex vivo or biopsies 1 hr post - ct , the loss of [ c]thymidine detected in the site grafted with dead cells was very similar , respectively , at 86.6% 7.4% and 86.2% 6.4% ( indicated by arrows ) . the bars correspond to the mean with the sd except for those used for the 100% reference , which were fixed to that value . to verify the accuracy of [ c ] thymidine quantification to reveal the grafted - cell death , a cell pellet from those prepared for transplantation was submitted to three cycles of freezing and thawing to kill the cells in four monkeys ( table 1 ) , and it was injected similarly as the live cells at one site that was sampled 1 day later . the results in cpm/10 cells ( figure 1b ) showed again no significant differences between muscle fragments injected ex vivo ( 4,111 803 cpm/10 cells ) and biopsies 1 hr post - ct in vivo ( 3,745 456 cpm/10 cells ) . although substantially reduced , there was some radioactivity in the biopsies of the sites injected with dead cells ( 516 233 cpm/10 cells ) . in figure 1c we compared the radiolabel remaining in the site injected with dead cells , taking as reference both the muscle fragments injected ex vivo and the biopsies 1 hr post - ct . the remaining radiolabel was , respectively , 13.4% 7.4% and 13.8% 6.4% . therefore , we chose the biopsies at 1 hr post - ct as a reference to analyze the loss of radiolabel , since the cell injection and muscle - sampling conditions were the same as the other biopsies in the post - ct follow - up . there were four different immuno - transplantation conditions in the study ( table 1 ) , which we consider separately . note that ( 1 ) and ( 2 ) were done in the same monkeys , which received autologous scdms on one side of the muscle and allogeneic scdms on the other side.(1)allogeneic ct into non - immunosuppressed rhesus monkeys ( figure 2a ) . the radioactivity at post - ct day 1 was 92.3% 13.7% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 17.4% 8.3% ( significant difference with day 1 ) , and at day 7 was 3.1% 1.6% ( significant difference with day 3).figure 2quantification of [ c]thymidine in the four immuno - transplantation conditions of the studythe arrows indicate the percentage of radiolabel loss between the means in the bars indicated by the fractional lines . note that ( a)(c ) correspond to the same monkeys , which received autografts in one side of the muscles and allografts in the other side . ( a ) allogeneic transplantation of non--gal - labeled cells into non - immunosuppressed recipients ( monkeys 15 ) . ( b and c ) autologous transplantation of non--gal - labeled cells into non - immunosuppressed recipients ( monkeys 15 ) . in ( b ) we represent the five monkeys used for this condition , while in ( c ) we represent in black bars the mean and sd of monkeys 14 , overlapping the results of monkey 5 in gray bars , to show the striking difference of this monkey with the others . ( d ) allogeneic transplantation of -gal - labeled cells into non - immunosuppressed recipients ( monkeys 610 ) . ( e ) allogeneic transplantation of -gal - labeled cells into immunosuppressed recipients ( monkeys 1113 ) . ( f ) comparison of the values of each immuno - transplantation condition within each period post - ct . when not indicated , there were no significant differences within each post - ct period . there are significant differences only between some values at post - ct day 3 , as indicated by brackets ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 ; n / s , not significant ) . the bars correspond to the mean with the sd except for those used for the 100% reference , which were fixed to that value.(2)autologous ct into non - immunosuppressed rhesus monkeys ( figures 2b and 2c ) . the radioactivity at post - ct day 1 was 102.9% 15.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 40.3% 29.4% ( significant difference with day 1 ) , and at day 7 was 14.3% 20% ( significant difference with day 3 ) ( figure 2b ) . the sd was wide at post - ct days 3 and 7 because in monkey 5 the radioactivity at those periods was much higher than in the others monkeys : 91% at post - ct day 3 and 49.9% at post - ct 7 . this was not due to a problem with the reference at 1 hr post - ct , given that in the muscle fragment injected ex vivo the radiolabel was 105.3% of 1 hr post - ct , that is , quite similar . excluding monkey 5 , the mean and sd of the other four monkeys are closer to those of the sites of allo - transplantation ( figure 2c ) : the radioactivity at day 1 was 98.9% 14.7% of 1 hr post - ct ( no significant difference ) , at day 3 was 27.7% 9.1% ( significant difference with day 1 ) , and at day 7 was 5.4% 2.2% ( significant difference with day 3 ) . given this striking difference of monkey 5 , figure 2c shows the mean of monkeys 14 , overlapping monkey 5 as gray bars.(3)allogeneic ct ( lacz labeled ) into non - immunosuppressed cynomolgus monkeys ( figure 2d ) . we tested scdms labeled with lacz because this is the standard in our ct studies in macaques . the radioactivity at day 1 was 87.9% 19.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 52.1% 18.1% ( significant difference with day 1 ) , and at day 7 was 4.4% 2.5% ( significant difference with day 3).(4)allogeneic ct ( lacz labeled ) into immunosuppressed cynomolgus and rhesus monkeys ( figure 2e ) . the radioactivity at day 3 was 55.3% 11.4% of that detected 1 hr post - ct ( significant difference ) , at day 7 was 8.4% 6.5% ( significant difference with day 3 ) , and at 3 weeks was 11.5% 7.9% ( no significant difference with day 7 ) . the radioactivity at post - ct day 1 was 92.3% 13.7% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 17.4% 8.3% ( significant difference with day 1 ) , and at day 7 was 3.1% 1.6% ( significant difference with day 3).figure 2quantification of [ c]thymidine in the four immuno - transplantation conditions of the studythe arrows indicate the percentage of radiolabel loss between the means in the bars indicated by the fractional lines . note that ( a)(c ) correspond to the same monkeys , which received autografts in one side of the muscles and allografts in the other side . ( a ) allogeneic transplantation of non--gal - labeled cells into non - immunosuppressed recipients ( monkeys 15 ) . ( b and c ) autologous transplantation of non--gal - labeled cells into non - immunosuppressed recipients ( monkeys 15 ) . in ( b ) we represent the five monkeys used for this condition , while in ( c ) we represent in black bars the mean and sd of monkeys 14 , overlapping the results of monkey 5 in gray bars , to show the striking difference of this monkey with the others . ( d ) allogeneic transplantation of -gal - labeled cells into non - immunosuppressed recipients ( monkeys 610 ) . ( e ) allogeneic transplantation of -gal - labeled cells into immunosuppressed recipients ( monkeys 1113 ) . ( f ) comparison of the values of each immuno - transplantation condition within each period post - ct . when not indicated , there were no significant differences within each post - ct period . there are significant differences only between some values at post - ct day 3 , as indicated by brackets ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 ; n / s , not significant ) . the bars correspond to the mean with the sd except for those used for the 100% reference , which were fixed to that value . quantification of [ c]thymidine in the four immuno - transplantation conditions of the study the arrows indicate the percentage of radiolabel loss between the means in the bars indicated by the fractional lines . note that ( a)(c ) correspond to the same monkeys , which received autografts in one side of the muscles and allografts in the other side . ( a ) allogeneic transplantation of non--gal - labeled cells into non - immunosuppressed recipients ( monkeys 15 ) . ( b and c ) autologous transplantation of non--gal - labeled cells into non - immunosuppressed recipients ( monkeys 15 ) . in ( b ) we represent the five monkeys used for this condition , while in ( c ) we represent in black bars the mean and sd of monkeys 14 , overlapping the results of monkey 5 in gray bars , to show the striking difference of this monkey with the others . ( d ) allogeneic transplantation of -gal - labeled cells into non - immunosuppressed recipients ( monkeys 610 ) . ( e ) allogeneic transplantation of -gal - labeled cells into immunosuppressed recipients ( monkeys 1113 ) . ( f ) comparison of the values of each immuno - transplantation condition within each period post - ct . when not indicated , there were no significant differences within each post - ct period . there are significant differences only between some values at post - ct day 3 , as indicated by brackets ( * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 ; n / s , not significant ) . the bars correspond to the mean with the sd except for those used for the 100% reference , which were fixed to that value . the radioactivity at post - ct day 1 was 102.9% 15.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 40.3% 29.4% ( significant difference with day 1 ) , and at day 7 was 14.3% 20% ( significant difference with day 3 ) ( figure 2b ) . the sd was wide at post - ct days 3 and 7 because in monkey 5 the radioactivity at those periods was much higher than in the others monkeys : 91% at post - ct day 3 and 49.9% at post - ct 7 . this was not due to a problem with the reference at 1 hr post - ct , given that in the muscle fragment injected ex vivo the radiolabel was 105.3% of 1 hr post - ct , that is , quite similar . excluding monkey 5 , the mean and sd of the other four monkeys are closer to those of the sites of allo - transplantation ( figure 2c ) : the radioactivity at day 1 was 98.9% 14.7% of 1 hr post - ct ( no significant difference ) , at day 3 was 27.7% 9.1% ( significant difference with day 1 ) , and at day 7 was 5.4% 2.2% ( significant difference with day 3 ) . given this striking difference of monkey 5 , figure 2c shows the mean of monkeys 14 , overlapping monkey 5 as gray bars . allogeneic ct ( lacz labeled ) into non - immunosuppressed cynomolgus monkeys ( figure 2d ) . we tested scdms labeled with lacz because this is the standard in our ct studies in macaques . the radioactivity at day 1 was 87.9% 19.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 52.1% 18.1% ( significant difference with day 1 ) , and at day 7 was 4.4% 2.5% ( significant difference with day 3 ) . allogeneic ct ( lacz labeled ) into immunosuppressed cynomolgus and rhesus monkeys ( figure 2e ) . the radioactivity at day 3 was 55.3% 11.4% of that detected 1 hr post - ct ( significant difference ) , at day 7 was 8.4% 6.5% ( significant difference with day 3 ) , and at 3 weeks was 11.5% 7.9% ( no significant difference with day 7 ) . comparing the values obtained for each immuno - transplantation condition in each post - ct period , there were significant differences between some values at post - ct day 3 , but not between values on day 1 or day 7 ( figure 2f ) . in addition to the sites injected with radiolabeled cells to quantify the cell death , a nearby site of ct was performed for histological analysis to detect potential acute rejection . clear evidence of ongoing acute rejection , in the form of dense accumulations of lymphocytes with an important constituent of cd8 cells ( 31.4 17.8 cd8 cells / mm ) , was observed at day 7 in monkeys transplanted with lacz - labeled scdms without immunosuppression . there was no histological evidence of acute rejection in the other groups , even in monkeys that were not immunosuppressed and received the allogeneic scdms without a reporter gene . in this case , a few cd8 lymphocytes were observed in the tissues at day 7 ( 2.7 1.8 cd8 cells / mm ) , scattered in the muscle and with a few small focal accumulations , generally perivascular . this was not significantly different from the muscles that received autologous scdms ( 1.9 2.3 cd8 cells / mm ) . we observed many -galactosidase ( -gal ) myofibers at 3 weeks post - ct in monkeys 1113 , with the pattern of bands of engraftment previously described in nhps ( figure 3 ) . this confirmed the consistency of the long - term results in this study in the context of previous studies of ct in nhps.figure 3histological graft result at 3 weekscross - section of a biopsy taken 3 weeks post - transplantation in a muscle region grafted with lacz - labeled scdms in a monkey of the immunosuppressed group , stained for -gal demonstration ( blue - greenish stain ) . many -gal myofibers are observed , following the pattern of bands of engraftment , which correspond to the original trajectories of the cell injections ( indicated by the red arrow ) . this was the area of the biopsy in which more -gal myofibers were observed : in the complete injected region , about 14% of the surface that was -gal . the inset is a higher magnification of the indicated region to more clearly show the myofiber profiles . histological graft result at 3 weeks cross - section of a biopsy taken 3 weeks post - transplantation in a muscle region grafted with lacz - labeled scdms in a monkey of the immunosuppressed group , stained for -gal demonstration ( blue - greenish stain ) . many -gal myofibers are observed , following the pattern of bands of engraftment , which correspond to the original trajectories of the cell injections ( indicated by the red arrow ) . this was the area of the biopsy in which more -gal myofibers were observed : in the complete injected region , about 14% of the surface that was -gal . the inset is a higher magnification of the indicated region to more clearly show the myofiber profiles . in vitro radiolabeling of dna is the most widely used method to evaluate the post - ct death of im - transplanted cells.27 , 28 , 31 , 33 , 37 , 38 with this method , the post - ct loss of radioactivity is extrapolated as cell death . since radiolabeled dna divides between daughter cells during mitosis , its quantification evidences the dynamics of cell death regardless of whether some surviving cells proliferate.27 , 33 this method does not immediately detect cell death because the radiolabeled dna can remain in the tissue as long as the dna of the dead cells is not eliminated , essentially by macrophages . therefore , the radiolabel loss reflects the magnitude and kinetics of grafted - cell death , but there could be a delay between the actual cell death and its demonstration by radiolabel loss . for this reason , we indicate remaining radiolabel instead of cell survival in the graphs . in this study , 1 day was sufficient to eliminate 86.2% 6.4% of the [ c]thymidine present in cells killed by freezing and thawing just before transplantation . this contrasts with that observed in mice , where 1 day was sufficient to essentially eliminate the entire radiolabel present in grafted cells killed by the same method . this difference could be explained by the faster elimination of necrotic tissues by macrophages in small rodents . the comparison of radioactivity in muscle fragments injected ex vivo and in biopsies taken 1 hr after in vivo ct coincides with our previous findings in mice , where there were no significant differences between muscles injected ex vivo and muscles sampled even 6 hr after in vivo ct . in fact , 6 hr post - ct in mice was not sufficient to detect cell death by this method , even when cells were killed before ct , most likely because 6 hr is too early for macrophage infiltration.28 , 40 overall , most of the radiolabel present in the grafted cells was eliminated from muscle biopsies during the first 7 days after ct , both in allogeneic and autologous conditions ( with the exception of autologous ct in monkey 5 , in which only approximately half was eliminated ) . the loss of the radiolabel was also independent of the following : ( 1 ) the use of cells after a few in vitro passages with no genetic manipulation or cells more proliferated in vitro after introduction of the lacz gene , ( 2 ) the use of immunosuppression or not , ( 3 ) the cell injection method , and ( 4 ) the presence or absence of acute rejection evidence at day 7 . in all cases , there was no difference between the radiolabel detected 1 hr and 1 day post - ct , there was a significant decrease between day 1 and day 3 ( with variations , sometimes significant , between the different experimental groups , range : 45%83% ) , and another significant decrease ( range : 80%92% , no significant differences between the different experimental groups ) detected between day 3 and day 7 . in the subsequent period , although the sample was not very large ( n = 3 ) and showed a wide variation , there was no significant difference between the radiolabel present at day 7 and 3 weeks post - ct . this relative homogeneity in post - ct cell death in nhps is noteworthy because in mice some differences in cell treatment greatly affected cell death , which was much faster and more intense using cloned scdms immortalized with t antigen than using primary scdms barely proliferated in vitro.28 , 40 scdms immortalized with t antigen were used in several studies of post - ct cell death in mice,25 , 26 , 27 , 33 , 40 , 41 which may be considered as one small animal model of ct among others . the extrapolation of these results in a clinical situation is questionable , due to the numerous differences in nature between models and their size , cell lines , and immunological contexts . the fact that there were no significant differences between the radiolabel detected at 7 days and 3 weeks post - ct can be explained by the fact that the vast majority of the grafted scdms had already fused at post - ct day 7 and were no longer mononuclear cells . since they became myonuclei , they were not exposed to the same threats to which grafted cells are exposed in the early post - ct period . indeed , long - term ct studies in monkeys ( up to a year and a half ) revealed that the only threat to graft - derived myonuclei is acute rejection under allogeneic conditions . although the scope of this study was not to investigate immunological aspects ( which will be studied more deeply later ) , it is interesting to observe that there was histological evidence of acute rejection at post - ct day 7 in non - immunosuppressed monkeys grafted with allogeneic cells labeled with lacz , but there was no histological evidence of acute rejection at the same period in non - immunosuppressed monkeys that received allogeneic scdms without a reporter gene . indeed , this can indicate that the expression of transgenic -gal provokes a stronger specific acquired immune reaction leading probably to a faster rejection . on the other hand , our observations confirm in nhps the observations made in mice that the acquired immune response is not responsible for the early death of the grafted scdms . increasing the amount of scdms transplanted in macaques increases the ct outcome until an optimal graft is achieved . therefore , the post - ct cell death does not prevent an optimal graft if a sufficient amount of cells is transplanted , as long as the cells are distributed to avoid the formation of large accumulations in which ischemia will develop . in mice , in fact , a part of the grafted cells always survives and partially or totally compensates the cell death.27 , 28 , 43 thus , a method to significantly reduce this post - ct cell death would have the advantage of grafting smaller amounts of cells , thereby reducing the resources and costs of cell culture ( which has practical importance ) , but it is not a sine qua non condition for the success of ct . of the various hypotheses on the cause of this cell death , initial studies in mice blamed acute inflammation,25 , 41 but specific depletion of macrophages , neutrophils , and natural killer cells did not prevent this post - ct cell death . hypoxia and anoikis were supposed to induce apoptosis in the grafted scdms , but experiments to control each produced only minimal survival improvements . an unresolved enigma is that , whatever the factor that causes the cell death , it never eliminates all grafted scdms . it was postulated that there is a specific subpopulation of scdms capable of evading the post - ct cell death,27 , 43 although without elucidating the causes of the cell death . only one mechanism of cell death was unequivocally confirmed following scdm transplantation in nhps : ischemic necrosis of the internal region of the cell accumulations , due to the limited diffusion of oxygen from the surrounding tissue . ischemic necrosis depends on the size of the cell collections , and this was the reason why in the present study we delivered small amounts of cells per injection . apart from this mechanism , the overall post - ct cell death is still not well understood , and further studies are needed to solve this problem . finally , it is important to remark that the present observations apply to ct in a muscle whose structure is preserved . it could be interesting to analyze what occurs in muscles that have partially or totally lost myofibers and were replaced by fibrous and/or adipose tissue , as is the case in several degenerative myopathies . the study included seven cynomolgus ( macaca fascicularis , six males and one female , age range 36 years , weight 3.97.9 kg ) and six rhesus ( macaca mulatta , males , age range 45 years 9 months , weight 810 kg ) macaques ( table 1 ) . for transplantations and biopsies , monkeys were kept under general anesthesia using isofluorane ( 1.5%2% in oxygen ) after induction with i m ketamine ( 10 mg / kg ) and glycopyrrolate ( 0.05 mg / kg ) . buprenorphine ( 0.01 mg / kg twice daily [ b.i.d . ] for 3 days ) was given i m for post - operative analgesia . due to the several muscle samples to be taken , euthanasia was done at the end of the experiments by intravenous pentobarbital overdose ( 120 mg / kg ) after anesthesia with i m ketamine ( 15 mg / kg ) . cynomolgus monkeys were transplanted with scdms from a primary cell line obtained previously from another cynomolgus monkey and labeled with a lacz reporter gene . most rhesus monkeys were transplanted with scdms of primary cell lines without genetic manipulation , produced from muscle biopsies taken in the same monkeys of the study . the exception was monkey 11 ( table 1 ) , for which the cells of another rhesus monkey were labeled with lacz . in all cases , muscle samples were minced with fine scissors into fragments of less than 1 mm and then dissociated with 0.2% collagenase ( sigma ) in hank s balanced salt solution ( hbss , gibco ) for 1 hr , followed by another dissociation in 0.125% trypsin ( gibco ) in hbss for 45 min . the cells were sub - cultured in mcdb-120 with 15% fetal bovine serum ( fbs , hyclone ) , 10 ng / ml basic fibroblast growth factor ( feldan ) , 0.5 mg / ml bsa ( sigma ) , 1.0 m dexamethasone ( sigma ) , and 5 g / ml human insulin ( sigma ) . lacz cells were produced by in vitro infection with a replication - defective retroviral vector lnpozc7 ( gift of dr . constance cepko , harvard university ) encoding a lacz reporter gene and a neomycin resistance gene , selected with 600 g / ml geneticin ( invitrogen ) , proliferated until confluence , and frozen for storage in liquid nitrogen . rhesus scdms without reporter genes were frozen for storage in liquid nitrogen after the first passage . in preparation for ct , the frozen cells were thawed and proliferated during one or two passages in culture . cell radiolabeling was performed by adding 0.25 ci / ml [ methyl - c ] thymidine ( amersham biosciences ) to the growth medium 24 hr prior to ct . for transplantation , the cells were detached from the flasks using 0.1% trypsin in hbss , washed three times with hbss , and resuspended in hbss for injection . the cell viability ( table 1 ) was verified with a trypan blue exclusion test , adding 0.1 ml 0.4% trypan blue ( sigma ) solution to 1 ml cell suspension to be examined in a hemocytometer . to verify the accuracy of the [ c ] thymidine quantification to reveal the grafted - cell death , cell pellets were submitted to three cycles of freezing and thawing to kill the cells , and they were injected similarly as the living cells in one site of four monkeys ( table 1 ) . the muscles used for ct were the biceps brachii , the quadriceps femoris , and , less frequently , the gastrocnemius . one muscle per period was used in each monkey , varying the muscle throughout the study . for radiolabeled cells , ct was performed by exposing the muscle after a skin incision , visually controlling the cell injection , and confirming that there were no leaks . in monkeys 110 , we delivered the cells in small superficial pockets to visually control that they went into the region to be sampled later . we used 50-l precision syringes ( hamilton ) and 27-gauge 1/2 length disposable needles , delicately injecting 20 or 30 l cell suspension at each site . in monkeys 1113 ( long - term follow - up under immunosuppression ) , we delivered the cells by matrices of parallel equidistant injections , which is our standard for long - term studies of ct in monkeys and humans . matrices were smaller than in our routine ct in monkeys ( 0.25 cm , 5 mm depth ) because we wanted to sample the entire grafted region in biopsies that did not exceed 1 cm . we used 100-l precision syringes ( hamilton ) attached to pb600 - 1 repeating dispensers ( hamilton ) and 27-gauge 1/2 length disposable needles . the inter - injection distance was 1 mm , delivering 2 l cell suspension per i m needle path . in addition , a nearby site of ct was performed for histological analysis to evidence potential acute rejection . the cells injected into these sites were not radiolabeled . to identify cell - grafted sites during biopsies , stitches of inert non - absorbable polypropylene 4.0 suture ( prolene , ethicon ) monkeys 1113 were immunosuppressed with an i m formulation of tacrolimus ( a generous gift from astellas pharma ) , using our standard protocol for ct studies in nhps . immunosuppression started 57 days before ct and was maintained until the end of the experiment . tacrolimus was injected i m once a day , beginning at 0.5 mg / kg / day and adjusting the dosage to target blood levels of > 50 g / l , as quantified in blood samples with an imx tacrolimus ii kit for micro - particle enzyme immunoassay ( abbott ) . muscle biopsies were performed in the ct sites 1 hr , 1 day , 3 days , and 7 days post - ct in non - immunosuppressed monkeys ( 110 ) and 1 hr , 3 days , 7 days , and 3 weeks post - ct in immunosuppressed monkeys ( 1113 ) . biopsies to quantify the radiolabel were placed in microtubes , frozen in liquid nitrogen , and stored at 80c until dna extraction . biopsies for histology were mounted in embedding medium , snap - frozen in liquid nitrogen , and stored at 80c until performing serial sections of 1015 m in a cryostat at 25c . to compare the radiolabel detection after cell injection performed ex vivo on a muscle fragment with in vivo ct biopsied 1 hr later , we froze a cell pellet similar to those used for in vivo ct in some monkeys . after euthanasia in these monkeys , we dissected a fragment of ungrafted muscle ( similar to biopsies of cell - grafted sites ) , thawed the frozen cells , and injected them into the muscle fragment , which was frozen in liquid nitrogen and stored at 80c until dna extraction . the amount of radiolabel in the muscle injected with the [ c]thymidine - labeled cells was measured in the total dna isolated from the muscle samples as in previous mouse studies . similar aliquots of the dna solution were mixed with 5 ml liquid scintillation counting cocktail ( sigma ) , and the radioactivity was measured in a wallac 1409 liquid scintillation counter ( perkinelmer ) as cpm . sections were stained with h&e for standard analysis . for -gal histochemical detection in muscles transplanted with lacz cells , sections were fixed 3 min in 0.25% glutaraldehyde , rinsed with pbs , and incubated 24 hr at room temperature in 0.4 mg / ml x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ) ( boehringer mannheim ) with 1 mm mgcl2 , 3 mm ke3f(cn)6,3 mm ke4f(cn)6 , and3h20 in pbs . to depict acute rejection , we immunodetected cd8 lymphocytes using a mouse anti - human / macaque cd8 monoclonal antibody ( bd biosciences ) , followed by a 30-min incubation with a biotinylated anti - mouse antibody ( 1/150 , dako ) and either a 30-min incubation with streptavidin - cy3 ( 1/700 , sigma ) or a 30-min incubation in streptavidin - peroxidase ( 1/200 , dako ) . peroxidase activity was revealed with 3,3 diaminobenzidine ( 0.25 mg / ml ) and 0.03% hydrogen peroxide . stained sections were analyzed using an axiophot microscope with epifluorescence and bright - field optics ( zeiss ) . to estimate the density of cd8 cells , we counted the number of cd8 cells per muscle section and the area of the section was measured using imagej software . for the bar graphs , we estimated the mean value of n = 310 monkeys 1 sd . a paired t test was used to assess the probability of significant differences between successive periods in the follow - up of the radiolabel loss ( i.e. , comparing each period with the precedent , as indicated in figures 1 and 2a2e ) , given that the number of values was similar for each period and each sequence of values corresponded to a same monkey . when comparing values from different experimental groups , we used an unpaired t test ( figure 2f ) . statistical significance was defined as p < 0.05 . conceptualization , methodology , validation , formal analysis , investigation , visualization , project administration , and writing original draft , d.s . ; writing review & editing , funding acquisition , and resources , d.s . and j.p.t .

cell therapy could be useful for the treatment of myopathies . a problem observed in mice , with different results and interpretations , is a significant death among the transplanted cells . we analyzed this problem in non - human primates , the animal model more similar to humans . autologous or allogeneic myoblasts ( with or without a reporter gene ) were proliferated in vitro , labeled with [ 14c]thymidine , and intramuscularly injected in macaques . some monkeys were immunosuppressed for long - term follow - up . cell - grafted regions were biopsied at different intervals and analyzed by radiolabel quantification and histology . most radiolabel was lost during the first week after injection , regardless of whether the cells were allogeneic or autologous , the culture conditions , and the use or not of immunosuppression . there was no significant difference between 1 hr and 1 day post - transplantation , a significant decrease between days 1 and 3 ( 45% to 83% ) , a significant decrease between days 3 and 7 ( 80% to 92% ) , and no significant differences between 7 days and 3 weeks . our results confirmed in non - human primates a progressive and significant death of the grafted myoblasts during the first week after administration , relatively similar to some observations in mice but with different kinetics .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

cell therapy ( ct ) in the form of intramuscular ( i m ) administration of satellite cell - derived myoblasts ( scdms ) is a potential treatment for muscle pathologies . its first indication was duchenne muscular dystrophy , but later clinical applications sought treatment of selectively affected muscles , such as the cricopharyngeal muscle in oculopharyngeal muscular dystrophy , the tibialis anterior in patients with fascioscapular muscular dystrophy , the urinary sphincter in urinary incontinence , and the external anal sphincter in anal incontinence . ( 1 ) when injected scdms fuse with the myofibers of the host tissue , graft - derived myonuclei allow gene complementation , which would be useful for the treatment of genetic myopathies . the fact that there is a significant death of the grafted cells was in fact reported in several types of cts , including transplantation of neurons , hepatocytes , cardiac cells , and islets . however , the observations in mice showed significant differences between different research teams , especially in relation to the dynamics and intensity of the cell death . however , this problem was never studied in non - human primates ( nhps ) , which are the optimal model for human extrapolation in preclinical transplantation research because of the narrow phylogenetic relationship and biological similitudes . therefore , we analyzed the post - ct death of scdms in macaques ( table 1 ) , both to obtain information more extrapolable to the human and to help clarify an issue in which there are conflicting interpretations . for this , we proliferated autologous or allogeneic scdms in vitro , which were radiolabeled with [ c]thymidine before i m administration to quantify the grafted - cell death by measuring the radioactivity in the dna isolated from the cell - grafted muscles.table 1monkeys included in the study , indicating details of the protocol and quantification of acute rejectionmonkeyspeciesistype of graftorigin of cellsareporter proteinamount of cells per sitecell death pre - ct ( % ) bfollow - updead cellscct ex vivodcd8 cells at post - ct day 7e1rhesus allomonkey 22 102.47 daysxx allomonkey 23.3 104.87 days x automonkey 43.5 1087 days allons19-gal1.5 1077 days+++11rhesusyesallomonkey 2-gal1.7 10133 weeks12cynoyesallons19-gal3 1053 weeks13cynoyesallons19-gal2.7 107.93 weeks-gal , -galactosidase ; allo , allogeneic ; auto , autologous ; ct , cell transplantation ; cyno , cynomolgus ; is , immunosuppression ; , the macaque female included in the study.ans19 : identification of the cynomolgus donor of the cells.bestimated by a trypan blue exclusion test.cinjection of cells killed by freezing and thawing.dcells injected into pieces of muscle taken at euthanasia.enumber of cd8 cells per mm of muscle section as follows : , < 3 ; + , 310 ; + + , 1030 ; + + + , > 30 . we tested whether there were differences in the radiolabel detection when the cells were injected ex vivo on a muscle fragment ( where we could ensure that all cells were inside the muscle sample before freezing ) compared to a cell - grafted site performed in vivo and biopsied 1 hr later ( we waited 1 hr to allow the saline of the cell suspension to be absorbed ) , which was our first choice as a reference for the radiolabel loss . the radioactivity measured in the muscle fragments injected ex vivo was 3,763 1,076 cpm/10 cells and in the biopsies sampled 1 hr post - ct in vivo was 3,324 1,139/10 cells , with no significant difference ( figure 1a).figure 1quantification of [ c]thymidine in validation tests(a ) comparing the radiolabel measured in the scintillation counter in cpm per 10 of grafted cells , there was no significant difference between muscle fragments injected with cells ex vivo and biopsies of the regions injected with cells in vivo and sampled 1 hr later . comparing the results in cpm per 10 cells ( b ) , there was again no significant difference between muscle fragments injected with cells ex vivo and biopsies 1 hr post - ct in vivo . quantification of [ c]thymidine in validation tests ( a ) comparing the radiolabel measured in the scintillation counter in cpm per 10 of grafted cells , there was no significant difference between muscle fragments injected with cells ex vivo and biopsies of the regions injected with cells in vivo and sampled 1 hr later . ( b and c ) since the presence or absence of radioactivity in the biopsies is extrapolated as survival or death of the grafted cells , we wanted to evaluate the effectiveness of the monkey organism in the present context to remove the radioactivity present in dead cells after 1 day of their administration . comparing the results in cpm per 10 cells ( b ) , there was again no significant difference between muscle fragments injected with cells ex vivo and biopsies 1 hr post - ct in vivo . to verify the accuracy of [ c ] thymidine quantification to reveal the grafted - cell death , a cell pellet from those prepared for transplantation was submitted to three cycles of freezing and thawing to kill the cells in four monkeys ( table 1 ) , and it was injected similarly as the live cells at one site that was sampled 1 day later . the results in cpm/10 cells ( figure 1b ) showed again no significant differences between muscle fragments injected ex vivo ( 4,111 803 cpm/10 cells ) and biopsies 1 hr post - ct in vivo ( 3,745 456 cpm/10 cells ) . the radioactivity at post - ct day 1 was 92.3% 13.7% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 17.4% 8.3% ( significant difference with day 1 ) , and at day 7 was 3.1% 1.6% ( significant difference with day 3).figure 2quantification of [ c]thymidine in the four immuno - transplantation conditions of the studythe arrows indicate the percentage of radiolabel loss between the means in the bars indicated by the fractional lines . the radioactivity at post - ct day 1 was 102.9% 15.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 40.3% 29.4% ( significant difference with day 1 ) , and at day 7 was 14.3% 20% ( significant difference with day 3 ) ( figure 2b ) . the sd was wide at post - ct days 3 and 7 because in monkey 5 the radioactivity at those periods was much higher than in the others monkeys : 91% at post - ct day 3 and 49.9% at post - ct 7 . this was not due to a problem with the reference at 1 hr post - ct , given that in the muscle fragment injected ex vivo the radiolabel was 105.3% of 1 hr post - ct , that is , quite similar . excluding monkey 5 , the mean and sd of the other four monkeys are closer to those of the sites of allo - transplantation ( figure 2c ) : the radioactivity at day 1 was 98.9% 14.7% of 1 hr post - ct ( no significant difference ) , at day 3 was 27.7% 9.1% ( significant difference with day 1 ) , and at day 7 was 5.4% 2.2% ( significant difference with day 3 ) . the radioactivity at day 1 was 87.9% 19.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 52.1% 18.1% ( significant difference with day 1 ) , and at day 7 was 4.4% 2.5% ( significant difference with day 3). the radioactivity at day 3 was 55.3% 11.4% of that detected 1 hr post - ct ( significant difference ) , at day 7 was 8.4% 6.5% ( significant difference with day 3 ) , and at 3 weeks was 11.5% 7.9% ( no significant difference with day 7 ) . the radioactivity at post - ct day 1 was 92.3% 13.7% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 17.4% 8.3% ( significant difference with day 1 ) , and at day 7 was 3.1% 1.6% ( significant difference with day 3).figure 2quantification of [ c]thymidine in the four immuno - transplantation conditions of the studythe arrows indicate the percentage of radiolabel loss between the means in the bars indicated by the fractional lines . the radioactivity at post - ct day 1 was 102.9% 15.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 40.3% 29.4% ( significant difference with day 1 ) , and at day 7 was 14.3% 20% ( significant difference with day 3 ) ( figure 2b ) . the sd was wide at post - ct days 3 and 7 because in monkey 5 the radioactivity at those periods was much higher than in the others monkeys : 91% at post - ct day 3 and 49.9% at post - ct 7 . excluding monkey 5 , the mean and sd of the other four monkeys are closer to those of the sites of allo - transplantation ( figure 2c ) : the radioactivity at day 1 was 98.9% 14.7% of 1 hr post - ct ( no significant difference ) , at day 3 was 27.7% 9.1% ( significant difference with day 1 ) , and at day 7 was 5.4% 2.2% ( significant difference with day 3 ) . the radioactivity at day 1 was 87.9% 19.6% of that detected 1 hr post - ct ( no significant difference ) , at day 3 was 52.1% 18.1% ( significant difference with day 1 ) , and at day 7 was 4.4% 2.5% ( significant difference with day 3 ) . the radioactivity at day 3 was 55.3% 11.4% of that detected 1 hr post - ct ( significant difference ) , at day 7 was 8.4% 6.5% ( significant difference with day 3 ) , and at 3 weeks was 11.5% 7.9% ( no significant difference with day 7 ) . comparing the values obtained for each immuno - transplantation condition in each post - ct period , there were significant differences between some values at post - ct day 3 , but not between values on day 1 or day 7 ( figure 2f ) . there was no histological evidence of acute rejection in the other groups , even in monkeys that were not immunosuppressed and received the allogeneic scdms without a reporter gene . in this case , a few cd8 lymphocytes were observed in the tissues at day 7 ( 2.7 1.8 cd8 cells / mm ) , scattered in the muscle and with a few small focal accumulations , generally perivascular . this confirmed the consistency of the long - term results in this study in the context of previous studies of ct in nhps.figure 3histological graft result at 3 weekscross - section of a biopsy taken 3 weeks post - transplantation in a muscle region grafted with lacz - labeled scdms in a monkey of the immunosuppressed group , stained for -gal demonstration ( blue - greenish stain ) . in vitro radiolabeling of dna is the most widely used method to evaluate the post - ct death of im - transplanted cells.27 , 28 , 31 , 33 , 37 , 38 with this method , the post - ct loss of radioactivity is extrapolated as cell death . the comparison of radioactivity in muscle fragments injected ex vivo and in biopsies taken 1 hr after in vivo ct coincides with our previous findings in mice , where there were no significant differences between muscles injected ex vivo and muscles sampled even 6 hr after in vivo ct . in fact , 6 hr post - ct in mice was not sufficient to detect cell death by this method , even when cells were killed before ct , most likely because 6 hr is too early for macrophage infiltration.28 , 40 overall , most of the radiolabel present in the grafted cells was eliminated from muscle biopsies during the first 7 days after ct , both in allogeneic and autologous conditions ( with the exception of autologous ct in monkey 5 , in which only approximately half was eliminated ) . the loss of the radiolabel was also independent of the following : ( 1 ) the use of cells after a few in vitro passages with no genetic manipulation or cells more proliferated in vitro after introduction of the lacz gene , ( 2 ) the use of immunosuppression or not , ( 3 ) the cell injection method , and ( 4 ) the presence or absence of acute rejection evidence at day 7 . in all cases , there was no difference between the radiolabel detected 1 hr and 1 day post - ct , there was a significant decrease between day 1 and day 3 ( with variations , sometimes significant , between the different experimental groups , range : 45%83% ) , and another significant decrease ( range : 80%92% , no significant differences between the different experimental groups ) detected between day 3 and day 7 . in the subsequent period , although the sample was not very large ( n = 3 ) and showed a wide variation , there was no significant difference between the radiolabel present at day 7 and 3 weeks post - ct . this relative homogeneity in post - ct cell death in nhps is noteworthy because in mice some differences in cell treatment greatly affected cell death , which was much faster and more intense using cloned scdms immortalized with t antigen than using primary scdms barely proliferated in vitro.28 , 40 scdms immortalized with t antigen were used in several studies of post - ct cell death in mice,25 , 26 , 27 , 33 , 40 , 41 which may be considered as one small animal model of ct among others . the fact that there were no significant differences between the radiolabel detected at 7 days and 3 weeks post - ct can be explained by the fact that the vast majority of the grafted scdms had already fused at post - ct day 7 and were no longer mononuclear cells . although the scope of this study was not to investigate immunological aspects ( which will be studied more deeply later ) , it is interesting to observe that there was histological evidence of acute rejection at post - ct day 7 in non - immunosuppressed monkeys grafted with allogeneic cells labeled with lacz , but there was no histological evidence of acute rejection at the same period in non - immunosuppressed monkeys that received allogeneic scdms without a reporter gene . on the other hand , our observations confirm in nhps the observations made in mice that the acquired immune response is not responsible for the early death of the grafted scdms . in mice , in fact , a part of the grafted cells always survives and partially or totally compensates the cell death.27 , 28 , 43 thus , a method to significantly reduce this post - ct cell death would have the advantage of grafting smaller amounts of cells , thereby reducing the resources and costs of cell culture ( which has practical importance ) , but it is not a sine qua non condition for the success of ct . of the various hypotheses on the cause of this cell death , initial studies in mice blamed acute inflammation,25 , 41 but specific depletion of macrophages , neutrophils , and natural killer cells did not prevent this post - ct cell death . apart from this mechanism , the overall post - ct cell death is still not well understood , and further studies are needed to solve this problem . the cells were sub - cultured in mcdb-120 with 15% fetal bovine serum ( fbs , hyclone ) , 10 ng / ml basic fibroblast growth factor ( feldan ) , 0.5 mg / ml bsa ( sigma ) , 1.0 m dexamethasone ( sigma ) , and 5 g / ml human insulin ( sigma ) . for transplantation , the cells were detached from the flasks using 0.1% trypsin in hbss , washed three times with hbss , and resuspended in hbss for injection . to verify the accuracy of the [ c ] thymidine quantification to reveal the grafted - cell death , cell pellets were submitted to three cycles of freezing and thawing to kill the cells , and they were injected similarly as the living cells in one site of four monkeys ( table 1 ) . in monkeys 1113 ( long - term follow - up under immunosuppression ) , we delivered the cells by matrices of parallel equidistant injections , which is our standard for long - term studies of ct in monkeys and humans . to identify cell - grafted sites during biopsies , stitches of inert non - absorbable polypropylene 4.0 suture ( prolene , ethicon ) monkeys 1113 were immunosuppressed with an i m formulation of tacrolimus ( a generous gift from astellas pharma ) , using our standard protocol for ct studies in nhps . muscle biopsies were performed in the ct sites 1 hr , 1 day , 3 days , and 7 days post - ct in non - immunosuppressed monkeys ( 110 ) and 1 hr , 3 days , 7 days , and 3 weeks post - ct in immunosuppressed monkeys ( 1113 ) . after euthanasia in these monkeys , we dissected a fragment of ungrafted muscle ( similar to biopsies of cell - grafted sites ) , thawed the frozen cells , and injected them into the muscle fragment , which was frozen in liquid nitrogen and stored at 80c until dna extraction . similar aliquots of the dna solution were mixed with 5 ml liquid scintillation counting cocktail ( sigma ) , and the radioactivity was measured in a wallac 1409 liquid scintillation counter ( perkinelmer ) as cpm . a paired t test was used to assess the probability of significant differences between successive periods in the follow - up of the radiolabel loss ( i.e.

pigment dispersion syndrome ( pds ) results from posterior bowing of the iris and rubbing between the lens zonules and epithelial layer of the iris , and usually affects myopic eyes in men during the third to fourth decade of life . the literature indicated that caucasians have the highest prevalence of pds among all races , with clinical characteristics including krukenberg spindle , homogeneous trabecular meshwork ( tm ) pigmentation , and spoke - like mid - peripheral iris transillumination defects ( itds ) , which are referred as a triad of pds . zonular and lenticular pigmentation on posterior lens surface are also common findings in pds patients after mydriasis . pigmentary glaucoma ( pg ) is usually inevitable if pds is not detected and interrupted at an early stage . gradual pigmentation on the tm may lead to increased aqueous humor outflow resistance , resultant elevated intraocular pressure ( iop ) , and glaucomatous neuropathy . tm pigmentation has been widely accepted as the cause of pg , which is resultant from pds . as secondary open - angle glaucoma , pg has much in common with primary open - angle glaucoma ( poag ) in clinical manifestations and treatment principle except for laser iridoctomy to eliminate the reverse pupillary block . trabeculectomy is commonly applied in advanced pg patients when laser treatment and/or antiglaucoma medication fail to arrest the high iop . the literature has numerous references addressing efficacy and safety of trabeculectomy on poag , while it has quite limited references on clinical outcomes of pg . because the prevalence of pg is much lower than that of poag , the number of pds / pg patients is rare in clinical practice . as a consequent , it is difficult to collect enough pg patients for scientific research in majority of hospitals . the aim of the current study was to summarize the long - term clinical outcomes of trabeculectomy in 18 consecutive chinese patients with pg in north china , which can provide a valuable reference for ophthalmologists in the management of pg . all outpatients presenting for care at the glaucoma specialty clinic at beijing tongren eye center , beijing from may 2006 to april 2007 were evaluated for signs of pds : corneal endothelial pigmentation , anterior iris stromal pigment dusting , itds , posterior iris bowing , increased tm pigmentation , and pigment granule dusting on lens zonules or peripheral posterior surface . detailed ophthalmic examinations included visual acuity ( va ) , best - corrected visual acuity ( bcva ) , iop measurement , slit lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey swedish interactive thresholding algorithm ( sita)-standard 30 - 2 visual field analysis ( vfa ) . systemic and ocular medical histories were also recorded . all clinicians attended lectures on the clinical features of pds in different racial groups and how to detect subtle clinical signs of pds in pigmented racial patients before the study . corneal endothelial pigment dusting was described as : krukenberg spindle , diffusive pattern , or none . if the patient had received antiglaucoma medication before the evaluation , the iop measured before medication was taken as the initial iop . slit - lamp examination , gonioscopy , and funduscopic examination of the optic disc of all subjects were performed and graded by the same doctor ( dr . guo - ping qing ) in order to avoid inter - physician bias . diagnostic criteria for pds in chinese patients included at least two of the following three signs : krukenberg spindle , homogenous moderate to heavy tm pigmentation ( scheie ii ) , and any degree of zonular and/or lenticular pigment granule dusting . patients with a history of uveitis , trauma , previous ocular surgery or anterior segment laser treatment , or any evidence of exfoliation material were excluded . diagnosis of pg was made if pds patient had two or more of the following findings : initial iop > 21 mmhg , glaucomatous optic nerve damage ( increased cupping or glaucomatous disk appearance ) , or visual field ( vf ) defect . we certified that all applicable institutional and governmental regulations concerning the ethical use of human subjects were followed during this research . informed consent was obtained from each subject after explanation of the nature and possible consequences of the study , which was approved by the institutional review board of beijing tongren hospital , capital medical university . eighteen consecutive pg patients were identified and received trabeculectomy in at least one eye for medically refractory glaucoma . baseline examination included va , bcva , iop , slit - lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey sita - standard 30 - 2 vfa . postoperative iop , va , bcva , vfa parameters , optic disc cupping , morphological changes in bleb , and characteristic clinical findings of pg were main outcome parameters and were compared to the baseline values . all statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) . the paired - sample t - test was applied to compare postoperative iop , va , bcva , and vfa parameters at the last follow - up visit with the baseline . all outpatients presenting for care at the glaucoma specialty clinic at beijing tongren eye center , beijing from may 2006 to april 2007 were evaluated for signs of pds : corneal endothelial pigmentation , anterior iris stromal pigment dusting , itds , posterior iris bowing , increased tm pigmentation , and pigment granule dusting on lens zonules or peripheral posterior surface . detailed ophthalmic examinations included visual acuity ( va ) , best - corrected visual acuity ( bcva ) , iop measurement , slit lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey swedish interactive thresholding algorithm ( sita)-standard 30 - 2 visual field analysis ( vfa ) . systemic and ocular medical histories were also recorded . all clinicians attended lectures on the clinical features of pds in different racial groups and how to detect subtle clinical signs of pds in pigmented racial patients before the study . corneal endothelial pigment dusting was described as : krukenberg spindle , diffusive pattern , or none . if the patient had received antiglaucoma medication before the evaluation , the iop measured before medication was taken as the initial iop . slit - lamp examination , gonioscopy , and funduscopic examination of the optic disc of all subjects were performed and graded by the same doctor ( dr . guo - ping qing ) in order to avoid inter - physician bias . diagnostic criteria for pds in chinese patients included at least two of the following three signs : krukenberg spindle , homogenous moderate to heavy tm pigmentation ( scheie ii ) , and any degree of zonular and/or lenticular pigment granule dusting . patients with a history of uveitis , trauma , previous ocular surgery or anterior segment laser treatment , or any evidence of exfoliation material were excluded . diagnosis of pg was made if pds patient had two or more of the following findings : initial iop > 21 mmhg , glaucomatous optic nerve damage ( increased cupping or glaucomatous disk appearance ) , or visual field ( vf ) defect . we certified that all applicable institutional and governmental regulations concerning the ethical use of human subjects were followed during this research . informed consent was obtained from each subject after explanation of the nature and possible consequences of the study , which was approved by the institutional review board of beijing tongren hospital , capital medical university . eighteen consecutive pg patients were identified and received trabeculectomy in at least one eye for medically refractory glaucoma . baseline examination included va , bcva , iop , slit - lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey sita - standard 30 - 2 vfa . postoperative iop , va , bcva , vfa parameters , optic disc cupping , morphological changes in bleb , and characteristic clinical findings of pg were main outcome parameters and were compared to the baseline values . all enrolled patients completed the 8-year follow - up after trabeculectomy . the paired - sample t - test was applied to compare postoperative iop , va , bcva , and vfa parameters at the last follow - up visit with the baseline . eighteen patients ( 12 males and 6 females ) out of 94 suspected pds in the glaucoma specialty clinic from may 2006 to april 2007 were identified as having pg in at least one eye , according to the diagnostic criteria . the mean age of the pg patients at enrollment was 35.5 7.0 years ( range : 2249 years ) . the mean ages for male and female subjects were 35.7 6.9 ( range : 2248 years ) and 35.2 8.0 years ( range : 2649 years ) , respectively . the initial iop of the research pg eyes were 30.8 10.4 mmhg ( range : 2241 mmhg , 1 mmhg=0.133 kpa ) before trabeculectomy , with mild to severe vf defect . humphrey vfa at enrollment showed that mean deviation ( md ) of the research eyes before surgery was 15.2 6.6 db ( ranging from 30.4 db to 2.6 db ) . all but two eyes of two patients had myopia of 0.5d or greater , with a mean refractive error of 5.20 5.79 ( ranging from 24.75 to + 0.5 ) spherical equivalent diopters . these two eyes had hyperopia of 0.75d and 1.25d , respectively . majority of the patients ( 14/18 ) had bilateral pg , while the other four patients had only one eye affected with pg . of patients with bilateral pg , nine ( 9/14 ) received trabeculectomy in both eyes for medically refractory glaucoma , and one eye was chosen randomly for research . of nine patients who were treated with trabeculectomy in only one eye with pg , all were enrolled for the study . eight years after trabeculectomy , all surgical pg eyes ( 18/18 ) had satisfactory iop control along with well - maintained va and visual function . the mean iop of research eyes was 13.7 2.5 mmhg ( range : 919 mmhg ) at the last follow - up visit , which was statistically lower than preoperation level ( t = 3.83 , p = 0.001 ) . three eyes were receiving adjunctive prostaglandin analogs ( pga ) eye drop daily to help maintain target iop . three eyes had worse va and bcva , and lost two lines or more of letters on snellen chart at the last follow - up visit ; among these three eyes , two of them were due to cataract formation , but did not need immediate cataract extraction ; the third one had dislocated lens and had to receive cataract surgery during the follow - up period , who was a 33-year - old female and complained a sudden blurring of vision in the morning when she rose up from a bending down position . biomicroscopy on unplanned visit revealed that the crystalline lens in the research eye had dislocated completely into the anterior chamber ( ac ) . she had improved vision after cataract surgery and no fluctuation of iop was detected after the cataract surgery . three of 18 pg eyes had slight enlargement of the glaucomatous optic cupping , verified through stereoscopic optic disc photography , in which vfa showed mild deterioration of vf defect . vf in the majority ( 15/18 ) of the research eyes did not worsen compared with the baseline 8 years after trabeculectomy . all but one of the pg eyes had various extent of posterior bowing of the mid - peripheral iris at enrollment , with a thin concave slit beam on the surface of the iris . the slit beam was projected vertically through the center of the pupil at 3045. seventeen pg eyes have obviously concave irides under slit lamp . only one pg eye had regular iris at initial diagnosis . none of the pg eyes exhibited diffuse anterior iris stromal pigment granule dusting , except for trace cluster of pigment granules on inferior surface of the iris in three pg eyes from three patients . after trabeculectomy , the concave iris became completely flat or regular in all research eyes . in the contralateral eyes of the enrolled patients , either trabeculectomy or laser peripheral iridoctomy was performed to eliminate reverse pupillary block , and the iris recovered to flat or regular . the change of iris configuration remained stable and unchanged throughout the postoperative period of study in both eyes of the patients . functional blebs , symbols of successful trabeculectomy , still existed in 12 pg eyes at the last follow - up visit , though they were smaller and more constricted than the original size . unanimously , pigment deposition in every part of the ac was found to be attenuated or decreased with time after trabeculectomy . such findings were especially obvious when comparing the endpoint state with the baseline at enrollment [ figure 1a1d ] . ( d ) decreased lenticular pigmentation at last follow - up visit ( 8 years after surgery ) . eleven patients ( 61.1% ) had krukenberg spindles , which were bilateral in eight and unilateral in three . the typical appearance of krukenberg spindle in these patients was somewhat more like a triangle , rather than a spindle [ figure 1e ] . of the remaining seven , three had trace diffuse corneal endothelial pigmentation and four had no corneal pigment dusting , suggesting that krukenberg spindle was not necessarily observed in all pg patients . during the 8-year follow - up period , it was commonly seen that the krukenberg spindle became ambiguous and vague with time after trabeculectomy [ figure 1f ] . typical spoke - like radial itds , described in white pds patients , were not discerned in any one of the patients . in two pg eyes of two enrolled patients , isolated short slit - like itds heavy homogeneous tm pigmentation and pigment granule dusting on lens zonules and/or peripheral posterior lens surface were seen in all research eyes . homogeneous tm pigmentation around the circumference of ac like a mascara line was visualized in all patients on gonioscopy , though the degrees were different . after mydriasis , the pg eyes showed different extent of pigment granule dusting on lens zonules and/or posterior peripheral surface referred to as zentmayer ring or scheie 's line . zonular and lenticular pigmentation were strong evidence for diagnosis of pds / pg in that they were demonstrating the existence of irido - zonular rubbing , cause of pigment dispersion . when compared with baseline , it was striking to find that pigmentation in all parts of the ac became attenuated and lighter in all patients , after the configuration of the iris returned to normal after trabeculectomy . to avoid inter - personal bias of the surgery all enrolled pg eyes had functional blebs in the first 6 months after the filtration surgery . at the end point of the study , 16 patients still had functional blebs with satisfactory iop control , with one receiving pga eye drop daily at night . the remaining two patients had a scarred bleb and both needed pga eye drop to keep iop at target level . no serious surgical complications needing immediate surgical intervention or leading to negative outcome were recorded in anyone of the pg eyes during or after the filtration procedure . leaking bleb was discovered in one pg eye and was successfully repaired through wearing a therapeutic soft contact lens . eighteen patients ( 12 males and 6 females ) out of 94 suspected pds in the glaucoma specialty clinic from may 2006 to april 2007 were identified as having pg in at least one eye , according to the diagnostic criteria . the mean age of the pg patients at enrollment was 35.5 7.0 years ( range : 2249 years ) . the mean ages for male and female subjects were 35.7 6.9 ( range : 2248 years ) and 35.2 8.0 years ( range : 2649 years ) , respectively . the initial iop of the research pg eyes were 30.8 10.4 mmhg ( range : 2241 mmhg , 1 mmhg=0.133 kpa ) before trabeculectomy , with mild to severe vf defect . humphrey vfa at enrollment showed that mean deviation ( md ) of the research eyes before surgery was 15.2 6.6 db ( ranging from 30.4 db to 2.6 db ) . all but two eyes of two patients had myopia of 0.5d or greater , with a mean refractive error of 5.20 5.79 ( ranging from 24.75 to + 0.5 ) spherical equivalent diopters . these two eyes had hyperopia of 0.75d and 1.25d , respectively . majority of the patients ( 14/18 ) had bilateral pg , while the other four patients had only one eye affected with pg . of patients with bilateral pg , nine ( 9/14 ) received trabeculectomy in both eyes for medically refractory glaucoma , and one eye was chosen randomly for research . of nine patients who were treated with trabeculectomy in only one eye with pg , all were enrolled for the study . eight years after trabeculectomy , all surgical pg eyes ( 18/18 ) had satisfactory iop control along with well - maintained va and visual function . the mean iop of research eyes was 13.7 2.5 mmhg ( range : 919 mmhg ) at the last follow - up visit , which was statistically lower than preoperation level ( t = 3.83 , p = 0.001 ) . three eyes were receiving adjunctive prostaglandin analogs ( pga ) eye drop daily to help maintain target iop . three eyes had worse va and bcva , and lost two lines or more of letters on snellen chart at the last follow - up visit ; among these three eyes , two of them were due to cataract formation , but did not need immediate cataract extraction ; the third one had dislocated lens and had to receive cataract surgery during the follow - up period , who was a 33-year - old female and complained a sudden blurring of vision in the morning when she rose up from a bending down position . biomicroscopy on unplanned visit revealed that the crystalline lens in the research eye had dislocated completely into the anterior chamber ( ac ) . she had improved vision after cataract surgery and no fluctuation of iop was detected after the cataract surgery . three of 18 pg eyes had slight enlargement of the glaucomatous optic cupping , verified through stereoscopic optic disc photography , in which vfa showed mild deterioration of vf defect . vf in the majority ( 15/18 ) of the research eyes did not worsen compared with the baseline 8 years after trabeculectomy . all but one of the pg eyes had various extent of posterior bowing of the mid - peripheral iris at enrollment , with a thin concave slit beam on the surface of the iris . the slit beam was projected vertically through the center of the pupil at 3045. seventeen pg eyes have obviously concave irides under slit lamp . only one pg eye none of the pg eyes exhibited diffuse anterior iris stromal pigment granule dusting , except for trace cluster of pigment granules on inferior surface of the iris in three pg eyes from three patients . after trabeculectomy , the concave iris became completely flat or regular in all research eyes . in the contralateral eyes of the enrolled patients , either trabeculectomy or laser peripheral iridoctomy was performed to eliminate reverse pupillary block , and the iris recovered to flat or regular . the change of iris configuration remained stable and unchanged throughout the postoperative period of study in both eyes of the patients . functional blebs , symbols of successful trabeculectomy , still existed in 12 pg eyes at the last follow - up visit , though they were smaller and more constricted than the original size . unanimously , pigment deposition in every part of the ac was found to be attenuated or decreased with time after trabeculectomy . such findings were especially obvious when comparing the endpoint state with the baseline at enrollment [ figure 1a1d ] . ( d ) decreased lenticular pigmentation at last follow - up visit ( 8 years after surgery ) . eleven patients ( 61.1% ) had krukenberg spindles , which were bilateral in eight and unilateral in three . the typical appearance of krukenberg spindle in these patients was somewhat more like a triangle , rather than a spindle [ figure 1e ] . of the remaining seven , three had trace diffuse corneal endothelial pigmentation and four had no corneal pigment dusting , suggesting that krukenberg spindle was not necessarily observed in all pg patients . during the 8-year follow - up period , it was commonly seen that the krukenberg spindle became ambiguous and vague with time after trabeculectomy [ figure 1f ] . typical spoke - like radial itds , described in white pds patients , were not discerned in any one of the patients . in two pg eyes of two enrolled patients , heavy homogeneous tm pigmentation and pigment granule dusting on lens zonules and/or peripheral posterior lens surface were seen in all research eyes . homogeneous tm pigmentation around the circumference of ac like a mascara line was visualized in all patients on gonioscopy , though the degrees were different . after mydriasis , the pg eyes showed different extent of pigment granule dusting on lens zonules and/or posterior peripheral surface referred to as zentmayer ring or scheie 's line . zonular and lenticular pigmentation were strong evidence for diagnosis of pds / pg in that they were demonstrating the existence of irido - zonular rubbing , cause of pigment dispersion . when compared with baseline , it was striking to find that pigmentation in all parts of the ac became attenuated and lighter in all patients , after the configuration of the iris returned to normal after trabeculectomy . to avoid inter - personal bias of the surgery all enrolled pg eyes had functional blebs in the first 6 months after the filtration surgery . at the end point of the study , 16 patients still had functional blebs with satisfactory iop control , with one receiving pga eye drop daily at night . the remaining two patients had a scarred bleb and both needed pga eye drop to keep iop at target level . no serious surgical complications needing immediate surgical intervention or leading to negative outcome were recorded in anyone of the pg eyes during or after the filtration procedure . leaking bleb was discovered in one pg eye and was successfully repaired through wearing a therapeutic soft contact lens . it has shown good iop - lowering efficacy and safety in primary glaucoma and has been widely accepted in clinical practice for treatment of secondary glaucoma . pg is a type of secondary open - angle glaucoma , in which medication , laser , and trabeculectomy are all treatment options depending on different stages of the disease . this study has summarized the long - term efficacy and safety of trabeculectomy on pg patients , which have not been reported in previous literature . the results have shown that trabeculectomy had a long iop - lowering effect on pg eyes and promised safety as well . the 8-year outcome of the trabeculectomy has demonstrated that the visual function in pg eyes can be well - preserved after the filtration surgery . yet , it is potentially sight - threatening with highly elevated iop leading to irreversible glaucomatous neuropathy quickly . early detection , treatment of reverse papillary block , and sufficient lowering of iop are critical in the management of pg . in trabeculectomy , all aspects of the treatment principle might be realized . the iridectomy breaks down the reverse pupillary block by communicating the ac and pc , after which the iris returns to be flat or regular and departs from the anterior surface of the crystalline lens and zonules , preventing more pigment granules to liberate from the ipe layer of the iris . at the same time , the artificial outflow pathway drains the aqueous humor out of the eyeball and reduces the elevated iop significantly . a functional bleb is an important indicator for the success of trabeculectomy and serves as a clue for postoperative iop level . in poag eyes , functional blebs seldom survive more than 5 years because of tissue scarring around the operation site . it was striking to find that , in the pg eyes , 15/18 of the functional blebs survived 8 years after the surgery . such high survival rate of functional blebs was accompanied with good iop - control and preservation of visual function . the reason for a long survival rate of functional blebs in pg eyes after filtration surgery is still unknown . the main difference between poag and pg eyes is that the latter has a lot of pigment granules deposited on anterior segment of the eyeball while the former does not . thus , it is not unreasonable to postulate that pigment granules have a positive effect in preserving functional blebs , and such effect lasts long enough to keep the blebs survive 8 years . as the results have shown , the pigment granules still existed at the last follow - up visit 8 years after trabeculectomy , though the extent decreased . the high survival rate of functional blebs was accompanied with sufficient iop - lowering in the pg eyes . eight - year outcome of trabeculectomy has shown that the treated eyes had well - controlled iop , mostly with no adjunctive antiglaucoma medication at the end point of the study . at the last follow - up visit , the mean iop of the treated eyes was 13.7 2.5 mmhg ( range : 919 mmhg ) . only three pg eyes were receiving adjunctive pga eye drop daily to help maintain the target iop . consequently , large majority of the pg eyes had stable va , bcva , and vf , as shown in the result section . there was no need for a second antiglaucoma surgery for any of the eyes during the study period . in the literature , the long - term iop - reducing efficacy of trabeculectomy on poag is not as promising yet . majority of poag eyes will face failure of trabeculectomy due to scarred bleb within 5 years . it is not uncommon that a second antiglaucoma surgery might be introduced if sufficient adjunctive antiglaucoma medication fails to reduce the iop to target level . the mechanism for elevating resistance of aqueous humor outflow and high iop in poag is more complicated and substantial than that in pg . when trabeculectomy is performed in poag , the artificial pass way assists the outflow of the aqueous humor and reduces the iop . such drainage assistance attenuates with time when tissue scarring occurs around the operation site , which results in a limited long - term iop - lowering efficacy of trabeculectomy on poag patients . the 8-year observation on pg eyes has demonstrated that tm pigmentation attenuated with time after the reverse pupillary block had been broken down after iridectomy , which prevented more pigment to disperse after the surgery . on the other hand , histological study of the tm tissue gained in trabeculectomy demonstrated that pigment granules in trabecular spaces might be phagocytozed by trabecular cells , which helped to clear the trabecular spaces . moreover , small pigment granules can be drained out of the tm with aqueous humor convection . such migration of pigment granule results in decreased number of pigment particles in the trabecular space and a compromised resistance of aqueous humor outflow . and such effect accumulates with time . as the results have shown [ figure 1 ] , pigmentation in the anterior segment of the all research eyes attenuated after trabeculectomy . both the long survival of functional blebs and the compromised tm pigmentation play an important role in the mechanism of iop - lowering in pg eyes and might also help explain why trabeculectomy has a longer iop - reducing efficacy in pg than poag . side - effects of trabeculectomy were another major concern of this study . in the present study , we have found that the intra- and post - operative side - effects were quite similar between pg and those in poag . no severe side - effects of the surgery were discovered on the follow - up visits of the pg eyes . a leaking bleb was found in one of the pg eyes and healed after wearing of therapeutic soft contact lens . in summary , we have characterized the long - term outcome of trabeculectomy on 18 pg eyes . functional blebs survived longer than that in poag patients , and visual function was well - protected 8 years after the surgery . trabeculectomy is a safe and effective treatment for pg eyes and should be introduced when medication and laser treatment fail to reduce the elevated iop . project supported by a grant from the national natural science foundation of china ( no . project supported by a grant from the national natural science foundation of china ( no .

background : though trabeculectomy is often performed on patients with medically refractive pigmentary glaucoma ( pg ) , the clinical outcomes of surgical treatment on pg remain unknown . the aim of this study was to summarize the long - term efficacy and safety of trabeculectomy on pg.methods:this was a prospective case series observational study . eighteen consecutive pg patients were followed up for 8 years after trabeculectomy from may 2006 to april 2007 . visual acuity ( va ) , best - corrected visual acuity ( bcva ) , slit lamp biomicroscopy , intraocular pressure ( iop ) measurement , humphrey visual field analysis ( vfa ) , and stereoscopic funduscopy were performed on admission and every 6 months after the surgery . postoperative iop , va , bcva , vfa , adjunctive anti - glaucoma medication , treatment - related side - effects , changes in blebs , and main clinical findings in the anterior segment of pg were recorded and compared with the baseline.results:eighteen pg eyes from 18 patients , with average preoperative iop of 34.5 4.7 mmhg ( range : 2147 mmhg , 1 mmhg=0.133 kpa ) were enrolled in this study . all enrolled patients completed the follow - up visits and required examinations . eight years after trabeculectomy , all surgical eyes ( 18/18 ) had satisfactory iop control with an average of 13.7 2.5 mmhg ( range : 919 mmhg ) , which was significantly lower than baseline ( p = 0.001 ) . majority ( 15/18 ) of the pg eyes had stable va , bcva , vfa , and optic disc cupping parameters . functional blebs still existed in 12/18 of the pg eyes at the last follow - up visit . unanimously , pigmentation in the anterior segment attenuated with time after surgical treatment . no severe side - effects were recorded in any of the surgical eyes.conclusions:all surgical pg eyes in this study had satisfactory iop control 8 years after the surgery with well - preserved visual function . the long - term efficacy and safety of trabeculectomy are promising in pg patients .

I M Patient enrollment and inclusion criteria Primary outcomes Statistical analysis R Patients demographic characteristics Intraocular pressure reduction and visual function preservation Postoperative changes in anterior segment Complications and side-effects D Financial support and sponsorship Conflicts of interest

pigment dispersion syndrome ( pds ) results from posterior bowing of the iris and rubbing between the lens zonules and epithelial layer of the iris , and usually affects myopic eyes in men during the third to fourth decade of life . the literature indicated that caucasians have the highest prevalence of pds among all races , with clinical characteristics including krukenberg spindle , homogeneous trabecular meshwork ( tm ) pigmentation , and spoke - like mid - peripheral iris transillumination defects ( itds ) , which are referred as a triad of pds . pigmentary glaucoma ( pg ) is usually inevitable if pds is not detected and interrupted at an early stage . gradual pigmentation on the tm may lead to increased aqueous humor outflow resistance , resultant elevated intraocular pressure ( iop ) , and glaucomatous neuropathy . the literature has numerous references addressing efficacy and safety of trabeculectomy on poag , while it has quite limited references on clinical outcomes of pg . because the prevalence of pg is much lower than that of poag , the number of pds / pg patients is rare in clinical practice . the aim of the current study was to summarize the long - term clinical outcomes of trabeculectomy in 18 consecutive chinese patients with pg in north china , which can provide a valuable reference for ophthalmologists in the management of pg . all outpatients presenting for care at the glaucoma specialty clinic at beijing tongren eye center , beijing from may 2006 to april 2007 were evaluated for signs of pds : corneal endothelial pigmentation , anterior iris stromal pigment dusting , itds , posterior iris bowing , increased tm pigmentation , and pigment granule dusting on lens zonules or peripheral posterior surface . detailed ophthalmic examinations included visual acuity ( va ) , best - corrected visual acuity ( bcva ) , iop measurement , slit lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey swedish interactive thresholding algorithm ( sita)-standard 30 - 2 visual field analysis ( vfa ) . slit - lamp examination , gonioscopy , and funduscopic examination of the optic disc of all subjects were performed and graded by the same doctor ( dr . diagnostic criteria for pds in chinese patients included at least two of the following three signs : krukenberg spindle , homogenous moderate to heavy tm pigmentation ( scheie ii ) , and any degree of zonular and/or lenticular pigment granule dusting . diagnosis of pg was made if pds patient had two or more of the following findings : initial iop > 21 mmhg , glaucomatous optic nerve damage ( increased cupping or glaucomatous disk appearance ) , or visual field ( vf ) defect . informed consent was obtained from each subject after explanation of the nature and possible consequences of the study , which was approved by the institutional review board of beijing tongren hospital , capital medical university . eighteen consecutive pg patients were identified and received trabeculectomy in at least one eye for medically refractory glaucoma . baseline examination included va , bcva , iop , slit - lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey sita - standard 30 - 2 vfa . postoperative iop , va , bcva , vfa parameters , optic disc cupping , morphological changes in bleb , and characteristic clinical findings of pg were main outcome parameters and were compared to the baseline values . the paired - sample t - test was applied to compare postoperative iop , va , bcva , and vfa parameters at the last follow - up visit with the baseline . all outpatients presenting for care at the glaucoma specialty clinic at beijing tongren eye center , beijing from may 2006 to april 2007 were evaluated for signs of pds : corneal endothelial pigmentation , anterior iris stromal pigment dusting , itds , posterior iris bowing , increased tm pigmentation , and pigment granule dusting on lens zonules or peripheral posterior surface . detailed ophthalmic examinations included visual acuity ( va ) , best - corrected visual acuity ( bcva ) , iop measurement , slit lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey swedish interactive thresholding algorithm ( sita)-standard 30 - 2 visual field analysis ( vfa ) . slit - lamp examination , gonioscopy , and funduscopic examination of the optic disc of all subjects were performed and graded by the same doctor ( dr . diagnosis of pg was made if pds patient had two or more of the following findings : initial iop > 21 mmhg , glaucomatous optic nerve damage ( increased cupping or glaucomatous disk appearance ) , or visual field ( vf ) defect . informed consent was obtained from each subject after explanation of the nature and possible consequences of the study , which was approved by the institutional review board of beijing tongren hospital , capital medical university . eighteen consecutive pg patients were identified and received trabeculectomy in at least one eye for medically refractory glaucoma . baseline examination included va , bcva , iop , slit - lamp biomicroscopy pre- and post - mydriasis , gonioscopy , funduscopic examination , and automated humphrey sita - standard 30 - 2 vfa . postoperative iop , va , bcva , vfa parameters , optic disc cupping , morphological changes in bleb , and characteristic clinical findings of pg were main outcome parameters and were compared to the baseline values . all enrolled patients completed the 8-year follow - up after trabeculectomy . the paired - sample t - test was applied to compare postoperative iop , va , bcva , and vfa parameters at the last follow - up visit with the baseline . eighteen patients ( 12 males and 6 females ) out of 94 suspected pds in the glaucoma specialty clinic from may 2006 to april 2007 were identified as having pg in at least one eye , according to the diagnostic criteria . the mean age of the pg patients at enrollment was 35.5 7.0 years ( range : 2249 years ) . the mean ages for male and female subjects were 35.7 6.9 ( range : 2248 years ) and 35.2 8.0 years ( range : 2649 years ) , respectively . the initial iop of the research pg eyes were 30.8 10.4 mmhg ( range : 2241 mmhg , 1 mmhg=0.133 kpa ) before trabeculectomy , with mild to severe vf defect . eight years after trabeculectomy , all surgical pg eyes ( 18/18 ) had satisfactory iop control along with well - maintained va and visual function . the mean iop of research eyes was 13.7 2.5 mmhg ( range : 919 mmhg ) at the last follow - up visit , which was statistically lower than preoperation level ( t = 3.83 , p = 0.001 ) . three eyes had worse va and bcva , and lost two lines or more of letters on snellen chart at the last follow - up visit ; among these three eyes , two of them were due to cataract formation , but did not need immediate cataract extraction ; the third one had dislocated lens and had to receive cataract surgery during the follow - up period , who was a 33-year - old female and complained a sudden blurring of vision in the morning when she rose up from a bending down position . three of 18 pg eyes had slight enlargement of the glaucomatous optic cupping , verified through stereoscopic optic disc photography , in which vfa showed mild deterioration of vf defect . vf in the majority ( 15/18 ) of the research eyes did not worsen compared with the baseline 8 years after trabeculectomy . all but one of the pg eyes had various extent of posterior bowing of the mid - peripheral iris at enrollment , with a thin concave slit beam on the surface of the iris . the slit beam was projected vertically through the center of the pupil at 3045. seventeen pg eyes have obviously concave irides under slit lamp . none of the pg eyes exhibited diffuse anterior iris stromal pigment granule dusting , except for trace cluster of pigment granules on inferior surface of the iris in three pg eyes from three patients . after trabeculectomy , the concave iris became completely flat or regular in all research eyes . in the contralateral eyes of the enrolled patients , either trabeculectomy or laser peripheral iridoctomy was performed to eliminate reverse pupillary block , and the iris recovered to flat or regular . functional blebs , symbols of successful trabeculectomy , still existed in 12 pg eyes at the last follow - up visit , though they were smaller and more constricted than the original size . unanimously , pigment deposition in every part of the ac was found to be attenuated or decreased with time after trabeculectomy . ( d ) decreased lenticular pigmentation at last follow - up visit ( 8 years after surgery ) . during the 8-year follow - up period , it was commonly seen that the krukenberg spindle became ambiguous and vague with time after trabeculectomy [ figure 1f ] . typical spoke - like radial itds , described in white pds patients , were not discerned in any one of the patients . in two pg eyes of two enrolled patients , isolated short slit - like itds heavy homogeneous tm pigmentation and pigment granule dusting on lens zonules and/or peripheral posterior lens surface were seen in all research eyes . after mydriasis , the pg eyes showed different extent of pigment granule dusting on lens zonules and/or posterior peripheral surface referred to as zentmayer ring or scheie 's line . when compared with baseline , it was striking to find that pigmentation in all parts of the ac became attenuated and lighter in all patients , after the configuration of the iris returned to normal after trabeculectomy . to avoid inter - personal bias of the surgery all enrolled pg eyes had functional blebs in the first 6 months after the filtration surgery . at the end point of the study , 16 patients still had functional blebs with satisfactory iop control , with one receiving pga eye drop daily at night . no serious surgical complications needing immediate surgical intervention or leading to negative outcome were recorded in anyone of the pg eyes during or after the filtration procedure . eighteen patients ( 12 males and 6 females ) out of 94 suspected pds in the glaucoma specialty clinic from may 2006 to april 2007 were identified as having pg in at least one eye , according to the diagnostic criteria . the mean age of the pg patients at enrollment was 35.5 7.0 years ( range : 2249 years ) . the mean ages for male and female subjects were 35.7 6.9 ( range : 2248 years ) and 35.2 8.0 years ( range : 2649 years ) , respectively . the initial iop of the research pg eyes were 30.8 10.4 mmhg ( range : 2241 mmhg , 1 mmhg=0.133 kpa ) before trabeculectomy , with mild to severe vf defect . eight years after trabeculectomy , all surgical pg eyes ( 18/18 ) had satisfactory iop control along with well - maintained va and visual function . the mean iop of research eyes was 13.7 2.5 mmhg ( range : 919 mmhg ) at the last follow - up visit , which was statistically lower than preoperation level ( t = 3.83 , p = 0.001 ) . three eyes had worse va and bcva , and lost two lines or more of letters on snellen chart at the last follow - up visit ; among these three eyes , two of them were due to cataract formation , but did not need immediate cataract extraction ; the third one had dislocated lens and had to receive cataract surgery during the follow - up period , who was a 33-year - old female and complained a sudden blurring of vision in the morning when she rose up from a bending down position . three of 18 pg eyes had slight enlargement of the glaucomatous optic cupping , verified through stereoscopic optic disc photography , in which vfa showed mild deterioration of vf defect . vf in the majority ( 15/18 ) of the research eyes did not worsen compared with the baseline 8 years after trabeculectomy . all but one of the pg eyes had various extent of posterior bowing of the mid - peripheral iris at enrollment , with a thin concave slit beam on the surface of the iris . the slit beam was projected vertically through the center of the pupil at 3045. seventeen pg eyes have obviously concave irides under slit lamp . only one pg eye none of the pg eyes exhibited diffuse anterior iris stromal pigment granule dusting , except for trace cluster of pigment granules on inferior surface of the iris in three pg eyes from three patients . after trabeculectomy , the concave iris became completely flat or regular in all research eyes . in the contralateral eyes of the enrolled patients , either trabeculectomy or laser peripheral iridoctomy was performed to eliminate reverse pupillary block , and the iris recovered to flat or regular . functional blebs , symbols of successful trabeculectomy , still existed in 12 pg eyes at the last follow - up visit , though they were smaller and more constricted than the original size . unanimously , pigment deposition in every part of the ac was found to be attenuated or decreased with time after trabeculectomy . ( d ) decreased lenticular pigmentation at last follow - up visit ( 8 years after surgery ) . during the 8-year follow - up period , it was commonly seen that the krukenberg spindle became ambiguous and vague with time after trabeculectomy [ figure 1f ] . typical spoke - like radial itds , described in white pds patients , were not discerned in any one of the patients . in two pg eyes of two enrolled patients , heavy homogeneous tm pigmentation and pigment granule dusting on lens zonules and/or peripheral posterior lens surface were seen in all research eyes . after mydriasis , the pg eyes showed different extent of pigment granule dusting on lens zonules and/or posterior peripheral surface referred to as zentmayer ring or scheie 's line . when compared with baseline , it was striking to find that pigmentation in all parts of the ac became attenuated and lighter in all patients , after the configuration of the iris returned to normal after trabeculectomy . to avoid inter - personal bias of the surgery all enrolled pg eyes had functional blebs in the first 6 months after the filtration surgery . at the end point of the study , 16 patients still had functional blebs with satisfactory iop control , with one receiving pga eye drop daily at night . no serious surgical complications needing immediate surgical intervention or leading to negative outcome were recorded in anyone of the pg eyes during or after the filtration procedure . pg is a type of secondary open - angle glaucoma , in which medication , laser , and trabeculectomy are all treatment options depending on different stages of the disease . this study has summarized the long - term efficacy and safety of trabeculectomy on pg patients , which have not been reported in previous literature . the 8-year outcome of the trabeculectomy has demonstrated that the visual function in pg eyes can be well - preserved after the filtration surgery . early detection , treatment of reverse papillary block , and sufficient lowering of iop are critical in the management of pg . in trabeculectomy , all aspects of the treatment principle might be realized . at the same time , the artificial outflow pathway drains the aqueous humor out of the eyeball and reduces the elevated iop significantly . it was striking to find that , in the pg eyes , 15/18 of the functional blebs survived 8 years after the surgery . the reason for a long survival rate of functional blebs in pg eyes after filtration surgery is still unknown . the main difference between poag and pg eyes is that the latter has a lot of pigment granules deposited on anterior segment of the eyeball while the former does not . thus , it is not unreasonable to postulate that pigment granules have a positive effect in preserving functional blebs , and such effect lasts long enough to keep the blebs survive 8 years . as the results have shown , the pigment granules still existed at the last follow - up visit 8 years after trabeculectomy , though the extent decreased . the high survival rate of functional blebs was accompanied with sufficient iop - lowering in the pg eyes . eight - year outcome of trabeculectomy has shown that the treated eyes had well - controlled iop , mostly with no adjunctive antiglaucoma medication at the end point of the study . at the last follow - up visit , the mean iop of the treated eyes was 13.7 2.5 mmhg ( range : 919 mmhg ) . consequently , large majority of the pg eyes had stable va , bcva , and vf , as shown in the result section . in the literature , the long - term iop - reducing efficacy of trabeculectomy on poag is not as promising yet . when trabeculectomy is performed in poag , the artificial pass way assists the outflow of the aqueous humor and reduces the iop . such drainage assistance attenuates with time when tissue scarring occurs around the operation site , which results in a limited long - term iop - lowering efficacy of trabeculectomy on poag patients . the 8-year observation on pg eyes has demonstrated that tm pigmentation attenuated with time after the reverse pupillary block had been broken down after iridectomy , which prevented more pigment to disperse after the surgery . as the results have shown [ figure 1 ] , pigmentation in the anterior segment of the all research eyes attenuated after trabeculectomy . both the long survival of functional blebs and the compromised tm pigmentation play an important role in the mechanism of iop - lowering in pg eyes and might also help explain why trabeculectomy has a longer iop - reducing efficacy in pg than poag . side - effects of trabeculectomy were another major concern of this study . in the present study , we have found that the intra- and post - operative side - effects were quite similar between pg and those in poag . no severe side - effects of the surgery were discovered on the follow - up visits of the pg eyes . in summary , we have characterized the long - term outcome of trabeculectomy on 18 pg eyes . functional blebs survived longer than that in poag patients , and visual function was well - protected 8 years after the surgery .

refers to the phenomenon of neurons and neural networks modifying their connections and/or behaviour in response to new information , sensory stimulation , development , damage , or dysfunction . the ultimate goal of neurorehabilitation is to induce neural plasticity in a manner that restores the full original function and potential of the injured brain ( neurological restoration ) , but a variety of other patterns of neural plasticity may also occur during recovery , including compensatory activity , use of redundant networks , or changes in behavioural or cognitive strategy . direct measures of such changes are critical to understanding how and when recovery from brain injury takes place and ultimately may lead to improved or novel rehabilitative treatments . one very popular modality used to measure neuroplasticity is task - based functional mri ( t - fmri ) . this technique infers from local changes in cerebral blood flow ( cbf ) to identify brain regions that are more active while subjects execute a task than during a comparison or resting state . for a more in - depth explanation of fmri when used to measure neuroplasticity , however , t - fmri suffers from a unique set of challenges that are not always fully acknowledged . with the accelerating development of neurorehabilitation strategies researchers need to be cognisant of the limitations of commonly used neuroimaging technologies , including t - fmri , in order to collect information capable of advancing our understanding of the neurorehabilitative process . in particular , it is critical that researchers can correctly interpret what a change in t - fmri signal reflects , if they are to understand the mechanisms of functional recovery . to aid researchers in this regard , this review explores two important questions : what are the challenges in interpreting changes in t - fmri signal as intervention - induced neuroplasticity ? and how can complementary information from other modalities aid such interpretations ? to contextualise our discussion , we define four basic criteria that we believe are essential for informative interpretation of any neuroimaging signal change in terms of brain changes . we propose that detected changes should ( 1 ) be moderately stable or evolve reliably , ( 2 ) be meaningfully distinguishable from day - to - day variation in brain activity , ( 3 ) offer biological insight into the recovery process , and ( 4 ) reliably relate to ( or influence ) clinical changes . these criteria are somewhat straightforward : to advance neurorehabilitative science , reported changes must be unambiguous , reliable , related to recovery , and clearly a direct or indirect effect of the intervention at hand . with this in mind , we begin this review by outlining t - fmri findings associated with intervention - induced neuroplasticity and discuss uncertainties surrounding their interpretations . we highlight that change in t - fmri activation patterns can be difficult to extrapolate to brain reorganisation and , in some cases , may be confounded by processing inherent to the technique . we follow this overview by offering supporting strategies , focussing on the supplementation of t - fmri findings with information from other modalities , such as structural mri or transcranial magnetic stimulation ( tms ) . examples are provided as to how incorporating such information can improve interpretation of t - fmri data , strengthening specific claims about intervention driven neuroplasticity . though some points made here may be generalised to other contexts , this commentary restricts discussion to studies targeting motor impairment and movement rehabilitation in patients with cerebral palsy ( cp ) or acquired brain injuries , such as traumatic brain injury ( tbi ) . as relevant literature describing therapy - driven brain reorganisation is limited in patients with acquired brain injuries , we also make reference to neuroimaging studies based on adult stroke populations and some nonlongitudinal studies . it must be kept in mind that while subject groups may all undergo neuroplasticity in response to rehabilitation , they may do so from a vastly different baseline , particularly due to the impacts of brain injury on early development . further , for the sake of brevity , discussion here is restricted to standard glm - analyses of t - fmri , as this is the dominant technique in published literature ; resting state fmri and other forms of fmri are not considered . there are three primary findings that are commonly reported in t - fmri studies of neurorehabilitation , summarised in figure 1 . altered ipsilesional activated - voxel counts , or heightened peak intensities , are commonly reported for patients with brain injuries who have received treatment , improved function , or when compared with controls ( figures 1(a ) , 1(b ) , and 2(a ) ) . heightened activation of motor regions has been reported for children with tbi and adolescents with cp when compared with controls . a recent systematic review reported seven longitudinal t - fmri studies of treatment interventions for unilateral cp , drawn from four unique subject cohorts . after therapy , area of activation of the ( most ) impaired hemisphere reportedly increased in a subset of subjects within each study . in tbi , one study of seven adult subjects with primarily - nonchronic injury showed changes in the activation volumes of several sensorimotor - related regions in response to motor rehabilitation . the location and relative changes in activation volumes varied greatly between subjects . increased ipsilesional premotor activation has been shown in response to constraint - induced movement therapy , alongside improvements in fugl meyer assessment scores , in a single adult with chronic traumatic damage to the primary sensorimotor cortex ( s1m1 ) . similarly , increased s1m1 activation has been found in two adult tbi subjects after robotic therapy . likewise , following adult stroke , regions of sensorimotor activation are reportedly larger in recovered patients than in partially recovered patients and can further enlarge with motor training . the second common t - fmri finding in patients with brain injuries is a shift in the hemispheric - balance of activation ( figure 1(e ) ) . in normal subjects , basic motor tasks overwhelmingly activate the contralateral s1m1 . both stroke and unilateral cp patients , however , regularly demonstrate robustly bilateral activation [ 12 , 20 , 21 ] . these balances of activation are typically calculated as laterality index ( li ) : ( 1)li=cic+i , where c and i are suprathreshold voxel counts or t - value sums ( weighted li , also referred to here as li for simplicity ) , for the contralateral and ipsilateral hemispheres , respectively . lis fall between 1 ( only ipsilateral activation ) and + 1 ( only contralateral activation ) . in stroke , s1m1 li values for the paretic hand are lowest in acute stroke , due to both decreased ipsilesional activity and increased contralesional activity . over time , these values become more positive [ 18 , 21 ] but do not typically return completely to normal values , even in well - recovered patients [ 17 , 23 ] . in chronic stroke patients , li values are often [ 24 , 25 ] , but not always , reported to shift toward the lesioned hemisphere in response to rehabilitative therapy . in children with unilateral cp , activation of ipsilateral sensorimotor regions can be evoked with active movements , passive movements , and tactile stimulation of the impaired limb , the patterns of which depend on their type of reorganisation [ 6 , 27 ] . small - scale studies of children with unilateral cp suggest that virtual reality and constraint - induced movement therapies can alter the balance of activation toward the contralateral hemisphere [ 13 , 28 ] . this may prove functionally beneficial : contralateral somatosensory activation during motor tasks appears to be associated with improved unimanual capacity . numerous studies have proposed that laterality shifts demonstrate an adaptive bihemispheric reorganisation of motor networks [ 22 , 23 , 25 , 28 , 29 ] . differences in intrahemispheric location of s1m1 activation , between either time points or subject groups , are also frequently reported as evidence of neurological reorganisation ( figures 1(c ) and 1(d ) ) . shifts in peak activation have been described 4 , 12 , and 24 months after stroke . different loci of activation have been reported between stroke and control subjects numerous times [ 17 , 31 ] . one study has reported a correlation between peak s1m1 activity location and motor impairment . it is clear that changes in t - fmri measures have been reported in a variety of studies and pathologies . this section identifies several challenges that make the interpretation of such results in terms of neuroplasticity particularly difficult . these issues include subject variability , biological ambiguity , methodological considerations , and confounds introduced by disease states . as we shall discuss , these factors impede informative interpretation of the t - fmri signal by obscuring two key facts : ( 1 ) whether neurological change has genuinely taken place and ( 2 ) if so , what type of change has been observed . possible solutions to reduce the impact of these variables are summarised within the final section of this review . these incorporate the use of information from other modalities within the study design , providing complementary support for t - fmri measured brain changes , to provide more robust evidence of neuroplasticity . one of the greatest challenges for t - fmri in studies of neurorehabilitation is the heterogeneity in findings , both within and between studies of patients with brain injury . relocations of activation , for example , are not always reported and have been variable even within studies , differing , for example , by patient subgroup or task performed . in addition , changes in activation patterns do not consistently correlate with behavioural improvements ( figure 2(b ) ) . distinct changes in activation patterns have been reported in rehabilitative studies of adult stroke ( postrehabilitation versus retention ) , hemispherectomy ( pre- versus postrehabilitation ) , and paediatric cp ( pre- versus postrehabilitation ) , despite subjects demonstrating stable motor scores . in unilateral cp , the degree of s1m1 activation for active and passive movements may not correlate with motor scores [ 12 , 20 ] , and results for sensory impairment are mixed [ 8 , 12 , 35 ] . similarly , for stroke , activation of the ipsilateral primary motor cortex has been associated with both good and poor behavioural outcomes . factors such as anatomical location , extent , type , and timing of insult can have profound influences on neurological impairments , response to treatment , and the type of neuroplasticity required for recovery . controlling for such factors can be very difficult . restricting a study to patients in the chronic stage of injury , for example , may not remove effects due to progressive wallerian degeneration and/or volumetric changes , which take place during the first few years following stroke and , potentially , tbi . response to treatment also appears to be subject to intact contralateral corticothalamic connections in stroke subjects and ipsilateral corticospinal connections in children with cp . such factors can dramatically alter the interpretation and biological significance of measures such as li , but their identification requires utilisation of additional modalities , such as tms or diffusion imaging . attempts to limit such variability is probably one reason why most t - fmri studies investigating neuroplasticity include only ~410 subjects with brain injury [ 13 , 21 ] . reproducibility studies have demonstrated that even well - controlled longitudinal t - fmri studies of normal subjects likely have a high degree of intrasubject measurement error and require at least 20 subjects per group to perform reliable and sensitive group analyses . the higher degree of variability seen within brain injury cohorts means that required numbers are likely to be substantially higher . it is common in the t - fmri literature to refer to activation differences as direct evidence of adaptive neuroplasticity . what is rarely addressed is the fact that activation differences , in isolation , do not allow researchers to differentiate between a variety of substantially different biological processes , many of which do not indicate regained , novel , or improved neurological capabilities and may not be positive or adaptive at all . one of the most obvious alternative explanations to adaptive neuroplasticity is that activation changes reflect normal system dynamics compensating for poor performance . it is already established that the brain can switch between apparently functionally equivalent sensorimotor representations in response to disrupted activity , for example , during a tumour removal operation , or reversibly within minutes of direct inactivation of motor areas . importantly , t - fmri alone is unable to determine whether such dynamics reflect a switch ( 1 ) to an equipotent area ( reflecting ongoing impairment ) , ( 2 ) back to the original area ( restored function ) , or ( 3 ) to an area previously incapable of such responsibility ( novel gain in function ) . given the three distinctively different take - home messages for the intervention investigated , there is a strong argument for researchers to seek secondary evidence ( e.g. , microstructural , conduction , or connectivity changes ) before assuming that an activation change necessarily indicates novel or regained function . rather than relying on neurological recovery , subjects can improve task performance by altering the role of muscle groups , improved motor planning , or better attending to feedback . some adult stroke patients rely more heavily on proprioceptive feedback than healthy subjects , for example . adult stroke patients have also been shown to adopt compensatory movement patterns , including atypical - muscle use for pointing and reaching tasks , during rehabilitation [ 46 , 47 ] . importantly , such compensation can result in improved motor scores , despite unimproved motor capabilities , and is associated with poorer recovery . in addition , studies combining tms and t - fmri have revealed that attention , anticipation , and/or the forward - planning of motor movements dramatically alters cortical excitability in button pressing tasks [ 48 , 49 ] . given these points , it is not unreasonable to surmise that subtly different behavioural strategies may underlie subtle changes in t - fmri activation patterns . while it could be argued that learning is a form , or the result , of neuroplasticity , again the usability of information becomes limited if one can not differentiate between working around ongoing disability and neurological restoration . one argument is that controlling for differential performance is essential to avoid different workloads or feedback confounding results ( figure 2(b ) ) . in order to perform similarly to controls , however , impaired patients have to apply more effort or execute different strategies , such as a more heavy reliance on feedback , which can increase recruitment of s1m1 , attentional networks , and/or supplementary areas . these sustained attentional demands are also more difficult for brain injured subjects to meet [ 5153 ] and may influence activation of some sensorimotor areas , independently of motor output . increased cognitive fatigue may also result in more frequent head movement , which can impact analyses . to avoid this issue , the equivalency of perceived effort can , instead , be controlled for ( e.g. , by modulating the range of motion or force exerted ) . subjects performing different tasks , however , may use different task strategies , musculature , and/or receive different somatosensory feedback , all of which may alter activation patterns . in some instances , it may be possible to conduct two tasks , one controlling for perceived effort and another where performance is controlled between participants . these two sets of functional results can then be interpreted in the context of one another and the behavioural observations noted during scanning . researchers should carefully consider their participants before selecting this course of action as the attention required to perform multiple tasks without head movement may be beyond the means of young children , people with moderate - to - severe disability , and participants with acute brain injury ( such as concussion ) . another option is the use of trivial tasks with limited cognitive load , for which perceived task difficulty and performance are likely to be identical across sessions . scans using these tasks , however , may be insensitive to subtle reorganisation and would require exclusion of most moderately impaired patients , for whom no task is trivial . passive movements of the impaired limb are a final option but may miss genuine activity and reorganisation associated with motor planning and execution . as such , most rehabilitation studies that incorporate fmri of motor tasks are best positioned by accepting the task equivalency problem , choosing a simple / stable task , and making claims in the context of secondary , independent evidence of neurological change . shifts in li toward the contralesional hemisphere have been previously interpreted as neuroplastic compensation for a damaged sensorimotor cortex . at least in stroke , however , contralesional activation does not appear to be a good predictor of functional recovery . given that the motor cortices inhibit one another in normal subjects , an alternative explanation is interhemispheric disinhibition ( figure 3 ) : damage to the lesioned hemisphere reduces its inhibitory ability , leading to contralesional hyperactivation . tms and fmri + tms studies have provided direct evidence for this hypothesis in subjects with cp , tbi , and stroke [ 17 , 59 ] . contralesional activity may even have a net - negative influence : direct inhibition of such activity with transcranial direct current stimulation can improve motor scores and motor - skill acquisition in adults with chronic stroke . unknown anatomy and functional dynamics can further undermine interpretation of changes in li . in cp , preserved ipsilateral corticospinal connections may exist , which t - fmri - only studies are unable to discern . in stroke , one fmri + tms study revealed that contralesional dorsal - premotor - cortex activity was correlated with poorer clinical scores , facilitating the ipsilesional motor cortex in impaired patients but inhibiting it in patients exhibiting good recovery . these results highlight how difficult correctly interpreting t - fmri activation differences can be in subjects with impairment . activations may be adaptive , maladaptive , pathological , excitatory , inhibitory , and/or net - neutral . which interpretation is correct is something that can not be determined by t - fmri alone . there are methodological considerations to consider when evaluating changes , or differences , in the spatial extent and location of t - fmri activation peaks . smoothing of voxel intensities is a common step in t - fmri analyses and varies greatly between studies , often without supplied justification . smoothing can have dramatic nonlinear effects on voxel variances which can alter the volume and shape of activation , as well as the location of peaks ( figure 2(a ) ) . even kernels as small as 4 mm can shift peak - intensity localisation of motor centres by several millimetres . such effects should be kept in mind when inferring from activation characteristics , especially with larger smoothing kernels , which are more optimal for the small cohort sizes seen in this field . when conducting group analyses , it is typical to nonlinearly register scans to a standardised normal brain template . this normalisation step can , however , inappropriately distort the location of tissues surrounding brain lesions . this may lead to shifts in activation location and activation - size differences between groups in damaged hemispheres . performing affine - only registration , cost - function marking , or unified segmentation may reduce such effects but does not guarantee their elimination . these effects should be given consideration when interpreting group - wise analyses , especially given that reported location differences are typically in the millimetre range and derived from small sample sizes . care must also be taken with interpretation of cluster analyses , which comprise the majority of recent t - fmri analyses [ 62 , 67 ] . a cluster of voxels discovered through a cluster analysis does not infer that all voxels within that cluster were significantly active during the task . a cluster indicates a region that meets a minimum size requirement , somewhere inside of which there is evidence against the null hypothesis [ 6870 ] . a consequence of this is that the spatial specificity of these analyses is typically low , especially with larger clusters , and one can not make specific inferences about particular voxels within the cluster . when studying neuroplasticity , an enlarged cluster does not , thus , necessarily mean that neurons on the periphery of that region are newly utilised for a task . similarly , a cluster that has changed shape or shifted slightly may still only have the true activation in the same location . this is of particular concern when liberal primary voxel - level thresholds ( e.g. , p < 0.01 ) are used , as these further dilute the ability to make claims about spatial location of activation . use of liberal thresholds is not uncommon : a recent review of 814 cluster - based fmri studies published in high - impact journals described use of liberal thresholds as both endemic and detrimental to the neuroimaging field . beyond their most obvious motor impairment , subjects with brain injuries may also present with a number of complicating factors that can not easily be controlled for between groups or time points and may impact t - fmri analyses in unexpected ways . in acute and subacute stages of brain injury , fmri evolution of activity patterns during this time may also simply demonstrate the temporary effects of a regressing oedema , mass effect , and/or inflammation , all of which are expected to acutely impact function . as such , special care should be taken not to misconstrue t - fmri changes during early disease states as neuroplasticity , without secondary evidence ruling out such causes . standard bold analyses rely on a number of assumptions , including that neurovascular coupling ( 1 ) is consistently overcompensatory , ( 2 ) is adequately regionally invariant , and ( 3 ) has a sufficiently standard time - course between regions and subjects . these assumptions may be invalidated by the substantial cerebrovascular damage that is associated with many forms of stroke , tbi , and congenital hemiplegia . altered cbf has been reported for all clinical stages of both stroke [ 7274 ] and tbi [ 71 , 75 ] . stroke patients ' haemodynamics may be additionally impacted nonglobally by concurrent vascular disease caused by risk factors such as advanced age , smoking , hypertension , and diabetes mellitus . as normal haemodynamic responses overcompensate for metabolic needs , reduced cerebrovascular reactivity can present as a diminished bold signal , despite unaltered levels of neural activity . as such , in longitudinal designs involving nonchronic patients , it may be impossible to differentiate between changes in neural activation and cerebrovascular reactivity using t - fmri alone . of particular concern , several studies have found that the haemodynamic response near a lesioned site is more strongly impacted by injury than nonlesioned regions , even in chronic disease states [ 71 , 73 , 74 ] . finally , there is evidence that aspects of cerebrovascular reactivity may be correlated with motor performance in certain stroke patients , even in the absence of marked vascular disease . it is noteworthy that dynamic causal modelling , a more advanced fmri analysis method , may be more robust to haemodynamic inhomogeneities by modelling haemodynamics in a region - wise fashion [ 78 , 79 ] although movement between frames can be reversed through reslicing , there are other sources of signal changes associated with movement ( e.g. , spin history effects ) that will remain . even after statistical adjustment , submillimetre rms movement can lead to measurably reduced statistical power . such movement is more likely in subjects with movement disorders ( i.e. , dystonia ) or reduced cognitive abilities or who find the task difficult . movement artefacts can be reduced by excluding subjects or censoring frames with movements , but this may systematically reduce the statistical power for one subject group and can lead to sampling biases . the aim of this review is not to critique t - fmri per se nor to blanket - prescribe a specific method by which to quantify functional images when measuring neuroplasticity . rather , we wish to make researchers and clinicians aware of the systematic and methodological challenges affecting common t - fmri study designs , which are often not addressed or acknowledged , and elucidate how these issues can be mitigated through a multimodal approach . to summarise our case so far , even if confounds such as movement , acute effects , and haemodynamic differences are eliminated , it is still possible that some findings may be explainable by unavoidable data processing steps , such as smoothing and spatial normalisation . these issues are particularly concerning given the vast patient variability and low subject numbers seen in this field . even when overcoming such issues , assumptions of brain plasticity based on t - fmri evidence alone are problematic due to difficulties in differentiating between recovery , compensation , use of preexisting redundancies , changes in strategy , and maladaptive processes . in studies of neurorehabilitation , it is critical that researchers can correctly interpret what a change in t - fmri signal actually means in order to understand the mechanisms of functional recovery . in this review our basic criteria for informative interpretation required that signal changes were moderately stable , meaningfully distinguishable from day - to - day variation , reliably related to clinical changes , and offered biological insight into the recovery process . the first , imperative , step to meeting these criteria with t - fmri is to relate changes to valid and reliable measures of motor function . planning longitudinal studies can also provide certainty that any activation changes seen are not due to patient heterogeneity . to overcome the remaining challenges , firstly , multimodal information can allow more homogenous cohorts to be selected or subgroups identified for analysis . secondly , by providing contextual information , other modalities can narrow down which biological process t - fmri may have indexed . relatedly , additional modalities can quantify potentially influential covariates , such as haemodynamic differences , to assess their impact on t - fmri . finally , when uncertainties and/or ambiguities are still prevalent , change measured through an independent method can provide confidence that t - fmri is genuinely indexing a stable functional change . many multimodal configurations are available that have already proven valuable in helping studies meet these criteria ; examples are listed in table 1 . structural mri allows measurement of cortical thickness : essentially an index of locally or globally available grey - matter . while cortical thickness can be challenging to measure precisely , especially in patient cohorts presenting with cortical lesions or malformations , such analyses are typically automated , simple to visually assess , and can be easily overlaid with t - fmri statistical parametric maps . adequate structural images are routinely acquired within fmri - scan sessions and usually simple to acquire motion - free . while structural imaging is probably less sensitive to change than t - fmri , these methods share few sources of uncertainty and provide one another with useful contexts for plausible interpretation . in particular , as locally increased grey - matter thickness likely reflects newly ongoing utilisation of that tissue , increases in this measure may indicate that any accompanying t - fmri activation increases are moderately stable and reflect some form of gain - in - function rather than , for example , a switch to an unchanged backup network . changes seen in cortical thickness are particularly beneficial to studies with limited subject numbers , where well - powered group analyses , which can rule out day - to - day variability in neural or vascular dynamics , are difficult or impossible to perform . analyses of structural images and diffusion mri ( below ) can also quantify potential covariates ( such as degeneration , regressing oedema , or developmental maturation ) that may affect t - fmri metrics longitudinally and are likely to vary by subject - cohort and time - point . tms is unique in its ability to directly characterise structural - functional connectivity , including intercortical inhibition , corticospinal tract conductivity , and motor thresholds . tms may prove particularly useful for studies that need to characterise the functional meaning of t - fmri determined li changes . tms has been used in multiple studies to differentiate between subject subgroups , allowing researchers to understand the biological significance of bilateral fmri activation patterns in cp [ 6 , 8 ] and reveal correlations between fmri changes and long term outcomes in stroke . magnetoencephalography ( meg ) and electroencephalography ( eeg ) can improve certainty in t - fmri changes by providing direct measures of net neuronal activity that are not likely to be impacted by factors such as haemodynamics , or the aforementioned methodological considerations . the very high temporal resolution of these methods can also allow researchers to distinguish between stages of processing , such as motor planning and execution . concurrent eeg + fmri is now possible , although caution may be advised in cohorts for whom head movement is an issue , as concurrent artefacts may result in plausible type - i errors . eeg and meg information can profoundly change the interpretation of changes in t - fmri metrics , such li or activation volume , and elucidate whether comparisons between subject groups are valid . for example , meg has been used in conjunction with fmri and tms to demonstrate that , in some subjects with cp , bilateral t - fmri s1m1 activation reflects contralateral somatosensory processing alongside ipsilateral ( reorganised ) motor processing . this illustrates clearly how categorisation of such subjects into homogeneous subgroups can be critical for t - fmri metrics to be appropriately interpreted ( figure 3 ) . [ d]efinitively disentangling such bilateral activation is only possible when complementary methods are used , like tms and meg . positron emission tomography and arterial spin labelling are neuroimaging methods that can provide measurements of regional cbf , and so reveal whether haemodynamic differences are affecting fmri measurements . arterial spin labelling is a contrast - agent - free mri technique that can be carried out in ~10 minutes , during the same session as an fmri . pet is advantageous in that it can additionally provide direct measures of glucose metabolism in brain tissue but requires access to pet imaging equipment and associated radiopharmaceutical facilities . because both of these methods provide quantitative measures of local haemodynamics , they can quantify precisely how fmri measurements in each region are affected by factors such as angiogenesis or vascular impairments . this may provide certainty in situations involving lesions , suggest adjustment of haemodynamic parameters , provide guidance on study design ( i.e. , indicate whether a block - design should be chosen over an event - related design ) , or shed light on otherwise - unclear findings . in one illustrative study of healthy adults , increases in t - fmri activation volumes were shown in the supplementary motor area and m1 after two weeks of motor training . these volumes subsequently declined to near - baseline values during the following two weeks of training , despite ongoing improvements in motor performance . pet scans showed that regional cbf increased between all time points , revealing that fmri decreases were probably due to increased blood flow at rest , rather than actual decreases in brain activity during task execution . diffusion mri ( dmri ) measures the directional diffusivity of water in tissue and can provide a variety of useful metrics . in subacute head injury or stroke , dmri can be used to ensure that t - fmri differences reflect more than inflammation or oedema . microstructural integrity indices , such as fractional anisotropy and mean diffusivity , can provide evidence that t - fmri changes represent ongoing changes in brain activity outside of the scanner : these metrics correlate with , and are sensitive to , myelination , which increases in response to ongoing electrical activity . advanced analyses can identify white matter pathways , calculate their intra - axonal volumes , and index the physical connection strength between cortical and subcortical regions . these measures correlate with functional measures in cp and may help provide a more complete picture when interpreting changes between balances of activation between brain regions . another form of dmri , neurite orientation dispersion and density imaging , provides the opportunity to reveal whether shifts or enlargements of t - fmri activation reflect local network changes in , for example , the cortex or thalamus . diffusion mri data are easily acquired in the same session as an fmri scan , usually in 812 minutes . as dmri is acquired at rest , overt movement is easier to avoid than with t - fmri and is unlikely to be correlated with factors such as ability . standard preprocessing methods can also correct or scrub moderately ( 10% ) motion - corrupted dmri data without compromising the final result . finally , a promising alternative approach is to not infer directly from t - fmri activation patterns , but rather to use t - fmri to identify functionally important regions - of - interest in which other modalities should make measurements ( figure 4 ) . this fusion of information can avoid some pitfalls of overanalysing changes in activation patterns , while considerably improving the sensitivity and interpretability of other modalities [ 5 , 88 ] . one fusion method which is being progressively adopted is the use of t - fmri activation patterns to guide diffusion tractography , allowing this method to focus microstructural and structural - connectivity measurements on functionally relevant areas [ 4 , 5 , 88 ] . asking focussed questions ( e.g. , how does rehabilitation alter s1m1 connectivity ? ) are not only inherently more testable than very broad questions ( e.g. , what does rehabilitation change in the brain ? ) , but can also provide guidance on which study design is appropriately powerful , which modalities and behavioural measures can contribute to the overall picture , and how t - fmri metrics may require supplementation or disambiguation with additional information . for example , investigations into somatosensory processing may require teasing apart t - fmri activation using temporally precise signals ( meg ) and/or information about integrity of the corticothalamic tracts ( tms or dmri ) . with a specific question in place , one should then consider which factors may primarily impair t - fmri interpretability . modalities that can minimise such issues are those which can either quantify their extent or provide supplementary evidence that is unaffected by such issues . for example , if a difference in t - fmri activation volume is expected between two groups , but one group may have impaired haemodynamics , quantifying regional - cbf with arterial spin labelling , or directly measuring brain activity with meg , may be of great benefit . as a contrasting example , a t - fmri study of subjects displaying dyskinesia is unlikely to benefit greatly from dmri , as both may be confounded by movement artefacts . studies with very low subject numbers , particularly cross - sectional studies , may see limited benefit from modalities that are less sensitive to change or have high intersubject variance . in such cases , resources may be better spent on boosting subject numbers or collecting additional behavioural information than on additional neuroimaging . in addition , studies that are unable to collect relevant and reliable clinical measures have limited abilities to discern the relevance of neuroplastic changes , regardless of how many imaging measures are taken . for measures of neuroplasticity in subjects with brain injuries , the reliability and interpretability of t - fmri is hampered by a unique set of systematic and methodological challenges . multimodal imaging provides the opportunity for t - fmri results to be interpreted with more confidence and biological specificity , ultimately providing greater understanding of the rehabilitative process . which complementary imaging modality offers the most benefit depends on the study question and subjects selected . many of these modalities have a minimal time and financial cost for acquisition while still offering exciting , novel opportunities to explore the relationship between structure , function , and clinical outcome which simply can not be investigated in any other way .

direct measurement of recovery from brain injury is an important goal in neurorehabilitation , and requires reliable , objective , and interpretable measures of changes in brain function , referred to generally as neuroplasticity . one popular imaging modality for measuring neuroplasticity is task - based functional magnetic resonance imaging ( t - fmri ) . in the field of neurorehabilitation , however , assessing neuroplasticity using t - fmri presents a significant challenge . this commentary reviews t - fmri changes commonly reported in patients with cerebral palsy or acquired brain injuries , with a focus on studies of motor rehabilitation , and discusses complexities surrounding their interpretations . specifically , we discuss the difficulties in interpreting t - fmri changes in terms of their underlying causes , that is , differentiating whether they reflect genuine reorganisation , neurological restoration , compensation , use of preexisting redundancies , changes in strategy , or maladaptive processes . furthermore , we discuss the impact of heterogeneous disease states and essential t - fmri processing steps on the interpretability of activation patterns . to better understand therapy - induced neuroplastic changes , we suggest that researchers utilising t - fmri consider concurrently acquiring information from an additional modality , to quantify , for example , haemodynamic differences or microstructural changes . we outline a variety of such supplementary measures for investigating brain reorganisation and discuss situations in which they may prove beneficial to the interpretation of t - fmri data .

1. Introduction 2. Common Findings 3. The Challenge 4. Disease Confounds 5. Summary and Recommendations 6. Conclusion

refers to the phenomenon of neurons and neural networks modifying their connections and/or behaviour in response to new information , sensory stimulation , development , damage , or dysfunction . the ultimate goal of neurorehabilitation is to induce neural plasticity in a manner that restores the full original function and potential of the injured brain ( neurological restoration ) , but a variety of other patterns of neural plasticity may also occur during recovery , including compensatory activity , use of redundant networks , or changes in behavioural or cognitive strategy . direct measures of such changes are critical to understanding how and when recovery from brain injury takes place and ultimately may lead to improved or novel rehabilitative treatments . one very popular modality used to measure neuroplasticity is task - based functional mri ( t - fmri ) . for a more in - depth explanation of fmri when used to measure neuroplasticity , however , t - fmri suffers from a unique set of challenges that are not always fully acknowledged . with the accelerating development of neurorehabilitation strategies researchers need to be cognisant of the limitations of commonly used neuroimaging technologies , including t - fmri , in order to collect information capable of advancing our understanding of the neurorehabilitative process . in particular , it is critical that researchers can correctly interpret what a change in t - fmri signal reflects , if they are to understand the mechanisms of functional recovery . to aid researchers in this regard , this review explores two important questions : what are the challenges in interpreting changes in t - fmri signal as intervention - induced neuroplasticity ? to contextualise our discussion , we define four basic criteria that we believe are essential for informative interpretation of any neuroimaging signal change in terms of brain changes . these criteria are somewhat straightforward : to advance neurorehabilitative science , reported changes must be unambiguous , reliable , related to recovery , and clearly a direct or indirect effect of the intervention at hand . with this in mind , we begin this review by outlining t - fmri findings associated with intervention - induced neuroplasticity and discuss uncertainties surrounding their interpretations . we highlight that change in t - fmri activation patterns can be difficult to extrapolate to brain reorganisation and , in some cases , may be confounded by processing inherent to the technique . we follow this overview by offering supporting strategies , focussing on the supplementation of t - fmri findings with information from other modalities , such as structural mri or transcranial magnetic stimulation ( tms ) . examples are provided as to how incorporating such information can improve interpretation of t - fmri data , strengthening specific claims about intervention driven neuroplasticity . though some points made here may be generalised to other contexts , this commentary restricts discussion to studies targeting motor impairment and movement rehabilitation in patients with cerebral palsy ( cp ) or acquired brain injuries , such as traumatic brain injury ( tbi ) . as relevant literature describing therapy - driven brain reorganisation is limited in patients with acquired brain injuries , we also make reference to neuroimaging studies based on adult stroke populations and some nonlongitudinal studies . it must be kept in mind that while subject groups may all undergo neuroplasticity in response to rehabilitation , they may do so from a vastly different baseline , particularly due to the impacts of brain injury on early development . further , for the sake of brevity , discussion here is restricted to standard glm - analyses of t - fmri , as this is the dominant technique in published literature ; resting state fmri and other forms of fmri are not considered . there are three primary findings that are commonly reported in t - fmri studies of neurorehabilitation , summarised in figure 1 . altered ipsilesional activated - voxel counts , or heightened peak intensities , are commonly reported for patients with brain injuries who have received treatment , improved function , or when compared with controls ( figures 1(a ) , 1(b ) , and 2(a ) ) . a recent systematic review reported seven longitudinal t - fmri studies of treatment interventions for unilateral cp , drawn from four unique subject cohorts . in tbi , one study of seven adult subjects with primarily - nonchronic injury showed changes in the activation volumes of several sensorimotor - related regions in response to motor rehabilitation . increased ipsilesional premotor activation has been shown in response to constraint - induced movement therapy , alongside improvements in fugl meyer assessment scores , in a single adult with chronic traumatic damage to the primary sensorimotor cortex ( s1m1 ) . the second common t - fmri finding in patients with brain injuries is a shift in the hemispheric - balance of activation ( figure 1(e ) ) . both stroke and unilateral cp patients , however , regularly demonstrate robustly bilateral activation [ 12 , 20 , 21 ] . these balances of activation are typically calculated as laterality index ( li ) : ( 1)li=cic+i , where c and i are suprathreshold voxel counts or t - value sums ( weighted li , also referred to here as li for simplicity ) , for the contralateral and ipsilateral hemispheres , respectively . small - scale studies of children with unilateral cp suggest that virtual reality and constraint - induced movement therapies can alter the balance of activation toward the contralateral hemisphere [ 13 , 28 ] . it is clear that changes in t - fmri measures have been reported in a variety of studies and pathologies . this section identifies several challenges that make the interpretation of such results in terms of neuroplasticity particularly difficult . these issues include subject variability , biological ambiguity , methodological considerations , and confounds introduced by disease states . as we shall discuss , these factors impede informative interpretation of the t - fmri signal by obscuring two key facts : ( 1 ) whether neurological change has genuinely taken place and ( 2 ) if so , what type of change has been observed . possible solutions to reduce the impact of these variables are summarised within the final section of this review . these incorporate the use of information from other modalities within the study design , providing complementary support for t - fmri measured brain changes , to provide more robust evidence of neuroplasticity . one of the greatest challenges for t - fmri in studies of neurorehabilitation is the heterogeneity in findings , both within and between studies of patients with brain injury . relocations of activation , for example , are not always reported and have been variable even within studies , differing , for example , by patient subgroup or task performed . in addition , changes in activation patterns do not consistently correlate with behavioural improvements ( figure 2(b ) ) . distinct changes in activation patterns have been reported in rehabilitative studies of adult stroke ( postrehabilitation versus retention ) , hemispherectomy ( pre- versus postrehabilitation ) , and paediatric cp ( pre- versus postrehabilitation ) , despite subjects demonstrating stable motor scores . restricting a study to patients in the chronic stage of injury , for example , may not remove effects due to progressive wallerian degeneration and/or volumetric changes , which take place during the first few years following stroke and , potentially , tbi . attempts to limit such variability is probably one reason why most t - fmri studies investigating neuroplasticity include only ~410 subjects with brain injury [ 13 , 21 ] . reproducibility studies have demonstrated that even well - controlled longitudinal t - fmri studies of normal subjects likely have a high degree of intrasubject measurement error and require at least 20 subjects per group to perform reliable and sensitive group analyses . it is common in the t - fmri literature to refer to activation differences as direct evidence of adaptive neuroplasticity . what is rarely addressed is the fact that activation differences , in isolation , do not allow researchers to differentiate between a variety of substantially different biological processes , many of which do not indicate regained , novel , or improved neurological capabilities and may not be positive or adaptive at all . it is already established that the brain can switch between apparently functionally equivalent sensorimotor representations in response to disrupted activity , for example , during a tumour removal operation , or reversibly within minutes of direct inactivation of motor areas . importantly , t - fmri alone is unable to determine whether such dynamics reflect a switch ( 1 ) to an equipotent area ( reflecting ongoing impairment ) , ( 2 ) back to the original area ( restored function ) , or ( 3 ) to an area previously incapable of such responsibility ( novel gain in function ) . some adult stroke patients rely more heavily on proprioceptive feedback than healthy subjects , for example . in addition , studies combining tms and t - fmri have revealed that attention , anticipation , and/or the forward - planning of motor movements dramatically alters cortical excitability in button pressing tasks [ 48 , 49 ] . given these points , it is not unreasonable to surmise that subtly different behavioural strategies may underlie subtle changes in t - fmri activation patterns . while it could be argued that learning is a form , or the result , of neuroplasticity , again the usability of information becomes limited if one can not differentiate between working around ongoing disability and neurological restoration . in order to perform similarly to controls , however , impaired patients have to apply more effort or execute different strategies , such as a more heavy reliance on feedback , which can increase recruitment of s1m1 , attentional networks , and/or supplementary areas . subjects performing different tasks , however , may use different task strategies , musculature , and/or receive different somatosensory feedback , all of which may alter activation patterns . researchers should carefully consider their participants before selecting this course of action as the attention required to perform multiple tasks without head movement may be beyond the means of young children , people with moderate - to - severe disability , and participants with acute brain injury ( such as concussion ) . another option is the use of trivial tasks with limited cognitive load , for which perceived task difficulty and performance are likely to be identical across sessions . scans using these tasks , however , may be insensitive to subtle reorganisation and would require exclusion of most moderately impaired patients , for whom no task is trivial . as such , most rehabilitation studies that incorporate fmri of motor tasks are best positioned by accepting the task equivalency problem , choosing a simple / stable task , and making claims in the context of secondary , independent evidence of neurological change . at least in stroke , however , contralesional activation does not appear to be a good predictor of functional recovery . unknown anatomy and functional dynamics can further undermine interpretation of changes in li . these results highlight how difficult correctly interpreting t - fmri activation differences can be in subjects with impairment . which interpretation is correct is something that can not be determined by t - fmri alone . there are methodological considerations to consider when evaluating changes , or differences , in the spatial extent and location of t - fmri activation peaks . this normalisation step can , however , inappropriately distort the location of tissues surrounding brain lesions . care must also be taken with interpretation of cluster analyses , which comprise the majority of recent t - fmri analyses [ 62 , 67 ] . use of liberal thresholds is not uncommon : a recent review of 814 cluster - based fmri studies published in high - impact journals described use of liberal thresholds as both endemic and detrimental to the neuroimaging field . beyond their most obvious motor impairment , subjects with brain injuries may also present with a number of complicating factors that can not easily be controlled for between groups or time points and may impact t - fmri analyses in unexpected ways . as such , special care should be taken not to misconstrue t - fmri changes during early disease states as neuroplasticity , without secondary evidence ruling out such causes . these assumptions may be invalidated by the substantial cerebrovascular damage that is associated with many forms of stroke , tbi , and congenital hemiplegia . as such , in longitudinal designs involving nonchronic patients , it may be impossible to differentiate between changes in neural activation and cerebrovascular reactivity using t - fmri alone . the aim of this review is not to critique t - fmri per se nor to blanket - prescribe a specific method by which to quantify functional images when measuring neuroplasticity . rather , we wish to make researchers and clinicians aware of the systematic and methodological challenges affecting common t - fmri study designs , which are often not addressed or acknowledged , and elucidate how these issues can be mitigated through a multimodal approach . to summarise our case so far , even if confounds such as movement , acute effects , and haemodynamic differences are eliminated , it is still possible that some findings may be explainable by unavoidable data processing steps , such as smoothing and spatial normalisation . even when overcoming such issues , assumptions of brain plasticity based on t - fmri evidence alone are problematic due to difficulties in differentiating between recovery , compensation , use of preexisting redundancies , changes in strategy , and maladaptive processes . in studies of neurorehabilitation , it is critical that researchers can correctly interpret what a change in t - fmri signal actually means in order to understand the mechanisms of functional recovery . in this review our basic criteria for informative interpretation required that signal changes were moderately stable , meaningfully distinguishable from day - to - day variation , reliably related to clinical changes , and offered biological insight into the recovery process . the first , imperative , step to meeting these criteria with t - fmri is to relate changes to valid and reliable measures of motor function . secondly , by providing contextual information , other modalities can narrow down which biological process t - fmri may have indexed . relatedly , additional modalities can quantify potentially influential covariates , such as haemodynamic differences , to assess their impact on t - fmri . finally , when uncertainties and/or ambiguities are still prevalent , change measured through an independent method can provide confidence that t - fmri is genuinely indexing a stable functional change . while cortical thickness can be challenging to measure precisely , especially in patient cohorts presenting with cortical lesions or malformations , such analyses are typically automated , simple to visually assess , and can be easily overlaid with t - fmri statistical parametric maps . while structural imaging is probably less sensitive to change than t - fmri , these methods share few sources of uncertainty and provide one another with useful contexts for plausible interpretation . in particular , as locally increased grey - matter thickness likely reflects newly ongoing utilisation of that tissue , increases in this measure may indicate that any accompanying t - fmri activation increases are moderately stable and reflect some form of gain - in - function rather than , for example , a switch to an unchanged backup network . analyses of structural images and diffusion mri ( below ) can also quantify potential covariates ( such as degeneration , regressing oedema , or developmental maturation ) that may affect t - fmri metrics longitudinally and are likely to vary by subject - cohort and time - point . tms may prove particularly useful for studies that need to characterise the functional meaning of t - fmri determined li changes . magnetoencephalography ( meg ) and electroencephalography ( eeg ) can improve certainty in t - fmri changes by providing direct measures of net neuronal activity that are not likely to be impacted by factors such as haemodynamics , or the aforementioned methodological considerations . eeg and meg information can profoundly change the interpretation of changes in t - fmri metrics , such li or activation volume , and elucidate whether comparisons between subject groups are valid . for example , meg has been used in conjunction with fmri and tms to demonstrate that , in some subjects with cp , bilateral t - fmri s1m1 activation reflects contralateral somatosensory processing alongside ipsilateral ( reorganised ) motor processing . this illustrates clearly how categorisation of such subjects into homogeneous subgroups can be critical for t - fmri metrics to be appropriately interpreted ( figure 3 ) . in one illustrative study of healthy adults , increases in t - fmri activation volumes were shown in the supplementary motor area and m1 after two weeks of motor training . diffusion mri ( dmri ) measures the directional diffusivity of water in tissue and can provide a variety of useful metrics . in subacute head injury or stroke , dmri can be used to ensure that t - fmri differences reflect more than inflammation or oedema . microstructural integrity indices , such as fractional anisotropy and mean diffusivity , can provide evidence that t - fmri changes represent ongoing changes in brain activity outside of the scanner : these metrics correlate with , and are sensitive to , myelination , which increases in response to ongoing electrical activity . another form of dmri , neurite orientation dispersion and density imaging , provides the opportunity to reveal whether shifts or enlargements of t - fmri activation reflect local network changes in , for example , the cortex or thalamus . as dmri is acquired at rest , overt movement is easier to avoid than with t - fmri and is unlikely to be correlated with factors such as ability . finally , a promising alternative approach is to not infer directly from t - fmri activation patterns , but rather to use t - fmri to identify functionally important regions - of - interest in which other modalities should make measurements ( figure 4 ) . this fusion of information can avoid some pitfalls of overanalysing changes in activation patterns , while considerably improving the sensitivity and interpretability of other modalities [ 5 , 88 ] . one fusion method which is being progressively adopted is the use of t - fmri activation patterns to guide diffusion tractography , allowing this method to focus microstructural and structural - connectivity measurements on functionally relevant areas [ 4 , 5 , 88 ] . , but can also provide guidance on which study design is appropriately powerful , which modalities and behavioural measures can contribute to the overall picture , and how t - fmri metrics may require supplementation or disambiguation with additional information . for example , investigations into somatosensory processing may require teasing apart t - fmri activation using temporally precise signals ( meg ) and/or information about integrity of the corticothalamic tracts ( tms or dmri ) . with a specific question in place , one should then consider which factors may primarily impair t - fmri interpretability . for example , if a difference in t - fmri activation volume is expected between two groups , but one group may have impaired haemodynamics , quantifying regional - cbf with arterial spin labelling , or directly measuring brain activity with meg , may be of great benefit . as a contrasting example , a t - fmri study of subjects displaying dyskinesia is unlikely to benefit greatly from dmri , as both may be confounded by movement artefacts . in addition , studies that are unable to collect relevant and reliable clinical measures have limited abilities to discern the relevance of neuroplastic changes , regardless of how many imaging measures are taken . for measures of neuroplasticity in subjects with brain injuries , the reliability and interpretability of t - fmri is hampered by a unique set of systematic and methodological challenges . multimodal imaging provides the opportunity for t - fmri results to be interpreted with more confidence and biological specificity , ultimately providing greater understanding of the rehabilitative process . which complementary imaging modality offers the most benefit depends on the study question and subjects selected . many of these modalities have a minimal time and financial cost for acquisition while still offering exciting , novel opportunities to explore the relationship between structure , function , and clinical outcome which simply can not be investigated in any other way .

asthma affects up to 12% of adults and 25% of children in australia and there is a significant undiagnosed cohort . although we have a range of medications that are effective in their own right , there is a need to further improve the management of this disease . this involves better clinical programs and an improved understanding of the basic mechanisms of asthma so that new ways can be introduced which work synergistically with conventional asthma medications to modify airway inflammation . asthma is a disease characterized by both bronchiolar smooth muscle constriction and a chronic airway inflammation . some of the features of the disease are modelled in the well - characterized acute and chronic models of ova - induced allergic airway inflammation in mice [ 2 , 3 ] . the inflammatory component of asthma is generally thought of as a th2-driven process , involving eosinophil recruitment to the airways and consequent damage , including airway epithelial cell death . damage and repair eventually lead to a remodelling of the airways which increases the sensitivity of the airway muscle to cholinergic agonists and further restricts airflow . however , other inflammatory signalling pathways may also play a significant role in the pathogenesis and progression of the disease . of particular interest is the role of the il-1 family of cytokines that are key components of the innate immune response . il-1 and il-18 are activated by cytosolic multiprotein complexes called inflammasomes [ 6 , 7 ] . inflammasomes have been best characterized in the monocyte - macrophage cell lineage , but recent evidence indicates that gingival ( and perhaps other ) types of epithelial cells may also contain these structures . their generic structure includes ( i ) a member of the nucleotide - binding oligomerization domain- ( nod- ) like receptor ( nlr ) family of pattern recognition molecules specific for each type of inflammasome , ( ii ) apoptosis - associated speck - like protein containing a caspase - recruitment domain ( asc ) , and ( iii ) caspase-1 . other inflammatory caspases or caspase - regulatory molecules such as x - linked or neuronal inhibitor of apoptosis proteins , xiap and niap , respectively , may also be recruited . different types of inflammasome ( e.g. , nlrp1 , nlrp3 , ipaf , and aim2 ) have been identified based on the nlr component ( or the non - nlr equivalent ) which forms the complex . of these the best characterized is the nlrp3 inflammasome which plays a predominant role in il-1 and il-18 production . in macrophages , binding of ligands such as lipopolysaccharide ( lps ) to membrane toll - like receptor 4 ( tlr-4 ) triggers the synthesis of pro - il-1 while a number of danger signals including molecules released from necrotic cells ( e.g. , atp and uric acid ) promote assembly of the nlrp3 inflammasome complex , activation of caspase-1 from its precursor , processing of il-1 to its active form , and release of il-1 from the cells . an early protective role for inflammasomes might be predicted by the hygiene hypothesis , whereby exposure to microbes and their products ( such as lps ) early in life is thought to protect against development of asthma , perhaps by a skewing of the immune response away from one dominated by th2 cytokines . however , current evidence would favour a proinflammatory role for il-1 since ( i ) there are increased levels of serum , balf , and bronchial epithelial il-1 in human asthmatics , compared to healthy subjects [ 1012 ] , ( ii ) increase in serum il-1 has also been reported in primates , ( iii ) il-1 levels were decreased 2-fold in the bronchial epithelium following inhalation of beclomethasone dipropionate ( as measured by an immunohistological technique ) , and ( iv ) administration of tnf- and il-1 induces airway hyper - reactivity , a feature of asthma [ 15 , 16 ] . il-18 , another potent pro - inflammatory cytokine whose maturation requires activation of caspase-1 on the inflammasome , is typically considered as a th1 cytokine due to its effects associated with ifn-. increased serum il-18 has also been described in asthmatics [ 1719 ] . finally , danger signal molecules such as uric acid and extracellular atp have been shown to mediate inflammasome - dependent inflammation in experimental rodent models of lung injury and asthma , respectively . in addition to providing a physical barrier to inhaled pathogens , allergens , and other foreign agents , the airway mucosa ( epithelium and secretions ) has a number of potential mechanisms by which it can contribute to innate immune defences in the lung . loss of the integrity of the airway epithelium is widely thought to be a critical component in the pathogenesis of asthma . to our knowledge , there has been no systematic study of the inflammasome in airway epithelium . in this study , we have looked for evidence of nlrp3 inflammasome involvement in ova - induced airway inflammation in mice , as a model of human asthma , as well as in primary bronchial epithelial cultures . rabbit polyclonal antibodies ( abs ) to il-1 , nlrp3 , total caspase-1 ; goat abs to caspase-1 p10 and p20 active subunits ; and mouse monoclonal ab ( mab ) to sp - d were from santa cruz biotechnology ( santa cruz , ca , usa ) . other rabbit abs were asc from abcam ( cambridge , uk ) , ccr3 from epitomics ( burlingame , cl , usa ) , and il-18 from rockland ( gilbertsville , pennsylvania , usa ) . secondary abs were sheep f(ab)2 anti - rabbit igg ( cy3 ) , goat f(ab)2 anti - rabbit igg ( fitc ) , rabbit anti - goat igg ( cy3 ) , sheep f(ab)2 anti - mouse igg ( cy3 ) , ( sigma - aldrich chemicals , st louis , mo , usa ) , and a donkey f(ab)2 anti - mouse igg dylight594 ( jackson immunoresearch , west grove , pa , usa ) . a classical mouse model that induces strong eosinophilic inflammation , airway hyper - responsiveness , and vascular and parenchymal changes after sensitisation and challenge with ova was used to induce inflammation . all experiments were performed under the university of adelaide animal ethics committee approval number m-57 - 2007 , and in compliance with principles of animal care publication number 86 - 23 of the national institute of health and the australian code of practice for the care and use of animals for scientific purposes , 6th edition . female balb / c mice ( age 46 weeks ; specific pathogen free ) were purchased from the university of adelaide , adelaide , australia . three mice were set aside for analysis of inflammasome components in the absence of any stimulus . the remaining mice were divided into two experimental groups ( n = 8 in each group ) and housed in plastic cages ( 38 25 cm ) at 21c with a 14 h light/10-h dark cycle . ova - treated mice received 50 g of chicken ova in 1 ml of alhydrogel ( csl , parkville , australia ) in 0.9% sterile saline , i.p . on days 0 and 14 . mice sensitized to ova were then aerochallenged with 10 mg / ml ova in 0.9% saline from day 22 to day 32 , for 30 min , three times a day , every 2nd day , using a side - stream nebulizer , which produced particles of 13 m ( fisher and paykel , sydney , australia ) . dose of pentobarbitone sodium ( 50 mg kg ) , 22 h after the last nebulization . the trachea was cannulated with a blunted 19-gauge needle and bronchoalveolar lavage fluid ( balf ) was collected by lavaging the lungs three times by instilling and withdrawing the same volume ( 1 ml ) of ice - cold hanks buffered salt solution ( hbss , ph 7.4 ) . one part of the balf sample was immediately cytospun onto slides and the other part was centrifuged to obtain supernatant for cytokine and uric acid assay . tissue samples were archived as appropriate for various downstream measurements , including standard assessments of airway and tissue eosinophilia and lung histopathology . samples of mouse lung tissue were immediately placed into rna later ( applied biosystems / ambion , usa ) . tissue was homogenized using a tissueruptor ( qiagen pty ltd , australia ) and rna extracted using an rneasy mini kit ( qiagen pty ltd , australia ) . an on - column dnase treatment was performed using an rnase - free dnase set ( qiagen pty ltd , australia ) . rna quality was checked using an experion rna stdsens kit on the experion electrophoresis station ( biorad laboratories inc , australia ) . mouse rna samples obtaining rqi of 710 ( green ) were used in the subsequent array analysis . rna content was quantified using a nano drop 1000 spectrophotometer ( thermo scientific , usa ) . 1000 ng of rna was reverse transcribed into cdna using the rt first strand kit ( qiagen pty ltd , australia ) . the incubation steps for the reaction were performed on a mycycler thermal cycler ( biorad laboratories inc , australia ) . mouse cdna samples were combined with rt sybr green / fluorescein qpcr master mix with nuclease - free sterile h2o ( qiagen pty ltd , australia ) and then added across an entire rt2 profiler pcr mouse inflammasome array ( pamm-097a ) 96-well plate . each mouse sample ( n = 8 from each group ) was tested on a separate 96-well array plate containing a panel of 84 wells pertaining to different genes of the inflammasome pathway , 5 house keeping control wells , 1 genomic dna control well , 3 reverse transcription control wells , and three positive pcr control wells . pcr was performed using an icycler with iq5 software ( biorad laboratories inc , australia ) and cq data was then analysed by the ct method using the rt profilier array data spreadsheet ( qiagen pty ltd , australia ) . serum cytokines were measured by bioplex as per the manufacturer 's instructions ( biorad laboratories inc , australia ) . in brief , the concentration of total protein per sample was determined by mini bradford assay ( biorad laboratories inc , australia ) using standards of bovine serum albumin ( sigma - aldrich chemicals , st louis , mo , usa ) . samples were normalised to 100 g total protein and analysed in duplicate using a bioplex promouse cytokine 8 plex magnetic bead array . plate data was collected using a bioplex 200 suspension array system and analysed using the bioplex manager 5 software ( biorad laboratories inc , australia ) . paraffin tissue sections of 5 m thickness were treated for antigen retrieval by microwave heating in 10 mm citrate buffer ph 6.0 unless otherwise stated . tissue sections were also heated in 10 mm tris edta buffer ph 9.0 for costaining of sp - d with caspase-1 , and digested with 1 mg / ml proteinase k ( promega , fitchburg , wisconsin , usa ) for staining of p10 and p20 caspase-1 subunits . sections were blocked with a serum - free protein blocking solution ( dakocytomation inc . , carpinteria , ca , usa ) , incubated overnight at 4c with primary abs , and 1 h at room temperature with secondary abs . immunofluorescence was detected and imaged with a zeiss microscope equipped with hbo 100 illuminating system , axiocam mrn digital camera and axiovision 4.8.1 software ( carl zeiss gmbh , goettingen , germany ) . cell death was detected in situ using a fluorescence tunel kit from promega ( fitchburg , wisconsin , usa ) following the manufacturer 's protocol . uric acid was measured in balf according to manufacturer 's instructions using the amplex red uric acid / uricase assay kit ( a22181 , invitrogen aust pty ltd , mulgrave , australia ) . fluorescence measurements were made using a fluorescence plate reader ( optima fluostar , bmg labtech , gmbh , offenburg , germany ) with excitation at 530 nm and emission read at 590 nm . normal human bronchial epithelial ( nhbe ) cells and bronchial epithelial growth medium ( begm ) were obtained from lonza ( lonza australia pty ltd , mt waverley , vic , australia ) . cells at 6070% confluence were stimulated for 18 h with e. coli lps ( 5 g / ml , sigma - aldrich chemicals , st louis , mo , usa ) , ifn- ( 50 ng / ml , sigma - aldrich chemicals , st louis , mo , usa ) , or combination of these two , in 8-well chamber slides ( bd biosciences , franklin lakes , nj , usa ) . cells were washed twice with phosphate - buffered saline ( pbs ) and then fixed with 2.5% formalin / pbs for 10 minutes . cells were washed 4 times with tbst ( tris - buffered saline , ph7.5 added with 0.05% tween-20 ) , air - dried overnight at room temperature , and stored at 20c until use . sds / pbs at room temperature , then washed 5 times with tbst to remove sds . following 1 h blocking incubation with a serum - free protein blocker ( dakocytomation inc . , carpinteria , ca , usa ) , cells were incubated with a mixture of primary antibodies ( see antibody section of methods ) including mouse monoclonal antibody to human nlrp3 and a rabbit polyclonal anti - human il-1 , overnight at 4c . next , following 5 washes with tbst , cells were incubated 1 h at room temperature with a mixture of secondary antibodies including a donkey f(ab)2 anti - mouse igg dylight594 and goat f(ab)2 anti - rabbit igg - fitc . for quantitation of fluorescence intensity , multiple microphotos were acquired randomly under a 20x objective . images were then blinded and analysed using a morphometric software package ( imagej , nih , bethesda , md , usa ) . in keeping with previous descriptions of the acute allergic airway inflammation model [ 2 , 23 ] , the airway inflammation in ova - challenged mice was characterized by dense peribronchial and perivascular infiltrates of leucocytes ( mostly eosinophils ) and oedema of bronchial epithelium . epithelial swelling was more prominent in large bronchi that were sometimes occluded by thickened epithelium . typically , there was a 7-fold increase in total balf cell count from 15.6 11.1 10 ( median 2.5% ci ) cells / ml in sal controls to 117.7 82.7 10 cells / ml in ova - treated mice ( p < 0.0001 ) . this was largely due to eosinophilia ( > 85% ) , although numbers of neutrophils , monocytes , and lymphocytes were also significantly increased ( p < 0.05 , data not shown ) . to test the hypothesis that the normal airway epithelium possesses the ability to assemble inflammasome complexes , we used immunofluorescence to analyse formalin - fixed lung tissue sections obtained from healthy balb / c mice for expression of inflammasome common components nlrp3 , asc and caspase-1 , and the substrate cytokines il-1 and il-18 . n = 3 ) and sal mice ( n = 8) that were sham - treated with saline i.p . injections and saline nebulisation and used as controls in the experimental murine airway inflammation model . serial sections of control mouse lungs ( n = 8) were examined by immunohistological labelling for caspase-1 and various other inflammasome - related proteins . figures 1(a)1(c ) show colocalization of caspase-1 and nlrp3 in the epithelia of three bronchioles . the fluorescence staining for caspase-1 however , omission of primary antibody or section incubation with normal rabbit igg ( at matched igg concentrations ) resulted in negligible fluorescence ( figure 1(f ) ) . colocalization of caspase-1 and nlrp3 with il-1 , il-18 , and asc in bronchiolar epithelia was shown ( figures 1(d)1(k ) ) . it should be noted that the antibodies to caspase-1 , il-1 , and il-18 did not distinguish between their precursor and mature forms . after performing immunofluorescence , some sections were washed and restained with hematoxylin and eosin ( h&e ) to examine morphology . figures 2(a ) and 2(b ) show low and high magnification images of a section of the normal mouse lung immunolabelled for caspase-1 , while figures 2(c ) and 2(d ) show the corresponding h&e restaining . the caspase-1 positive - staining cells around the alveoli were identified as both alveolar macrophages localised in the alveolar air space ( e.g. , arrowed cell in figure 2(d1 ) ) and type 2 alveolar cells ( atii ) typically seen at alveolar junctions and showing lamellar bodies in the cytoplasm ( figures 2(d2 ) and 2(d3 ) ) . the identity of these two cell types was confirmed by dual labelling of caspase-1 with either f4/80 ( a marker of mouse macrophages , figure 2(e ) ) or sp - d ( a marker of atii , figure 2(f ) ) . thus , the normal murine airway epithelium ( bronchial epithelium and atii ) expresses , at the protein level , inflammasome components nlrp3 , asc , and caspase-1 , as well as the substrate cytokines il-1 and il-18 . the antibody for caspase-1 used in the previous series of experiments did not differentiate between the inactive precursor and active form . using antibodies specific for the cleaved ends of p10 and p20 subunits of caspase-1 , we were able to demonstrate very low levels or absence of active caspase-1 in the control mouse lung epithelium ( figure 3(a ) ) . in contrast , distinct patterns of active caspase-1 staining were detected in the epithelium of the inflamed airways of ova - treated animals ( figures 3(b)3(d ) ) . in adjacent serial sections of inflamed lung , the two different antibodies p10 and p20 revealed the same punctate patterns of caspase-1 activation near the epithelial apical surface ( figures 3(c ) and 3(d ) ) . preabsorption with the relevant immunogen peptides reduced the labelling of active caspase-1 in the inflamed airway epithelium to a level comparable to that of the conjugate alone ( figures 3(e)3(g ) ) . because there are inherent problems in trying to quantify actual changes in fluorescence intensity where there is a redistribution of the label within the treatment accompanying the induction of inflammation , we have not attempted to quantify changes in intensity of immunofluorescence for the various proteins in these sections . these results suggest that inflammation of the airways in mice is accompanied by conversion of zymogen caspase 1 to its active form and a more apical distribution of inflammasome proteins . as mentioned earlier , our antibody panel did not differentiate between the inactive precursors and the mature ( active ) forms of il-1 and il-18 cytokines . however , there were different patterns of il-1 and il-18 distribution demonstrated by immunofluorescence between the epithelia of ova - treated and control mice ( figure 4 ) . in the healthy epithelium , both cytokines distributed more or less homogeneously in the cytoplasm ( figures 4(a ) and 4(b ) ) . in contrast , in the inflamed airways , the immunofluorescence of the il-1 cytokines had a more speckled appearance at the apical surface of the epithelium ( figures 4(c ) and 4(d ) arrows ) . furthermore , immunofluorescence of both il-1 and il-18 could be detected in the lumen ( figures 4(c ) and 4(d ) , arrowheads ) . similarly , luminal staining near the apical surface was also detected for caspase-1 ( figures 4(e ) and 4(k ) ) , nlrp3 ( figure 4(i ) ) , and asc ( not shown ) . when labelled sections of inflamed mouse lungs were restained with h&e ( figures 4(g)4(l ) ) , part of the luminal fluorescence of the cytokines and inflammasome proteins was localized to infiltrating leukocytes ( mostly eosinophils ) or well preserved cell bodies ( ~610 m in diameter ) often seen embedded in the mucus near the epithelial apex ( figure 4(h ) and 4(l ) insets ) . tissues were immediately fixed in formalin for paraffin embedding and the luminal staining was consistently observed in tissue sections , including serial adjacent sections ( e.g. , figures 3(c ) and 3(d ) , figures 4(c)4(e ) , with figure 4(f ) as negative control ) , in all of the mice tissues analysed . immunostaining of consecutive adjacent tissue sections with caspase-1 and ccr3 , a marker of eosinophils , showed colocalization of ccr3 with caspase-1 , detected by antibodies to either the total or the active forms of the caspase ( not shown ) . as ccr3 is not a specific marker of eosinophils but can also be expressed in some other cell types , immunolabelled sections were restained by h&e to identify the type of infiltrating cells positively stained for inflammasome components . in accordance with our previous results > 80% of these cells were eosinophils which infiltrated both the submucosal tissue and the airway lumen ( figures 4(h)4(l ) ) . another cell type which might contribute to inflammasome activation in the ova - induced airway inflammation model is the vascular endothelium . whereas in saline - treated mice the vascular endothelium stained weakly for total caspase-1 , it often stained strongly in the lungs of ova - treated mice ( data not shown ) . unfortunately , we could not apply the antibodies to active subunits of caspase-1 to study activation of endothelial caspase-1 , due to the nonspecific binding of these antibodies to red blood cells , which was not blocked by the specific peptides ( data not shown ) . the expression of total caspase-1 protein in inflamed but not healthy endothelium was completely removed by blocking with specific peptide ( data not shown ) . to look at the effect of airway inflammation on expression of gene products with relevance to the inflammasome , we analysed mrna from lungs of sal and ova - treated mice by superarray ( table 1 ) . typically , there were large increases ( 810 fold ) in chemokines ( ccl12 , ccl7 , and cxcl3 ) known to be important in the mouse airway inflammation model . there were also significant increases with ova - treatment in expression of a th2 cytokine il-6 . for the il-1-like family members , there was a 2.5-fold increase in gene expression for il-33 and 2- and 11-fold decreases in those for il-1 and il-18 , respectively . of the gene products involved in inflammasome regulation , the largest change was an 8-fold increase in the neuronal inhibitor of apoptosis protein naip , followed by an almost 4-fold decrease in nlrp3 expression . there were no significant changes in expression of nlrp1 and nlrp4 ( table 1 ) . in accordance with our previous data , using the tunel assay we confirmed an increased cell death in the lung sections of ova- versus sal - treated animals ( data not shown ) . an analysis of balf in the asthmatic mice demonstrated a significant increase in uric acid ( figure 5(a ) ) , which is known as both a product of nucleic acid catabolism in dead cells and a stimulus for nlrp3 inflammasome activation . we were unable to detect il-1 and il-18 in the balf of either saline- or ova - treated mice ( data not shown ) . this may be due to excessive dilution of the balf during the lavage or choosing the wrong time - point ( 22 hr after final challenge ) for lavaging . analysis of the sera showed high levels ( > 200 pg / ml ) of il-1 in 4 of the 8 ova - treated mice but in none of the 8 saline - treated control mice . the serum levels of il-1 were tightly correlated with those of tnf- ( r = 0.95 ) , suggesting systemic effects in this model of allergic airway inflammation ( figure 5(b ) ) . commercially available passage 4 primary cultured normal human bronchial epithelial ( nhbe ) cells were studied for expression of the nlrp3 and il-1 proteins , before and after priming with lps ( 5 ug / ml ) or ifn- ( 50 ng / ml ) . nlrp3 and il-1 proteins were quantified in the same cell by dual - labelling with the respective antibodies ( see methods ) and using imagej software to quantify fluorescence intensities . between 26 and 51 cells ( pooled from three randomly acquired images for each treatment ) were analysed simultaneously for both proteins . cells grown either in the absence of both agents or with ifn- alone expressed low levels of intracellular nlrp3 and il-1 as detected by immunofluorescence ( and quantified using imagej software ) . overnight stimulation with lps or lps plus ifn- resulted in significant increase ( p < 0.05 ) of both nlrp3 ( ~2 fold ) and il-1 ( 4 - 5 fold , figure 6 ) . this study provides the first evidence for presence of precursor components of the nrlp3 inflammasome in normal murine airway epithelium and some other resident or inflammatory airway cells . it also shows qualitative changes in the distribution of these markers , including appearance of active caspase-1 , during airway inflammation in the acute mouse ova model . further studies are warranted to establish the functional significance of inflammasome activation as well as links between inflammasome regulation and other known mechanisms in asthma such as th2 responses , ige production , and its fixation on innate immune cell surfaces , eosinophilia , and airway tissue remodelling . our findings suggest a role for inflammasomes in innate immune responses of the airway epithelium and reinforce the hypothesis that airway epithelium plays a sentinel role in the innate immune response to inhaled microbes , allergens , and other pathogens . in particular , we have shown that the components necessary for mounting of a rapid protective inflammatory response via inflammasome activation are all expressed in the murine airway epithelium prior to inflammatory stimulation . of interest , il-1 and il-18 a similar expression of il-1 was detected in rat lung epithelium tissue ( data not shown ) . the failure to detect il-1 in the circulation of the control mice suggests either that airway epithelium contributes very little to blood il-1 levels or that signals leading to release of il-1 ( and probably also il-18 ) are absent from normal airway epithelium . our finding of intracellular il-1 and il-18 is therefore in keeping with the hypothesis that the airway epithelium is an important sentinel in the innate immune response and is primed for a rapid response when exposed to danger signals but has yet to undergo inflammasome activation . studies in cells of monocytic lineage have shown that to mount a proinflammatory response , the inflammasome pathway works in synergy with receptors for pathogen - associated molecular patterns ( pamps ) and danger - associated molecular patterns ( damps ) , the best characterized of which are tlrs . thus , cell lines in their normal state usually do not express precursors of il-1 and il-18 ; production , maturation , and extracellular release of these potent proinflammatory cytokines require at least two signals . ligation of tlrs leads to enhanced transcription of the il-1 gene and accumulation of an immature form of il-1 in the cytoplasm . stimulation with substances such as extracellular atp or uric acid leads to a second signal that results in caspase-1 activation , processing of pro - il-1 , and release of the mature cytokine . using antibodies that detect the presence of neoepitopes in the caspase-1 subunits which become accessible only upon cleavage / activation of the proenzyme and a blocking peptide , we were able to provide qualitative evidence for presence of active caspase-1 in the airway epithelium of the ova - treated mice . we also found , in these ova - treated mice , a potentially interesting and novel phenomenon of subcellular translocation for epithelial il-1 , il-18 , and caspase-1 , whereby these molecules were seen to be concentrated at the epithelial apical surface and apparently shed into the airway lumen in the form of cell bodies . the significance of this is unclear but may provide a mechanism for release of inflammasome - relevant molecules into the mucosa . il-1 and il-18 are proteins without a signal sequence . the precursors accumulate in the cytosol following translation and a second signal is required for caspase-1-mediated cleavage of the precursors to the mature form that is released from the cell . various mechanisms for release of the active il-1 cytokines ( and caspase-1 , itself ) have been postulated , including preexport into secretory lysosomes or encapsulation in microvesicles or exosomes . evidence for release as cell bodies or smaller vesicles has been provided by studies in monocytes [ 24 , 26 ] , microglia , and dendritic cells . the clinical relevance of the luminal shedding of the il-1 cytokines is yet to be explored . our findings support the notion that the airway epithelium can itself mount a proinflammatory response via activation of its inflammasome complexes and consequent release of il-1 and il-18 . we were able to detect high levels of il-1 in the circulation but not in the balf . the reason for failure to detect the il-1-like cytokines in balf may be a consequence of dilution of apical secretions during our lavage procedure ( total wash volume was 3 ml ) or may be because the time interval between final ova challenge and balf collection ( 22 hr ) resulted in missing the peak cytokine response . relevant to this , 4 of the 8 ova - treated mice had high levels of il-1 and tnf- in their serum while the other 4 mice ( like all of the control mice ) had very low levels of both cytokines , despite all 8 ova - treated mice having high levels of airway inflammation as evidenced by eosinophilia in balf and tissues as well as epithelial swelling and cell death . our immunofluorescence studies also suggested that other potential sources of il-1-like cytokines are eosinophils , alveolar macrophages , type ii alveolar cells , and endothelium . relevant to our finding of nrlp3 in the normal mouse bronchiolar epithelium is the recent observation reporting nrlp3 and two other nlrs ( nod1 and nod2 ) in upper airway human tissues including normal nasal mucosa , nasal polyps , tonsils , and adenoids . it is likely therefore that inflammasome components are present along the entire length of the airways and constitute a front - line defence . their involvement in allergic and inflammatory diseases of the airways such as chronic rhinosinusitis , rhinitis , and asthma warrants further study . a major limitation with studies of the whole lung is that the lung is a complex organ comprising a number of tissues . during airway inflammation it is therefore difficult to interpret changes in gene expression at the level of a single cell type in the lung . because of this we were driven from the start at establishing the techniques to show the presence of inflammasome components in specific cell types of the lung at a histochemical level . our initial attempt to look at changes in expression of inflammasome - related genes in the context of murine airway inflammation showed a large increase in expression of the caspase inhibitor and inflammasome - binding protein naip as well as decreases in expression of il-18 and nrlp3 . decreases in nrlp3 and il18 gene expression during airway inflammation may indicate the existence of a negative feedback mechanism . since naip is also able to substitute for nrlp3 and other nrls in formation of the inflammasome complex , there may be a switch from nrlp3 to naip as a result of the inflammation . discordance between mrna and protein expression of p2x7 receptor , an important up - stream regulator of the nrlp3 inflammasome , has also been reported in inflamed intestinal epithelium from patients with inflammatory bowel disease . in that study , in which p2x7 receptor engagement was required for production and release of il-1 , mrna levels for p2x7 receptor were increased while protein levels were strongly decreased . the decrease in protein levels was much greater in patients with active disease compared to those with quiescent disease and correlated with the degree of polymorphonuclear infiltration into the epithelium ( transepithelial migration ) . the authors speculated that the decrease in protein expression may help to protect the intestinal epithelial cells from excessive activation of p2x7 receptor and subsequent cell death during the neutrophil transmigration . downregulation of nrlp3 protein may have a similar protective role against inflammation - associated cell death during episodes of asthma . dissection of the mechanisms involved in airway epithelial inflammasome regulation and activation will require the use of in vitro airway epithelial cultures . we have shown that the presence of nrlp3 and il-1 proteins in the commercially available normal human bronchial epithelial primary cells and the 25-fold upregulation of these proteins following priming with lps indicates involvement of tlr4 in the priming . another proinflammatory stimulus inf which acts via a distinct signalling pathway was ineffective . in conclusion , we have demonstrated the presence of precursor components of the nrlp3 inflammasome in healthy murine airway epithelium as well as changes in the subcellular distribution of these components and appearance of active caspase-1 in the epithelium of inflamed airways . at this stage we know little about the extent to which the innate immune system and inflammasome activation in particular exert protective or detrimental effects in asthma . further studies using both lung biopsies from patients and mechanistic studies in polarized cultures of human airway epithelium are warranted and may reveal new insight into the disease mechanism and additional targets for therapeutic and/or diagnostic applications in asthma .

little is known about innate immunity and components of inflammasomes in airway epithelium . this study evaluated immunohistological evidence for nlrp3 inflammasomes in normal and inflamed murine ( balb / c ) airway epithelium in a model of ovalbumin ( ova ) induced allergic airway inflammation . the airway epithelium of control mice exhibited strong cytoplasmic staining for total caspase-1 , asc , and nlrp3 , whereas the ova mice exhibited strong staining for active caspase-1 , with redistribution of caspase-1 , il-1 and il-18 , indicating possible activation of the nlrp3 inflammasome . active caspase-1 , nlrp3 , and other inflammasome components were also detected in tissue eosinophils from ova mice , and may potentially contribute to il-1 and il-18 production . in whole lung , inrna expression of naip and procaspase-1 was increased in ova mice , whereas nlrp3 , il-1 and il-18 decreased . some ova - treated mice also had significantly elevated and tightly correlated serum levels of il-1 and tnf. in cultured normal human bronchial epithelial cells , lps priming resulted in a significant increase in nlrp3 and ii - lp protein expression . this study is the first to demonstrate nlrp3 inflammasome components in normal airway epithelium and changes with inflammation . we propose activation and/or luminal release of the inflammasome is a feature of allergic airway inflammation which may contribute to disease pathogenesis .

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

asthma affects up to 12% of adults and 25% of children in australia and there is a significant undiagnosed cohort . this involves better clinical programs and an improved understanding of the basic mechanisms of asthma so that new ways can be introduced which work synergistically with conventional asthma medications to modify airway inflammation . asthma is a disease characterized by both bronchiolar smooth muscle constriction and a chronic airway inflammation . some of the features of the disease are modelled in the well - characterized acute and chronic models of ova - induced allergic airway inflammation in mice [ 2 , 3 ] . damage and repair eventually lead to a remodelling of the airways which increases the sensitivity of the airway muscle to cholinergic agonists and further restricts airflow . however , other inflammatory signalling pathways may also play a significant role in the pathogenesis and progression of the disease . of particular interest is the role of the il-1 family of cytokines that are key components of the innate immune response . il-1 and il-18 are activated by cytosolic multiprotein complexes called inflammasomes [ 6 , 7 ] . their generic structure includes ( i ) a member of the nucleotide - binding oligomerization domain- ( nod- ) like receptor ( nlr ) family of pattern recognition molecules specific for each type of inflammasome , ( ii ) apoptosis - associated speck - like protein containing a caspase - recruitment domain ( asc ) , and ( iii ) caspase-1 . , nlrp1 , nlrp3 , ipaf , and aim2 ) have been identified based on the nlr component ( or the non - nlr equivalent ) which forms the complex . of these the best characterized is the nlrp3 inflammasome which plays a predominant role in il-1 and il-18 production . , atp and uric acid ) promote assembly of the nlrp3 inflammasome complex , activation of caspase-1 from its precursor , processing of il-1 to its active form , and release of il-1 from the cells . however , current evidence would favour a proinflammatory role for il-1 since ( i ) there are increased levels of serum , balf , and bronchial epithelial il-1 in human asthmatics , compared to healthy subjects [ 1012 ] , ( ii ) increase in serum il-1 has also been reported in primates , ( iii ) il-1 levels were decreased 2-fold in the bronchial epithelium following inhalation of beclomethasone dipropionate ( as measured by an immunohistological technique ) , and ( iv ) administration of tnf- and il-1 induces airway hyper - reactivity , a feature of asthma [ 15 , 16 ] . il-18 , another potent pro - inflammatory cytokine whose maturation requires activation of caspase-1 on the inflammasome , is typically considered as a th1 cytokine due to its effects associated with ifn-. in addition to providing a physical barrier to inhaled pathogens , allergens , and other foreign agents , the airway mucosa ( epithelium and secretions ) has a number of potential mechanisms by which it can contribute to innate immune defences in the lung . loss of the integrity of the airway epithelium is widely thought to be a critical component in the pathogenesis of asthma . to our knowledge , there has been no systematic study of the inflammasome in airway epithelium . in this study , we have looked for evidence of nlrp3 inflammasome involvement in ova - induced airway inflammation in mice , as a model of human asthma , as well as in primary bronchial epithelial cultures . rabbit polyclonal antibodies ( abs ) to il-1 , nlrp3 , total caspase-1 ; goat abs to caspase-1 p10 and p20 active subunits ; and mouse monoclonal ab ( mab ) to sp - d were from santa cruz biotechnology ( santa cruz , ca , usa ) . all experiments were performed under the university of adelaide animal ethics committee approval number m-57 - 2007 , and in compliance with principles of animal care publication number 86 - 23 of the national institute of health and the australian code of practice for the care and use of animals for scientific purposes , 6th edition . female balb / c mice ( age 46 weeks ; specific pathogen free ) were purchased from the university of adelaide , adelaide , australia . ova - treated mice received 50 g of chicken ova in 1 ml of alhydrogel ( csl , parkville , australia ) in 0.9% sterile saline , i.p . each mouse sample ( n = 8 from each group ) was tested on a separate 96-well array plate containing a panel of 84 wells pertaining to different genes of the inflammasome pathway , 5 house keeping control wells , 1 genomic dna control well , 3 reverse transcription control wells , and three positive pcr control wells . tissue sections were also heated in 10 mm tris edta buffer ph 9.0 for costaining of sp - d with caspase-1 , and digested with 1 mg / ml proteinase k ( promega , fitchburg , wisconsin , usa ) for staining of p10 and p20 caspase-1 subunits . normal human bronchial epithelial ( nhbe ) cells and bronchial epithelial growth medium ( begm ) were obtained from lonza ( lonza australia pty ltd , mt waverley , vic , australia ) . in keeping with previous descriptions of the acute allergic airway inflammation model [ 2 , 23 ] , the airway inflammation in ova - challenged mice was characterized by dense peribronchial and perivascular infiltrates of leucocytes ( mostly eosinophils ) and oedema of bronchial epithelium . typically , there was a 7-fold increase in total balf cell count from 15.6 11.1 10 ( median 2.5% ci ) cells / ml in sal controls to 117.7 82.7 10 cells / ml in ova - treated mice ( p < 0.0001 ) . this was largely due to eosinophilia ( > 85% ) , although numbers of neutrophils , monocytes , and lymphocytes were also significantly increased ( p < 0.05 , data not shown ) . to test the hypothesis that the normal airway epithelium possesses the ability to assemble inflammasome complexes , we used immunofluorescence to analyse formalin - fixed lung tissue sections obtained from healthy balb / c mice for expression of inflammasome common components nlrp3 , asc and caspase-1 , and the substrate cytokines il-1 and il-18 . serial sections of control mouse lungs ( n = 8) were examined by immunohistological labelling for caspase-1 and various other inflammasome - related proteins . figures 1(a)1(c ) show colocalization of caspase-1 and nlrp3 in the epithelia of three bronchioles . colocalization of caspase-1 and nlrp3 with il-1 , il-18 , and asc in bronchiolar epithelia was shown ( figures 1(d)1(k ) ) . it should be noted that the antibodies to caspase-1 , il-1 , and il-18 did not distinguish between their precursor and mature forms . figures 2(a ) and 2(b ) show low and high magnification images of a section of the normal mouse lung immunolabelled for caspase-1 , while figures 2(c ) and 2(d ) show the corresponding h&e restaining . thus , the normal murine airway epithelium ( bronchial epithelium and atii ) expresses , at the protein level , inflammasome components nlrp3 , asc , and caspase-1 , as well as the substrate cytokines il-1 and il-18 . using antibodies specific for the cleaved ends of p10 and p20 subunits of caspase-1 , we were able to demonstrate very low levels or absence of active caspase-1 in the control mouse lung epithelium ( figure 3(a ) ) . in contrast , distinct patterns of active caspase-1 staining were detected in the epithelium of the inflamed airways of ova - treated animals ( figures 3(b)3(d ) ) . preabsorption with the relevant immunogen peptides reduced the labelling of active caspase-1 in the inflamed airway epithelium to a level comparable to that of the conjugate alone ( figures 3(e)3(g ) ) . because there are inherent problems in trying to quantify actual changes in fluorescence intensity where there is a redistribution of the label within the treatment accompanying the induction of inflammation , we have not attempted to quantify changes in intensity of immunofluorescence for the various proteins in these sections . as mentioned earlier , our antibody panel did not differentiate between the inactive precursors and the mature ( active ) forms of il-1 and il-18 cytokines . however , there were different patterns of il-1 and il-18 distribution demonstrated by immunofluorescence between the epithelia of ova - treated and control mice ( figure 4 ) . furthermore , immunofluorescence of both il-1 and il-18 could be detected in the lumen ( figures 4(c ) and 4(d ) , arrowheads ) . similarly , luminal staining near the apical surface was also detected for caspase-1 ( figures 4(e ) and 4(k ) ) , nlrp3 ( figure 4(i ) ) , and asc ( not shown ) . immunostaining of consecutive adjacent tissue sections with caspase-1 and ccr3 , a marker of eosinophils , showed colocalization of ccr3 with caspase-1 , detected by antibodies to either the total or the active forms of the caspase ( not shown ) . another cell type which might contribute to inflammasome activation in the ova - induced airway inflammation model is the vascular endothelium . whereas in saline - treated mice the vascular endothelium stained weakly for total caspase-1 , it often stained strongly in the lungs of ova - treated mice ( data not shown ) . unfortunately , we could not apply the antibodies to active subunits of caspase-1 to study activation of endothelial caspase-1 , due to the nonspecific binding of these antibodies to red blood cells , which was not blocked by the specific peptides ( data not shown ) . to look at the effect of airway inflammation on expression of gene products with relevance to the inflammasome , we analysed mrna from lungs of sal and ova - treated mice by superarray ( table 1 ) . there were also significant increases with ova - treatment in expression of a th2 cytokine il-6 . for the il-1-like family members , there was a 2.5-fold increase in gene expression for il-33 and 2- and 11-fold decreases in those for il-1 and il-18 , respectively . of the gene products involved in inflammasome regulation , the largest change was an 8-fold increase in the neuronal inhibitor of apoptosis protein naip , followed by an almost 4-fold decrease in nlrp3 expression . an analysis of balf in the asthmatic mice demonstrated a significant increase in uric acid ( figure 5(a ) ) , which is known as both a product of nucleic acid catabolism in dead cells and a stimulus for nlrp3 inflammasome activation . we were unable to detect il-1 and il-18 in the balf of either saline- or ova - treated mice ( data not shown ) . analysis of the sera showed high levels ( > 200 pg / ml ) of il-1 in 4 of the 8 ova - treated mice but in none of the 8 saline - treated control mice . the serum levels of il-1 were tightly correlated with those of tnf- ( r = 0.95 ) , suggesting systemic effects in this model of allergic airway inflammation ( figure 5(b ) ) . commercially available passage 4 primary cultured normal human bronchial epithelial ( nhbe ) cells were studied for expression of the nlrp3 and il-1 proteins , before and after priming with lps ( 5 ug / ml ) or ifn- ( 50 ng / ml ) . overnight stimulation with lps or lps plus ifn- resulted in significant increase ( p < 0.05 ) of both nlrp3 ( ~2 fold ) and il-1 ( 4 - 5 fold , figure 6 ) . this study provides the first evidence for presence of precursor components of the nrlp3 inflammasome in normal murine airway epithelium and some other resident or inflammatory airway cells . it also shows qualitative changes in the distribution of these markers , including appearance of active caspase-1 , during airway inflammation in the acute mouse ova model . our findings suggest a role for inflammasomes in innate immune responses of the airway epithelium and reinforce the hypothesis that airway epithelium plays a sentinel role in the innate immune response to inhaled microbes , allergens , and other pathogens . of interest , il-1 and il-18 a similar expression of il-1 was detected in rat lung epithelium tissue ( data not shown ) . the failure to detect il-1 in the circulation of the control mice suggests either that airway epithelium contributes very little to blood il-1 levels or that signals leading to release of il-1 ( and probably also il-18 ) are absent from normal airway epithelium . our finding of intracellular il-1 and il-18 is therefore in keeping with the hypothesis that the airway epithelium is an important sentinel in the innate immune response and is primed for a rapid response when exposed to danger signals but has yet to undergo inflammasome activation . thus , cell lines in their normal state usually do not express precursors of il-1 and il-18 ; production , maturation , and extracellular release of these potent proinflammatory cytokines require at least two signals . ligation of tlrs leads to enhanced transcription of the il-1 gene and accumulation of an immature form of il-1 in the cytoplasm . stimulation with substances such as extracellular atp or uric acid leads to a second signal that results in caspase-1 activation , processing of pro - il-1 , and release of the mature cytokine . using antibodies that detect the presence of neoepitopes in the caspase-1 subunits which become accessible only upon cleavage / activation of the proenzyme and a blocking peptide , we were able to provide qualitative evidence for presence of active caspase-1 in the airway epithelium of the ova - treated mice . we also found , in these ova - treated mice , a potentially interesting and novel phenomenon of subcellular translocation for epithelial il-1 , il-18 , and caspase-1 , whereby these molecules were seen to be concentrated at the epithelial apical surface and apparently shed into the airway lumen in the form of cell bodies . various mechanisms for release of the active il-1 cytokines ( and caspase-1 , itself ) have been postulated , including preexport into secretory lysosomes or encapsulation in microvesicles or exosomes . evidence for release as cell bodies or smaller vesicles has been provided by studies in monocytes [ 24 , 26 ] , microglia , and dendritic cells . our findings support the notion that the airway epithelium can itself mount a proinflammatory response via activation of its inflammasome complexes and consequent release of il-1 and il-18 . we were able to detect high levels of il-1 in the circulation but not in the balf . relevant to this , 4 of the 8 ova - treated mice had high levels of il-1 and tnf- in their serum while the other 4 mice ( like all of the control mice ) had very low levels of both cytokines , despite all 8 ova - treated mice having high levels of airway inflammation as evidenced by eosinophilia in balf and tissues as well as epithelial swelling and cell death . it is likely therefore that inflammasome components are present along the entire length of the airways and constitute a front - line defence . their involvement in allergic and inflammatory diseases of the airways such as chronic rhinosinusitis , rhinitis , and asthma warrants further study . a major limitation with studies of the whole lung is that the lung is a complex organ comprising a number of tissues . because of this we were driven from the start at establishing the techniques to show the presence of inflammasome components in specific cell types of the lung at a histochemical level . our initial attempt to look at changes in expression of inflammasome - related genes in the context of murine airway inflammation showed a large increase in expression of the caspase inhibitor and inflammasome - binding protein naip as well as decreases in expression of il-18 and nrlp3 . since naip is also able to substitute for nrlp3 and other nrls in formation of the inflammasome complex , there may be a switch from nrlp3 to naip as a result of the inflammation . discordance between mrna and protein expression of p2x7 receptor , an important up - stream regulator of the nrlp3 inflammasome , has also been reported in inflamed intestinal epithelium from patients with inflammatory bowel disease . in that study , in which p2x7 receptor engagement was required for production and release of il-1 , mrna levels for p2x7 receptor were increased while protein levels were strongly decreased . the authors speculated that the decrease in protein expression may help to protect the intestinal epithelial cells from excessive activation of p2x7 receptor and subsequent cell death during the neutrophil transmigration . dissection of the mechanisms involved in airway epithelial inflammasome regulation and activation will require the use of in vitro airway epithelial cultures . we have shown that the presence of nrlp3 and il-1 proteins in the commercially available normal human bronchial epithelial primary cells and the 25-fold upregulation of these proteins following priming with lps indicates involvement of tlr4 in the priming . in conclusion , we have demonstrated the presence of precursor components of the nrlp3 inflammasome in healthy murine airway epithelium as well as changes in the subcellular distribution of these components and appearance of active caspase-1 in the epithelium of inflamed airways . further studies using both lung biopsies from patients and mechanistic studies in polarized cultures of human airway epithelium are warranted and may reveal new insight into the disease mechanism and additional targets for therapeutic and/or diagnostic applications in asthma .

acute exacerbations of chronic obstructive pulmonary disease ( ae - copd ) account for a significant number of hospitalizations and therefore present an important economic burden in health care.1 ae - copd substantially contribute to loss in lung function , disease progression , and mortality.2 most patients presenting with ae - copd describe symptoms of respiratory infection.3 historically , bacteria have been considered to be the major cause of infectious exacerbations , but as bacteria are also found in clinically stable patients , the role of bacterial infection remains controversial.4 with the broad availability of molecular diagnostics like polymerase chain reaction ( pcr ) the prominent contribution of common viral agents of upper respiratory tract infection like human rhinovirus ( hrv ) and respiratory syncytial virus ( rsv ) to the etiology of ae - copd has been increasingly recognized.46 nevertheless , in about one third of severe exacerbations the cause remains unknown.1 in recent years novel respiratory viruses including the human metapneumovirus ( hmpv ) and several coronaviruses ( sars , nl63 , and hku1 ) have been discovered . their role in ae - copd has already been defined to some extent.7,8 human bocavirus ( hbov ) is a novel parvovirus initially discovered in 2005 in respiratory secretions of swedish children with symptoms of upper and lower respiratory tract infection with a frequency of 3.1%.9 proving its role as a causative agent of acute respiratory tract infections is challenging as hbov has not been propagated in cell culture and as animal models have not yet been established . hence , some of the modified postulates of koch are not applicable.10 numerous retrospective and prospective studies suggested an association between hbov and acute respiratory tract infections in children . clinical findings consisted of wheezing episodes , asthma exacerbations , acute obstructive bronchitis , bronchiolitis , croup , and pneumonia.1115 infants below the age of two years and patients with structural pulmonary diseases or immune disorders seem to be predominantly affected . preliminary evidence is provided that acute hbov infection may take a viremic course and may also be capable of persistence in the respiratory tract.16 hbov infections appear endemic worldwide.16 first studies on the seroepidemiology give evidence for protection by maternal antibodies within the first months of life.17 in a japanese study all of the 204 children examined had immunoglobulin g antibodies to the viral protein 1 ( vp1 ) by the age of 6 years , emphasizing its ubiquitous nature.18 taken together , these findings suggest that there is protective immunity after past infection which may explain why hbov is comparatively rare in adults.9,1922 frequencies of hbov detection in respiratory specimens of children described in the literature vary from 2% to 19%.11,23 recently , a hbov detection rate of 12% has been described among german children with symptoms of respiratory tract infections.24 to our knowledge the role of hbov in a well characterized population of adult patients with ae - copd has not been systematically studied yet . implications of a frequent detection in ae - copd are the introduction in clinical routine testing and a subsequent decrease of antimicrobial overuse . the aim of the present study was to determine the prevalence of hbov infection in hospitalized patients with ae - copd and to establish its potential clinical relevance by comparison to patients with stable copd hospitalized for other reasons than acute exacerbation . we retrospectively tested two groups of copd patients for the presence of hbov deoxyribonucleic acid ( dna ) . 212 patients , 141 ( 66.5% ) with ae - copd and a control group of 71 ( 33.5% ) copd patients with stable disease were selected for evaluation . these subjects had been enrolled between october 1999 and april 2003 in a prospective case - control manner in a 2:1 ratio . in order to prevent inclusion criteria for both groups were age of 18 to 85 years , exacerbated or stable copd , chronic airflow limitation ( forced expiratory volume in one second [ fev1 ] < 80% predicted ) , admission to hospital and written informed consent . exclusion criteria for both groups were bronchial asthma and dyspnea of other origin ( pneumonia , tuberculosis , pulmonary fibrosis , bronchiectasis , lung cancer , or congestive heart failure ) . the collection of demographic and clinical data , the standardized collection and processing of nasal lavage and induced sputum samples , the assessment of lung function , the evaluation of chest radiographs and underlying common definitions have been described in detail.25 briefly , copd and ae - copd were defined as by the global initiative for chronic obstructive lung disease ( gold).1 ae - copd was characterized by worsening of dyspnea , cough and/or sputum expectoration beyond normal day - to - day variations and was acute in onset . patients with stable copd did not experience an exacerbation within the last 30 days prior to hospital admission , had no changes in inhaled or oral medication within the last 14 days and had been admitted for other conditions into medical departments other than pulmonary care . the following variables were selected for data analysis : age , gender , body mass index , smoking behavior , smoking history in pack years , oral and inhaled steroid medication before admission , dose of oral steroid medication , where applicable , c - reactive protein and white blood count values , clinical symptoms on admission , comorbidity , and copd status . spirometric data were assessed on admission for both groups and before discharge in patients with ae - copd : fev1 in liters and % predicted , forced vital capacity ( fvc in liters and % predicted ) and fev1/fvc . both nasal lavage and induced sputum samples had been obtained from each patient between 1999 and 2003 and stored at 70 c until dna extraction was performed . viral coinfections with other common respiratory viruses ( hmpv , hrv , influenza viruses a and b , parainfluenza virus 3 , and rsv ) were determined by sensitive real - time pcr assays for all patients in nasal lavage and induced sputum samples as previously published.7,2528 dna was extracted from 250 l of induced sputum and nasal lavage samples using qiaamp dna blood mini kit ( qiagen , hilden , germany ) according to the instructions of the manufacturer . hbov dna amplification was performed with the nonstructural protein 1 ( np-1 ) primers bov188f ( 5'-gagctctgtaagtactattac-3 ' ) and bov542r ( 5'-ctctgtgttgactgaatacag-3 ' ) described by allander and colleagues9 using the hotstartaq dna polymerase ( qiagen , hilden , germany ) . pcr reactions were carried out in a 50 l volume consisting of 5 l extracted dna , 1 qiagen hotstar buffer , dntps at a final concentration of 200 m each , 200 pmol of each primer , and 1.5 u of taq polymerase as previously described by weissbrich and colleagues.29 cycling conditions were 50 cycles ( 94 c 30 s , 53 c 40 s and 1 min at 72 c ) after a preheating step of 10 min at 95 c pcr products were visualized by staining with ethidium bromide by agarose gel electrophoresis considering a positive pcr reaction when a band of the expected size of 354 base pairs ( bp ) was detected . sequence specificity was confirmed by sequencing all positive pcr products in both directions using big dye terminator chemistry and the abi prism 3100 ( applied biosystems , darmstadt , germany ) . a plasmid containing 5182 bp of hbov dna , isolate stockholm 2 ( st2 ) [ genbank : dq000496 ] cloned in the vector pcr4-topo ( invitrogen , paisley , uk ) was used as positive control . sensitivity of the hbov pcr assay was assessed by dilution series and was determined to a single copy of viral genome per reaction . categorical data were compared by pearson s chi - squared or fisher s exact test , where appropriate . smirnov test and differences were subsequently determined either with student s t - test or mann whitney u test . all p values reported were calculated two - sided with statistical significance set to p 0.05 . the study protocol was approved by the ethics committee of the ruhr university , bochum , germany . we retrospectively tested two groups of copd patients for the presence of hbov deoxyribonucleic acid ( dna ) . 212 patients , 141 ( 66.5% ) with ae - copd and a control group of 71 ( 33.5% ) copd patients with stable disease were selected for evaluation . these subjects had been enrolled between october 1999 and april 2003 in a prospective case - control manner in a 2:1 ratio . in order to prevent inclusion criteria for both groups were age of 18 to 85 years , exacerbated or stable copd , chronic airflow limitation ( forced expiratory volume in one second [ fev1 ] < 80% predicted ) , admission to hospital and written informed consent . exclusion criteria for both groups were bronchial asthma and dyspnea of other origin ( pneumonia , tuberculosis , pulmonary fibrosis , bronchiectasis , lung cancer , or congestive heart failure ) . the collection of demographic and clinical data , the standardized collection and processing of nasal lavage and induced sputum samples , the assessment of lung function , the evaluation of chest radiographs and underlying common definitions have been described in detail.25 briefly , copd and ae - copd were defined as by the global initiative for chronic obstructive lung disease ( gold).1 ae - copd was characterized by worsening of dyspnea , cough and/or sputum expectoration beyond normal day - to - day variations and was acute in onset . patients with stable copd did not experience an exacerbation within the last 30 days prior to hospital admission , had no changes in inhaled or oral medication within the last 14 days and had been admitted for other conditions into medical departments other than pulmonary care . the following variables were selected for data analysis : age , gender , body mass index , smoking behavior , smoking history in pack years , oral and inhaled steroid medication before admission , dose of oral steroid medication , where applicable , c - reactive protein and white blood count values , clinical symptoms on admission , comorbidity , and copd status . spirometric data were assessed on admission for both groups and before discharge in patients with ae - copd : fev1 in liters and % predicted , forced vital capacity ( fvc in liters and % predicted ) and fev1/fvc . both nasal lavage and induced sputum samples had been obtained from each patient between 1999 and 2003 and stored at 70 c until dna extraction was performed . viral coinfections with other common respiratory viruses ( hmpv , hrv , influenza viruses a and b , parainfluenza virus 3 , and rsv ) were determined by sensitive real - time pcr assays for all patients in nasal lavage and induced sputum samples as previously published.7,2528 dna was extracted from 250 l of induced sputum and nasal lavage samples using qiaamp dna blood mini kit ( qiagen , hilden , germany ) according to the instructions of the manufacturer . hbov dna amplification was performed with the nonstructural protein 1 ( np-1 ) primers bov188f ( 5'-gagctctgtaagtactattac-3 ' ) and bov542r ( 5'-ctctgtgttgactgaatacag-3 ' ) described by allander and colleagues9 using the hotstartaq dna polymerase ( qiagen , hilden , germany ) . pcr reactions were carried out in a 50 l volume consisting of 5 l extracted dna , 1 qiagen hotstar buffer , dntps at a final concentration of 200 m each , 200 pmol of each primer , and 1.5 u of taq polymerase as previously described by weissbrich and colleagues.29 cycling conditions were 50 cycles ( 94 c 30 s , 53 c 40 s and 1 min at 72 c ) after a preheating step of 10 min at 95 c pcr products were visualized by staining with ethidium bromide by agarose gel electrophoresis considering a positive pcr reaction when a band of the expected size of 354 base pairs ( bp ) was detected . sequence specificity was confirmed by sequencing all positive pcr products in both directions using big dye terminator chemistry and the abi prism 3100 ( applied biosystems , darmstadt , germany ) . a plasmid containing 5182 bp of hbov dna , isolate stockholm 2 ( st2 ) [ genbank : dq000496 ] cloned in the vector pcr4-topo ( invitrogen , paisley , uk ) was used as positive control . sensitivity of the hbov pcr assay was assessed by dilution series and was determined to a single copy of viral genome per reaction . categorical data were compared by pearson s chi - squared or fisher s exact test , where appropriate . smirnov test and differences were subsequently determined either with student s t - test or mann whitney u test . all p values reported were calculated two - sided with statistical significance set to p 0.05 . the study protocol was approved by the ethics committee of the ruhr university , bochum , germany . from october 1999 to april 2003 induced sputum and nasal lavage samples from 212 hospitalized copd patients , 141 with acute exacerbation and 71 with stable disease , had been obtained in a case - control setup in a 2:1 ratio . of those , pcr results for the presence of hbov dna were available both for induced sputum and nasal lavage specimens in 202 patients , 134 with acute exacerbation and 68 with stable disease , who constituted the actual study population ( figure 1 ) . ten subjects were excluded from data analysis as two induced sputum and eight nasal lavage samples were not sufficient for pcr experiments . the two groups were comparable in terms of age , sex , body mass index , smoking behavior , pack years , and frequency of inhaled and oral steroid medication . the age distribution in the ae - copd group ranged from 41 to 83 and from 43 to 81 years in the control group . significant differences were apparent for increased airflow limitation on admission ( fev1 in liters and % predicted ) , higher inflammatory laboratory parameters indicating infection ( crp and leukocytes ) and higher doses of administered oral steroids before admission in the ae - copd group . spirometric data before discharge were available for 84 of 134 patients ( 62.7% ) with ae - copd , had significantly improved after treatment for acute exacerbation and were comparable to the baseline airflow in the control group ( table 1 ) . three of 202 patients ( 1.5% ) tested for hbov dna were found to be positive by pcr and subsequent sequencing . of these subjects only one had an acute exacerbation , the other two had stable , but ( very ) severe copd ( table 2 ) . thus , the frequency of hbov detection demonstrated to be low in both ae - copd ( 1/134 , 0.8% ) and stable copd ( 2/68 , 2.9% ) . in two out of 202 patients ( 1.0% ) hbov was detected in induced sputum samples , whereas only one nasal lavage sample ( 1/202 , 0.5% ) tested positive for hbov . the clinical features of the three patients positive for hbov are shown in table 2 . hbov was found in two sputum samples and one nasal lavage sample , while corresponding nasal lavage and sputum samples were negative , respectively . all patients were male and presented in the winter / spring season ( march and november 2000 , january 2002 ) . they showed no infiltrations on chest x - ray , had moderately to very severely impaired lung function according to gold stages ii iv and reported various comorbidities ( arterial hypertension , cor pulmonale , obstructive sleep apnea , cardiomyopathy and ischemic stroke ) . two subjects had stable copd and reported cough , whitish expectoration and dyspnea on exertion without any recent changes in severity . the individual with ae - copd complained of progressive cough , purulent expectoration as well as dyspnea at rest and symptoms of acute gastroenteritis . the two patients with gold stages ii and iii had not experienced ae - copd within the last 12 months preceding the study inclusion . sputum microbiology was sterile in two subjects , while candida albicans was detected in the other subject . viral coinfections with rsv ( 6.5 10 viral copies , detected in the induced sputum of the ae - copd patient ) and rsv and influenza virus a ( 3.2 10 and 3.8 10 viral copies , respectively , both detected in nasal lavage samples ) were existent in two of the three patients . sequence determination of the three positive hbov pcr products revealed a > 99% sequence identity with the swedish strains st1 and st2 and between the nucleotide sequences obtained from the years 2000 and 2002 . nucleotide sequences of amplified pcr products were deposited into genbank as a result of this study [ genbank : fj156726-fj156728 ] . from october 1999 to april 2003 induced sputum and nasal lavage samples from 212 hospitalized copd patients , 141 with acute exacerbation and 71 with stable disease , had been obtained in a case - control setup in a 2:1 ratio . of those , pcr results for the presence of hbov dna were available both for induced sputum and nasal lavage specimens in 202 patients , 134 with acute exacerbation and 68 with stable disease , who constituted the actual study population ( figure 1 ) . ten subjects were excluded from data analysis as two induced sputum and eight nasal lavage samples were not sufficient for pcr experiments . the two groups were comparable in terms of age , sex , body mass index , smoking behavior , pack years , and frequency of inhaled and oral steroid medication . the age distribution in the ae - copd group ranged from 41 to 83 and from 43 to 81 years in the control group . significant differences were apparent for increased airflow limitation on admission ( fev1 in liters and % predicted ) , higher inflammatory laboratory parameters indicating infection ( crp and leukocytes ) and higher doses of administered oral steroids before admission in the ae - copd group . spirometric data before discharge were available for 84 of 134 patients ( 62.7% ) with ae - copd , had significantly improved after treatment for acute exacerbation and were comparable to the baseline airflow in the control group ( table 1 ) . three of 202 patients ( 1.5% ) tested for hbov dna were found to be positive by pcr and subsequent sequencing . of these subjects only one had an acute exacerbation , the other two had stable , but ( very ) severe copd ( table 2 ) . thus , the frequency of hbov detection demonstrated to be low in both ae - copd ( 1/134 , 0.8% ) and stable copd ( 2/68 , 2.9% ) . in two out of 202 patients ( 1.0% ) hbov was detected in induced sputum samples , whereas only one nasal lavage sample ( 1/202 , 0.5% ) tested positive for hbov . the clinical features of the three patients positive for hbov are shown in table 2 . hbov was found in two sputum samples and one nasal lavage sample , while corresponding nasal lavage and sputum samples were negative , respectively . all patients were male and presented in the winter / spring season ( march and november 2000 , january 2002 ) . they showed no infiltrations on chest x - ray , had moderately to very severely impaired lung function according to gold stages ii iv and reported various comorbidities ( arterial hypertension , cor pulmonale , obstructive sleep apnea , cardiomyopathy and ischemic stroke ) . two subjects had stable copd and reported cough , whitish expectoration and dyspnea on exertion without any recent changes in severity . the individual with ae - copd complained of progressive cough , purulent expectoration as well as dyspnea at rest and symptoms of acute gastroenteritis . the two patients with gold stages ii and iii had not experienced ae - copd within the last 12 months preceding the study inclusion . sputum microbiology was sterile in two subjects , while candida albicans was detected in the other subject . viral coinfections with rsv ( 6.5 10 viral copies , detected in the induced sputum of the ae - copd patient ) and rsv and influenza virus a ( 3.2 10 and 3.8 10 viral copies , respectively , both detected in nasal lavage samples ) were existent in two of the three patients . sequence determination of the three positive hbov pcr products revealed a > 99% sequence identity with the swedish strains st1 and st2 and between the nucleotide sequences obtained from the years 2000 and 2002 . nucleotide sequences of amplified pcr products were deposited into genbank as a result of this study [ genbank : fj156726-fj156728 ] . in this retrospective study of a prospectively recruited , case - controlled cohort we found hbov detection to be infrequent in adult hospitalized patients with acute exacerbation and stable copd during the years 1999 to 2003 in the urban region of central ruhr area , germany . in total , it was found both in acute exacerbation ( 0.8% , 1/134 ) and stable disease ( 2.9% , 2/68 ) as well as induced sputum and nasal lavage samples . these findings suggest that hbov may not be of clinical relevance in adult patients with ae - copd and routine testing for hbov in this population may not be warranted . data on hbov infections in adults is limited.9,13,1923,30 screening for respiratory viruses is not generally recommended for adults with symptoms of lower respiratory tract infection,1,31 thus material for large - scale retrospective analysis is hardly available . the very low frequency of overall hbov detection found in our study is in agreement with other studies in adults . the initial investigation by allander and colleagues did not detect hbov in 112 adults with respiratory symptoms at all.9 a large thai study detected hbov in 0.7% of subjects ( 6/834 ) aged 20 years or older ( including 126 controls ) only.13 two italian studies found frequencies of 1.2% ( 1/84 between the years 2000 and 2006)21 and 1.4% ( 2/143)19 in adult patients despite the use of different pcr assays ( real - time and single - round pcr , respectively ) . notably , a very recent us - american study found the identical frequency of 1.5% in 273 adults.22 the only previous study explicitly including patients with ae - copd found hbov in one of 126 ( 0.8% ) adult patients with ae - copd or pneumonia , a 71-year - old canadian male who had received oral steroids for the treatment of his exacerbation.20 unfortunately this report covered one winter season ( 2002/2003 ) only and neither specified the proportion of ae - copd patients included nor provided any clinical details on the population . in contrast to the reports mentioned above , which predominantly studied specimens representing the upper respiratory tract ( nasal swabs or nasopharyngeal aspirates ) , our study investigated both induced sputum and nasal lavage samples . in a previous pcr - based study we demonstrated that ae - copd requiring hospital admission was significantly associated with the presence of common respiratory viruses . induced sputum , which may more reliably reflect the lower respiratory tract , had a higher viral yield than upper respiratory tract specimens in patients with ae - copd.25 hrv ( 36% ) , influenza virus a ( 25% ) , and rsv ( 22% ) were most frequently detected , while in a similar study we recently found another emerging respiratory virus , hmpv , to be infrequent in ae - copd as well ( 2.3% , 3/130 , overall prevalence 1.3% , 3/229 ) . however all of the three hmpv detections occurred in the ae - copd group , making asymptomatic carriage unlikely.7 the present study investigated the frequency of hbov infection in four consecutive years with special care on avoidance of a seasonal selection bias by monthly recruitment of both groups in a 2:1 case - control setup . we used an optimized hbov pcr assay performing 50 cycles of single - round , hot - start pcr,29 which was sensitive enough to detect a single hbov dna copy per reaction . sequencing of pcr products revealed a nucleotide identity of > 99% between different samples and years . this finding of a limited genetic variation is in agreement with previous reports.9,23 in the present study viral coinfections , which were assessed by sensitive pcr assays , were existent in two of three hbov - positive subjects ( although specimens were not evaluated for the presence of some less common respiratory viruses like adenovirus , coronaviruses nl63 and hku1 , and parainfluenza viruses 1 and 2 ) . this finding is consistent with the hypothesis that hbov is carried at low copy numbers in some individuals and that its detection is enhanced , or the virus reactivated , by another respiratory infection.16 due to the very low detection rate in our study , an analysis of a potential association between hbov infection and distinct clinical features was not possible . remarkably , the patient with ae - copd who tested positive for hbov suffered from concomitant acute gastroenteritis . hbov has also been proposed to be a causative pathogen in acute gastroenteritis , but so far its etiologic role remains controversial.32,33 this is the first controlled study on the prevalence of hbov in adult hospitalized patients with ae - copd . we found hbov at a very low frequency in patients with acute exacerbation as well as stable copd , comparable to what has been reported in other adult study populations . thus , a major role of hbov infection as a common trigger of ae - copd is unlikely .

objectivehuman bocavirus ( hbov ) is a recently discovered parvovirus associated with acute respiratory tract infections in children . the objective of the present study was to determine the frequency and clinical relevance of hbov infection in adult patients with acute exacerbation of chronic obstructive pulmonary disease ( ae - copd).methodswe retrospectively tested 212 copd patients , 141 ( 66.5% ) with ae - copd and 71 ( 33.5% ) with stable disease , of whom nasal lavage and induced sputum had been obtained for the presence of hbov deoxyribonucleic acid ( dna ) . the specificity of positive polymerase chain reaction results was confirmed by sequencing.resultstwo hundred two of 212 patients for whom pcr results were available both for nasal lavage and induced sputum samples were eligible for data analysis . hbov dna was detected in three patients ( 1.5% ) . of those , only one patient had ae - copd . thus , the frequency of hbov infection demonstrated to be low in both ae - copd ( 0.8% ) and stable copd ( 2.9% ) . hbov was found in two sputum and one nasal lavage sample in different patients , respectively . sequencing revealed > 99% sequence identity with the reference strain.conclusionhbov detection was infrequent . since we detected hbov in both upper and lower respiratory tract specimens and in ae - copd as well as stable disease , a major role of hbov infection in adults with ae - copd is unlikely .

Introduction Material and methods Study design, subjects, and samples HBoV PCR and sequencing Statistical analysis Results Study population Prevalence of HBoV infection Clinical features of HBoV-positive subjects Sequencing of positive PCR products Discussion Conclusion

acute exacerbations of chronic obstructive pulmonary disease ( ae - copd ) account for a significant number of hospitalizations and therefore present an important economic burden in health care.1 ae - copd substantially contribute to loss in lung function , disease progression , and mortality.2 most patients presenting with ae - copd describe symptoms of respiratory infection.3 historically , bacteria have been considered to be the major cause of infectious exacerbations , but as bacteria are also found in clinically stable patients , the role of bacterial infection remains controversial.4 with the broad availability of molecular diagnostics like polymerase chain reaction ( pcr ) the prominent contribution of common viral agents of upper respiratory tract infection like human rhinovirus ( hrv ) and respiratory syncytial virus ( rsv ) to the etiology of ae - copd has been increasingly recognized.46 nevertheless , in about one third of severe exacerbations the cause remains unknown.1 in recent years novel respiratory viruses including the human metapneumovirus ( hmpv ) and several coronaviruses ( sars , nl63 , and hku1 ) have been discovered . their role in ae - copd has already been defined to some extent.7,8 human bocavirus ( hbov ) is a novel parvovirus initially discovered in 2005 in respiratory secretions of swedish children with symptoms of upper and lower respiratory tract infection with a frequency of 3.1%.9 proving its role as a causative agent of acute respiratory tract infections is challenging as hbov has not been propagated in cell culture and as animal models have not yet been established . hence , some of the modified postulates of koch are not applicable.10 numerous retrospective and prospective studies suggested an association between hbov and acute respiratory tract infections in children . clinical findings consisted of wheezing episodes , asthma exacerbations , acute obstructive bronchitis , bronchiolitis , croup , and pneumonia.1115 infants below the age of two years and patients with structural pulmonary diseases or immune disorders seem to be predominantly affected . preliminary evidence is provided that acute hbov infection may take a viremic course and may also be capable of persistence in the respiratory tract.16 hbov infections appear endemic worldwide.16 first studies on the seroepidemiology give evidence for protection by maternal antibodies within the first months of life.17 in a japanese study all of the 204 children examined had immunoglobulin g antibodies to the viral protein 1 ( vp1 ) by the age of 6 years , emphasizing its ubiquitous nature.18 taken together , these findings suggest that there is protective immunity after past infection which may explain why hbov is comparatively rare in adults.9,1922 frequencies of hbov detection in respiratory specimens of children described in the literature vary from 2% to 19%.11,23 recently , a hbov detection rate of 12% has been described among german children with symptoms of respiratory tract infections.24 to our knowledge the role of hbov in a well characterized population of adult patients with ae - copd has not been systematically studied yet . implications of a frequent detection in ae - copd are the introduction in clinical routine testing and a subsequent decrease of antimicrobial overuse . the aim of the present study was to determine the prevalence of hbov infection in hospitalized patients with ae - copd and to establish its potential clinical relevance by comparison to patients with stable copd hospitalized for other reasons than acute exacerbation . we retrospectively tested two groups of copd patients for the presence of hbov deoxyribonucleic acid ( dna ) . 212 patients , 141 ( 66.5% ) with ae - copd and a control group of 71 ( 33.5% ) copd patients with stable disease were selected for evaluation . the collection of demographic and clinical data , the standardized collection and processing of nasal lavage and induced sputum samples , the assessment of lung function , the evaluation of chest radiographs and underlying common definitions have been described in detail.25 briefly , copd and ae - copd were defined as by the global initiative for chronic obstructive lung disease ( gold).1 ae - copd was characterized by worsening of dyspnea , cough and/or sputum expectoration beyond normal day - to - day variations and was acute in onset . patients with stable copd did not experience an exacerbation within the last 30 days prior to hospital admission , had no changes in inhaled or oral medication within the last 14 days and had been admitted for other conditions into medical departments other than pulmonary care . spirometric data were assessed on admission for both groups and before discharge in patients with ae - copd : fev1 in liters and % predicted , forced vital capacity ( fvc in liters and % predicted ) and fev1/fvc . both nasal lavage and induced sputum samples had been obtained from each patient between 1999 and 2003 and stored at 70 c until dna extraction was performed . viral coinfections with other common respiratory viruses ( hmpv , hrv , influenza viruses a and b , parainfluenza virus 3 , and rsv ) were determined by sensitive real - time pcr assays for all patients in nasal lavage and induced sputum samples as previously published.7,2528 dna was extracted from 250 l of induced sputum and nasal lavage samples using qiaamp dna blood mini kit ( qiagen , hilden , germany ) according to the instructions of the manufacturer . hbov dna amplification was performed with the nonstructural protein 1 ( np-1 ) primers bov188f ( 5'-gagctctgtaagtactattac-3 ' ) and bov542r ( 5'-ctctgtgttgactgaatacag-3 ' ) described by allander and colleagues9 using the hotstartaq dna polymerase ( qiagen , hilden , germany ) . sequence specificity was confirmed by sequencing all positive pcr products in both directions using big dye terminator chemistry and the abi prism 3100 ( applied biosystems , darmstadt , germany ) . we retrospectively tested two groups of copd patients for the presence of hbov deoxyribonucleic acid ( dna ) . 212 patients , 141 ( 66.5% ) with ae - copd and a control group of 71 ( 33.5% ) copd patients with stable disease were selected for evaluation . the collection of demographic and clinical data , the standardized collection and processing of nasal lavage and induced sputum samples , the assessment of lung function , the evaluation of chest radiographs and underlying common definitions have been described in detail.25 briefly , copd and ae - copd were defined as by the global initiative for chronic obstructive lung disease ( gold).1 ae - copd was characterized by worsening of dyspnea , cough and/or sputum expectoration beyond normal day - to - day variations and was acute in onset . patients with stable copd did not experience an exacerbation within the last 30 days prior to hospital admission , had no changes in inhaled or oral medication within the last 14 days and had been admitted for other conditions into medical departments other than pulmonary care . spirometric data were assessed on admission for both groups and before discharge in patients with ae - copd : fev1 in liters and % predicted , forced vital capacity ( fvc in liters and % predicted ) and fev1/fvc . both nasal lavage and induced sputum samples had been obtained from each patient between 1999 and 2003 and stored at 70 c until dna extraction was performed . viral coinfections with other common respiratory viruses ( hmpv , hrv , influenza viruses a and b , parainfluenza virus 3 , and rsv ) were determined by sensitive real - time pcr assays for all patients in nasal lavage and induced sputum samples as previously published.7,2528 dna was extracted from 250 l of induced sputum and nasal lavage samples using qiaamp dna blood mini kit ( qiagen , hilden , germany ) according to the instructions of the manufacturer . hbov dna amplification was performed with the nonstructural protein 1 ( np-1 ) primers bov188f ( 5'-gagctctgtaagtactattac-3 ' ) and bov542r ( 5'-ctctgtgttgactgaatacag-3 ' ) described by allander and colleagues9 using the hotstartaq dna polymerase ( qiagen , hilden , germany ) . sequence specificity was confirmed by sequencing all positive pcr products in both directions using big dye terminator chemistry and the abi prism 3100 ( applied biosystems , darmstadt , germany ) . from october 1999 to april 2003 induced sputum and nasal lavage samples from 212 hospitalized copd patients , 141 with acute exacerbation and 71 with stable disease , had been obtained in a case - control setup in a 2:1 ratio . of those , pcr results for the presence of hbov dna were available both for induced sputum and nasal lavage specimens in 202 patients , 134 with acute exacerbation and 68 with stable disease , who constituted the actual study population ( figure 1 ) . ten subjects were excluded from data analysis as two induced sputum and eight nasal lavage samples were not sufficient for pcr experiments . significant differences were apparent for increased airflow limitation on admission ( fev1 in liters and % predicted ) , higher inflammatory laboratory parameters indicating infection ( crp and leukocytes ) and higher doses of administered oral steroids before admission in the ae - copd group . spirometric data before discharge were available for 84 of 134 patients ( 62.7% ) with ae - copd , had significantly improved after treatment for acute exacerbation and were comparable to the baseline airflow in the control group ( table 1 ) . three of 202 patients ( 1.5% ) tested for hbov dna were found to be positive by pcr and subsequent sequencing . of these subjects only one had an acute exacerbation , the other two had stable , but ( very ) severe copd ( table 2 ) . thus , the frequency of hbov detection demonstrated to be low in both ae - copd ( 1/134 , 0.8% ) and stable copd ( 2/68 , 2.9% ) . in two out of 202 patients ( 1.0% ) hbov was detected in induced sputum samples , whereas only one nasal lavage sample ( 1/202 , 0.5% ) tested positive for hbov . hbov was found in two sputum samples and one nasal lavage sample , while corresponding nasal lavage and sputum samples were negative , respectively . two subjects had stable copd and reported cough , whitish expectoration and dyspnea on exertion without any recent changes in severity . the individual with ae - copd complained of progressive cough , purulent expectoration as well as dyspnea at rest and symptoms of acute gastroenteritis . the two patients with gold stages ii and iii had not experienced ae - copd within the last 12 months preceding the study inclusion . sputum microbiology was sterile in two subjects , while candida albicans was detected in the other subject . viral coinfections with rsv ( 6.5 10 viral copies , detected in the induced sputum of the ae - copd patient ) and rsv and influenza virus a ( 3.2 10 and 3.8 10 viral copies , respectively , both detected in nasal lavage samples ) were existent in two of the three patients . sequence determination of the three positive hbov pcr products revealed a > 99% sequence identity with the swedish strains st1 and st2 and between the nucleotide sequences obtained from the years 2000 and 2002 . from october 1999 to april 2003 induced sputum and nasal lavage samples from 212 hospitalized copd patients , 141 with acute exacerbation and 71 with stable disease , had been obtained in a case - control setup in a 2:1 ratio . of those , pcr results for the presence of hbov dna were available both for induced sputum and nasal lavage specimens in 202 patients , 134 with acute exacerbation and 68 with stable disease , who constituted the actual study population ( figure 1 ) . ten subjects were excluded from data analysis as two induced sputum and eight nasal lavage samples were not sufficient for pcr experiments . significant differences were apparent for increased airflow limitation on admission ( fev1 in liters and % predicted ) , higher inflammatory laboratory parameters indicating infection ( crp and leukocytes ) and higher doses of administered oral steroids before admission in the ae - copd group . spirometric data before discharge were available for 84 of 134 patients ( 62.7% ) with ae - copd , had significantly improved after treatment for acute exacerbation and were comparable to the baseline airflow in the control group ( table 1 ) . three of 202 patients ( 1.5% ) tested for hbov dna were found to be positive by pcr and subsequent sequencing . of these subjects only one had an acute exacerbation , the other two had stable , but ( very ) severe copd ( table 2 ) . thus , the frequency of hbov detection demonstrated to be low in both ae - copd ( 1/134 , 0.8% ) and stable copd ( 2/68 , 2.9% ) . in two out of 202 patients ( 1.0% ) hbov was detected in induced sputum samples , whereas only one nasal lavage sample ( 1/202 , 0.5% ) tested positive for hbov . hbov was found in two sputum samples and one nasal lavage sample , while corresponding nasal lavage and sputum samples were negative , respectively . two subjects had stable copd and reported cough , whitish expectoration and dyspnea on exertion without any recent changes in severity . the individual with ae - copd complained of progressive cough , purulent expectoration as well as dyspnea at rest and symptoms of acute gastroenteritis . the two patients with gold stages ii and iii had not experienced ae - copd within the last 12 months preceding the study inclusion . sputum microbiology was sterile in two subjects , while candida albicans was detected in the other subject . viral coinfections with rsv ( 6.5 10 viral copies , detected in the induced sputum of the ae - copd patient ) and rsv and influenza virus a ( 3.2 10 and 3.8 10 viral copies , respectively , both detected in nasal lavage samples ) were existent in two of the three patients . sequence determination of the three positive hbov pcr products revealed a > 99% sequence identity with the swedish strains st1 and st2 and between the nucleotide sequences obtained from the years 2000 and 2002 . in this retrospective study of a prospectively recruited , case - controlled cohort we found hbov detection to be infrequent in adult hospitalized patients with acute exacerbation and stable copd during the years 1999 to 2003 in the urban region of central ruhr area , germany . in total , it was found both in acute exacerbation ( 0.8% , 1/134 ) and stable disease ( 2.9% , 2/68 ) as well as induced sputum and nasal lavage samples . these findings suggest that hbov may not be of clinical relevance in adult patients with ae - copd and routine testing for hbov in this population may not be warranted . data on hbov infections in adults is limited.9,13,1923,30 screening for respiratory viruses is not generally recommended for adults with symptoms of lower respiratory tract infection,1,31 thus material for large - scale retrospective analysis is hardly available . the very low frequency of overall hbov detection found in our study is in agreement with other studies in adults . the initial investigation by allander and colleagues did not detect hbov in 112 adults with respiratory symptoms at all.9 a large thai study detected hbov in 0.7% of subjects ( 6/834 ) aged 20 years or older ( including 126 controls ) only.13 two italian studies found frequencies of 1.2% ( 1/84 between the years 2000 and 2006)21 and 1.4% ( 2/143)19 in adult patients despite the use of different pcr assays ( real - time and single - round pcr , respectively ) . notably , a very recent us - american study found the identical frequency of 1.5% in 273 adults.22 the only previous study explicitly including patients with ae - copd found hbov in one of 126 ( 0.8% ) adult patients with ae - copd or pneumonia , a 71-year - old canadian male who had received oral steroids for the treatment of his exacerbation.20 unfortunately this report covered one winter season ( 2002/2003 ) only and neither specified the proportion of ae - copd patients included nor provided any clinical details on the population . in contrast to the reports mentioned above , which predominantly studied specimens representing the upper respiratory tract ( nasal swabs or nasopharyngeal aspirates ) , our study investigated both induced sputum and nasal lavage samples . in a previous pcr - based study we demonstrated that ae - copd requiring hospital admission was significantly associated with the presence of common respiratory viruses . induced sputum , which may more reliably reflect the lower respiratory tract , had a higher viral yield than upper respiratory tract specimens in patients with ae - copd.25 hrv ( 36% ) , influenza virus a ( 25% ) , and rsv ( 22% ) were most frequently detected , while in a similar study we recently found another emerging respiratory virus , hmpv , to be infrequent in ae - copd as well ( 2.3% , 3/130 , overall prevalence 1.3% , 3/229 ) . however all of the three hmpv detections occurred in the ae - copd group , making asymptomatic carriage unlikely.7 the present study investigated the frequency of hbov infection in four consecutive years with special care on avoidance of a seasonal selection bias by monthly recruitment of both groups in a 2:1 case - control setup . this finding of a limited genetic variation is in agreement with previous reports.9,23 in the present study viral coinfections , which were assessed by sensitive pcr assays , were existent in two of three hbov - positive subjects ( although specimens were not evaluated for the presence of some less common respiratory viruses like adenovirus , coronaviruses nl63 and hku1 , and parainfluenza viruses 1 and 2 ) . remarkably , the patient with ae - copd who tested positive for hbov suffered from concomitant acute gastroenteritis . hbov has also been proposed to be a causative pathogen in acute gastroenteritis , but so far its etiologic role remains controversial.32,33 this is the first controlled study on the prevalence of hbov in adult hospitalized patients with ae - copd . we found hbov at a very low frequency in patients with acute exacerbation as well as stable copd , comparable to what has been reported in other adult study populations . thus , a major role of hbov infection as a common trigger of ae - copd is unlikely .

all patients provided written informed consent according to german law as confirmed by the local committees ( 1442008 and 837.211.12 - 8312-f ) . glioblastoma tissue not required for neuropathological diagnostic procedures was obtained after surgical resection at the department of neurosurgery ( leipzig or mainz ) . an overview of the samples used in this study is given in table 1 . the tissue was transported to the laboratory in minimal essential medium ( mem ; invitrogen ) . slice cultures were prepared using a vibratome ( leica vt 1000 ) or a tissue chopper ( mcilwain tc752 ) at a thickness of 350 m under sterile conditions . before preparations , a standard razor blade was wiped with ethanol to remove any oil and then sterilized by autoclaving . additionally , a normal glass pipette and a pipette with the fine tip broken off were autoclaved . if needed , biopsy specimens were cut into appropriate - size pieces first to obtain evenly shaped slices of 5 5 mm . when the tissue chopper was used , the tissue was put on a stack of sterile filter membranes , cut , and then transferred carefully into ice - cold mem by forceps . in most of the preparations , the slices stuck together after cutting , so they were separated under a stereomicroscope with 2 scalpels without cutting into the tissue . slices were then transferred by the glass pipette with the wide opening onto membrane culture inserts ( millipore ) in 6-well plates at a maximum of 34 slices per insert depending on size . the cultivation medium consisted of mem ( gibco ) , 25% hank 's balanced salt solution ( with ca and mg ; gibco ) , 25% n - hydroxysuccinimide ( gibco ) , 1% l - glutamine ( braun ) , 1% glucose ( stock solution 45% , final concentration 0.45% ; braun ) , and 1% penicillin / streptomycin ( sigma ) . slices were cultivated on a liquid / air interface in a humidified incubator at 37c and 5% co2 . after time points ranging from 1 h to 4 weeks , slices were fixed in 4% paraformaldehyde and processed for paraffin embedding . paraffin sections ( 8 m ) were cut and stained with hematoxylin and eosin ( h&e ) for histology . histology of these sections was compared with the diagnostic histopathology of the same tumor to detect tissue culture induced changes . for immunocytochemistry , sections were dewaxed in xylene , rehydrated in a decreasing alcohol series , and ( if needed ) pretreated for antibody staining with citrate buffer ( ph 6 ) in a microwave . then , sections were washed in phosphate buffered saline ( pbs ) , permeabilized with 1.5% triton / pbs for 10 min , blocked with 10% normal goat serum in 1.5% triton / pbs for 1 h , and incubated overnight at 4c with primary antibodies against ki67 ( rabbit , 1:100 ; dcs ) , cleaved caspase 3 ( rabbit , 1:400 ; cell signaling ) , glial fibrillary acidic protein ( gfap ; dako , rabbit , 1:600 ; dako ) , nestin ( rabbit , 1:600 ; chemicon ) , vimentin ( mouse , 1:100 ; dako ) , neurofilament ( mouse , 1:100 ; dako ) , or h2ax ( mouse , 1:100 ; millipore ) . visualization was achieved by incubation either with appropriate fluorescent - labeled secondary antibodies ( goat anti - mouse or anti - rabbit alexa 488 ) or with biotinylated anti - rabbit immunoglobulin g followed by streptavidin - conjugated horseradish peroxidase and developing by adding diaminobenzidine for the color reaction . for fluorescent staining , photographs were taken using an olympus bx51 fluorescent microscope or a zeiss lsm 510 confocal microscope . in addition to the green fluorescent channel , the red channel was included because some of the samples exhibited a strong autofluorescence , which is normal in the nonjuvenile human brain . only cells devoid of red fluorescence were used for further analysis . for h&e and diaminobenzidine staining , a zeiss axioplan 2 microscope was used . table 1.glioblastoma samples used for slice culture experiments in this studysampleexperimentdata shown in03082010long - term culturefig . 112082010long - term culturefig . 125072012long - term culture and x - irradiationfigs . 1 and 312122011tmz treatmentfigs 505042011carbon ion irradiation and tmz treatmentfigs . 3 , 8 , 4 , and 611102011carbon ion irradiation and tmz treatmentfigs . glioblastoma samples used for slice culture experiments in this study all patients had a diagnosis of world health organization grade iv gbm . paraffin sections ( 8 m ) of slices were dewaxed as previously described here , and proliferating cells were stained with the ki67 antibody . , at least 12 images were acquired of 46 different sections per group and manually analyzed using imagej and the plugin cellcounter . the percentage of ki67-positive cells in relation to the total cell number was referred to as the proliferation index . glioblastoma tissue slices maintained on cell culture inserts were incubated with tmz ( dissolved in dimethyl sulfoxide ) at a final concentration of 50 or 200 m of the compound . control slices were incubated with the corresponding amount of dimethyl sulfoxide ( 0.2% v / v ) . after 72 h , tissue slices were removed from the membranes and incubated in 500 l of medium and 10 g / ml of hoechst 33342 ( sigma ) at 37c and 5% co2 for 1 h to allow for nuclear staining . the tissues were then transferred into 1 ml pbs containing 2 g / ml propidium iodide ( pi ; invitrogen ) and gently fixated between 2 glass coverslips . for confocal imaging and quantification of viable and dead cells within the tissues , an lsm-510 meta inverted laser scanning microscope and a 20/0.8 plan - apochromat objective ( carl zeiss microimaging ) were used . viable cells with hoechst - positive and pi - negative nuclei , and dead cells with hoechst - positive and pi - positive nuclei , were counted in 2 or 3 images of each tissue slice per group . one - way anova was used , and p < .05 was considered to be statistically significant . photon irradiation of slices was performed at the department for radiation therapy and radio - oncology , university of leipzig , with a 150-kv x - ray unit ( darpac 150-mc ) with an energy of 13.2 ma and a dose rate of 0.86 gy / min . cell culture plates were placed under a specially constructed plate device and irradiated until the desired dose was reached . alternatively , photon irradiation was performed using the gsi x - ray device ( ge isovolt titan 320 , 250 kv , 16 ma ) at a dose rate of 1.4 gy / min . hi irradiation with a carbon beam was performed at gsi ( gesellschaft fr schwerionenforschung ) , darmstadt , at the former patient irradiation site . the ion beam was generated at the sis18 synchrotron facility and delivered in a spread - out bragg peak ( sobp ) as used in carbon ion therapy . the dose applied to the slices was 2 or 4 gy in a 50-mm - width sobp corresponding to a linear energy transfer range of 5070 kev/m . with this method , then , the ion beam is directed at the 3-dimensional tumor volume , using active energy variation and the raster scanning technique . for experiments with slice cultures , the volume was defined as the area and the height of 1 well . before and after irradiation , slice cultures were kept in an incubator as previously described here and were removed for only about 15 min for transport and to place them on the irradiation belt . after irradiation , slices were fixed in 4% paraformaldehyde after one of several time points , washed in pbs , and further processed for paraffin embedding or cryosectioning . paraffin sections were prepared at 8 m , dried , and stored at room temperature . for staining of dna dsbs , cryosections were dried for 20 min at room temperature and then washed twice in pbs and incubated with 1.5% triton / pbs for 10 min . then sections were blocked with 10% normal goat serum in 1.5% triton / pbs for at least 1 h , followed by incubation overnight at 4c with h2ax primary antibody ( mouse monoclonal , 1:100 ; millipore ) . then , sections were washed 3 times with pbs and incubated with the secondary antibody ( goat anti - mouse 1:1000 ; alexa 488 , invitrogen ) for 1 h , washed again , counterstained with hoechst 33342 , and mounted with dako fluorescent mounting medium . z - stacks were taken using a zeiss lsm 510 confocal microscope at 400 magnification at intervals of 2 m . all patients provided written informed consent according to german law as confirmed by the local committees ( 1442008 and 837.211.12 - 8312-f ) . glioblastoma tissue not required for neuropathological diagnostic procedures was obtained after surgical resection at the department of neurosurgery ( leipzig or mainz ) . an overview of the samples used in this study is given in table 1 . the tissue was transported to the laboratory in minimal essential medium ( mem ; invitrogen ) . slice cultures were prepared using a vibratome ( leica vt 1000 ) or a tissue chopper ( mcilwain tc752 ) at a thickness of 350 m under sterile conditions . before preparations , a standard razor blade was wiped with ethanol to remove any oil and then sterilized by autoclaving . additionally , a normal glass pipette and a pipette with the fine tip broken off were autoclaved . if needed , biopsy specimens were cut into appropriate - size pieces first to obtain evenly shaped slices of 5 5 mm . when the tissue chopper was used , the tissue was put on a stack of sterile filter membranes , cut , and then transferred carefully into ice - cold mem by forceps . in most of the preparations , the slices stuck together after cutting , so they were separated under a stereomicroscope with 2 scalpels without cutting into the tissue . slices were then transferred by the glass pipette with the wide opening onto membrane culture inserts ( millipore ) in 6-well plates at a maximum of 34 slices per insert depending on size . the cultivation medium consisted of mem ( gibco ) , 25% hank 's balanced salt solution ( with ca and mg ; gibco ) , 25% n - hydroxysuccinimide ( gibco ) , 1% l - glutamine ( braun ) , 1% glucose ( stock solution 45% , final concentration 0.45% ; braun ) , and 1% penicillin / streptomycin ( sigma ) . slices were cultivated on a liquid / air interface in a humidified incubator at 37c and 5% co2 . after time points ranging from 1 h to 4 weeks , slices were fixed in 4% paraformaldehyde and processed for paraffin embedding . paraffin sections ( 8 m ) were cut and stained with hematoxylin and eosin ( h&e ) for histology . histology of these sections was compared with the diagnostic histopathology of the same tumor to detect tissue culture induced changes . for immunocytochemistry , sections were dewaxed in xylene , rehydrated in a decreasing alcohol series , and ( if needed ) pretreated for antibody staining with citrate buffer ( ph 6 ) in a microwave . then , sections were washed in phosphate buffered saline ( pbs ) , permeabilized with 1.5% triton / pbs for 10 min , blocked with 10% normal goat serum in 1.5% triton / pbs for 1 h , and incubated overnight at 4c with primary antibodies against ki67 ( rabbit , 1:100 ; dcs ) , cleaved caspase 3 ( rabbit , 1:400 ; cell signaling ) , glial fibrillary acidic protein ( gfap ; dako , rabbit , 1:600 ; dako ) , nestin ( rabbit , 1:600 ; chemicon ) , vimentin ( mouse , 1:100 ; dako ) , neurofilament ( mouse , 1:100 ; dako ) , or h2ax ( mouse , 1:100 ; millipore ) . visualization was achieved by incubation either with appropriate fluorescent - labeled secondary antibodies ( goat anti - mouse or anti - rabbit alexa 488 ) or with biotinylated anti - rabbit immunoglobulin g followed by streptavidin - conjugated horseradish peroxidase and developing by adding diaminobenzidine for the color reaction . for fluorescent staining , photographs were taken using an olympus bx51 fluorescent microscope or a zeiss lsm 510 confocal microscope . in addition to the green fluorescent channel , the red channel was included because some of the samples exhibited a strong autofluorescence , which is normal in the nonjuvenile human brain . only cells devoid of red fluorescence were used for further analysis . for h&e and diaminobenzidine staining , a zeiss axioplan 2 microscope was used . table 1.glioblastoma samples used for slice culture experiments in this studysampleexperimentdata shown in03082010long - term culturefig . 112082010long - term culturefig . 125072012long - term culture and x - irradiationfigs . 1 and 312122011tmz treatmentfigs 505042011carbon ion irradiation and tmz treatmentfigs . 3 , 8 , 4 , and 611102011carbon ion irradiation and tmz treatmentfigs . glioblastoma samples used for slice culture experiments in this study all patients had a diagnosis of world health organization grade iv gbm . paraffin sections ( 8 m ) of slices were dewaxed as previously described here , and proliferating cells were stained with the ki67 antibody . , at least 12 images were acquired of 46 different sections per group and manually analyzed using imagej and the plugin cellcounter . the percentage of ki67-positive cells in relation to the total cell number was referred to as the proliferation index . glioblastoma tissue slices maintained on cell culture inserts were incubated with tmz ( dissolved in dimethyl sulfoxide ) at a final concentration of 50 or 200 m of the compound . control slices were incubated with the corresponding amount of dimethyl sulfoxide ( 0.2% v / v ) . after 72 h , tissue slices were removed from the membranes and incubated in 500 l of medium and 10 g / ml of hoechst 33342 ( sigma ) at 37c and 5% co2 for 1 h to allow for nuclear staining . the tissues were then transferred into 1 ml pbs containing 2 g / ml propidium iodide ( pi ; invitrogen ) and gently fixated between 2 glass coverslips . for confocal imaging and quantification of viable and dead cells within the tissues , an lsm-510 meta inverted laser scanning microscope and a 20/0.8 plan - apochromat objective ( carl zeiss microimaging ) were used . pi was excited with the 543-nm laser line . viable cells with hoechst - positive and pi - negative nuclei , and dead cells with hoechst - positive and pi - positive nuclei , were counted in 2 or 3 images of each tissue slice per group . one - way anova was used , and p < .05 was considered to be statistically significant . photon irradiation of slices was performed at the department for radiation therapy and radio - oncology , university of leipzig , with a 150-kv x - ray unit ( darpac 150-mc ) with an energy of 13.2 ma and a dose rate of 0.86 gy / min . cell culture plates were placed under a specially constructed plate device and irradiated until the desired dose was reached . alternatively , photon irradiation was performed using the gsi x - ray device ( ge isovolt titan 320 , 250 kv , 16 ma ) at a dose rate of 1.4 gy / min . hi irradiation with a carbon beam was performed at gsi ( gesellschaft fr schwerionenforschung ) , darmstadt , at the former patient irradiation site . the ion beam was generated at the sis18 synchrotron facility and delivered in a spread - out bragg peak ( sobp ) as used in carbon ion therapy . the dose applied to the slices was 2 or 4 gy in a 50-mm - width sobp corresponding to a linear energy transfer range of 5070 kev/m . with this method , then , the ion beam is directed at the 3-dimensional tumor volume , using active energy variation and the raster scanning technique . for experiments with slice cultures , the volume was defined as the area and the height of 1 well . before and after irradiation , slice cultures were kept in an incubator as previously described here and were removed for only about 15 min for transport and to place them on the irradiation belt . after irradiation , slices were fixed in 4% paraformaldehyde after one of several time points , washed in pbs , and further processed for paraffin embedding or cryosectioning . paraffin sections were prepared at 8 m , dried , and stored at room temperature . for staining of dna dsbs , cryosections were dried for 20 min at room temperature and then washed twice in pbs and incubated with 1.5% triton / pbs for 10 min . then sections were blocked with 10% normal goat serum in 1.5% triton / pbs for at least 1 h , followed by incubation overnight at 4c with h2ax primary antibody ( mouse monoclonal , 1:100 ; millipore ) . then , sections were washed 3 times with pbs and incubated with the secondary antibody ( goat anti - mouse 1:1000 ; alexa 488 , invitrogen ) for 1 h , washed again , counterstained with hoechst 33342 , and mounted with dako fluorescent mounting medium . z - stacks were taken using a zeiss lsm 510 confocal microscope at 400 magnification at intervals of 2 m . slices were at first cut with a vibratome and survived well , with histological preservation of the main features of the original tumor for at least 16 days ( fig . 1 ) . at later stages , cell density appeared to decline in some tumor slices , whereas cells in other slices survived longer ( fig . , however , were difficult or even impossible to cut due to their viscous texture , which may have resulted from altered collagen expression . using a tissue chopper resolved this problem with equally good histological preservation and maximal survival time . histological examination of cultured gbm slices and comparison with the original neuropathology used for diagnosis confirmed striking maintenance of the general and individual hallmarks of the tumor ( pleomorphic nuclei , palisading , invading vessels / neovascularization , and necrotic areas ) . as expected , gbm stained positive for gfap , nestin ( intermediate filament protein , gbm stem cell marker ) , and vimentin ( mesenchymal intermediate filament protein ) and differed strongly in their cell density and content of necrotic areas ( fig . 2 ) . slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute ( a ) slices at 1 day or at 3 days ( b ) , 6 days ( c ) , or 12 days original h&e neuropathology ( e and i ) and h&e - stained paraffin - embedded sections ( 8 m ; f h and j l ) prepared from slices after various culture periods . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . slices were fixed after 7 days in culture and processed for paraffin sectioning ( 8 m ) . characteristic marker proteins were visualized by antibody staining for gfap ( a ) , nestin ( b ) , vimentin ( c ) , and neurofilament ( d ) . slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute ( a ) slices at 1 day or at 3 days ( b ) , 6 days ( c ) , or 12 days ( d ) in vitro . original h&e neuropathology ( e and i ) and h&e - stained paraffin - embedded sections ( 8 m ; f h and j l ) prepared from slices after various culture periods . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . slices were fixed after 7 days in culture and processed for paraffin sectioning ( 8 m ) . characteristic marker proteins were visualized by antibody staining for gfap ( a ) , nestin ( b ) , vimentin ( c ) , and neurofilament ( d ) . if dna repair can not be conducted properly , cell proliferation is arrested at a cell cycle checkpoint and either is inactivated ( eg , in postmitotic cells ) or proceeds into programmed cell death . here , we tested the dose- and time - dependent decrease of the proliferation index after sobp carbon or x - irradiation . gbm slices were fixed 6 or 24 h after irradiation and processed for paraffin sectioning . 3a d ) , which marks cells in every state of the cell cycle except the g0 resting phase . the percentage of ki67-positive cells in relation to the total number of nuclei was calculated to express the proliferation index . carbon ions did not significantly diminish proliferation at 6 h , but after 24 h a reduction of 40% was found ( p = .018 ; fig . this is the first demonstration of specific effects of hi on human gbm tissue ex vivo . photons also showed the anticipated time - dependent reduction of proliferation ( here , 50% after 24 h ; fig . thus , gbm - derived slice cultures can be irradiated and the biological effects of photons and hi on tumor cells and mechanisms of resistance can be studied in this model . 3.proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions . slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later . proliferating cells were then visualized in paraffin - embedded sections ( 8 m ) using a ki67 antibody ( green ) combined with nuclear counterstaining ( hoechst 33342 ; blue ) for quantitative analysis . ( a ) , non - irradiated ; ( b ) , irradiated with 4 gy carbon ions ; ( c ) , non - irradiated ; ( d ) , irradiated with 4 gy x - rays ; scale bar = 50 m ) . the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions . slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later . proliferating cells were then visualized in paraffin - embedded sections ( 8 m ) using a ki67 antibody ( green ) combined with nuclear counterstaining ( hoechst 33342 ; blue ) for quantitative analysis . ( a ) , non - irradiated ; ( b ) , irradiated with 4 gy carbon ions ; ( c ) , non - irradiated ; ( d ) , irradiated with 4 gy x - rays ; scale bar = 50 m ) . the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . after induction of dna dsbs by ionizing radiation , repair proteins are rapidly recruited to the sites of damage . in mammalian cells , this involves a cascade of proteins that ultimately allows repair of the breaks in the form of rejoining the loose dna ends . when we established the irradiation setup of gbm slice cultures , we wanted to test whether the slices were evenly hit by the beam , and therefore we used h2ax as an early dsb marker for the visualization of dna damage . h2ax describes a phosphorylation of histone 2ax at serine 139 around the region of the dsb , which allows binding of further repair proteins , such as mdc1 and 53bp1 . once the repair process is completed , the repair proteins dissociate and h2ax is dephosphorylated by a distinct phosphatase complex . slices were irradiated with 4 gy carbon ions in an sobp to match therapeutic conditions , fixed after 1 h , and embedded in paraffin , and sections were stained with an antibody to h2ax . h2ax was mostly absent in controls but appeared in a punctuated nuclear pattern in all sections , confirming induction of dna damage throughout the irradiated tissues ( fig . 4 ) . slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later . paraffin sections ( 8 m ) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody ( green ) and nuclei with hoechst ( blue ) . although h2ax was rarely detected in non - irradiated controls ( a ) , exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax ( b ) . slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later . paraffin sections ( 8 m ) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody ( green ) and nuclei with hoechst ( blue ) . although h2ax was rarely detected in non - irradiated controls ( a ) , exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax ( b ) . it is an alkylating agent that , in an aqueous solution at physiological ph , dissolves into its bioactive form mtic ( 5-(3-methyl-1-triazeno)imidazole-4-carboxamide ) , which is capable of penetrating the blood whether gbm tissue in culture responds to tmz treatment , slices were incubated with vehicle control or tmz ( 50 or 200 m ) . after 72 or 96 h , hoechst 33342 and pi were added to the cultures to visualize intact and dying nuclei using confocal live imaging . microimages were taken in which dying cells ( hoechst / pi double labeled ) were counted and their number related to the total number of nuclei ( hoechst single labeled ) , allowing calculation of a cell death rate . tmz - induced cell death was highly significant at 72 h , with little or no further increase until 96 h. this effect was more pronounced in slices treated with 200 m compared with 50 m of tmz ( fig . thus , chemotherapeutic effects can be mimicked in gbm slices and observed over time in situ . slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h ( a ) . after determination that an incubation of 72 h was sufficient , the experiment was repeated 3 times with 50 or 200 m tmz ( b ) . dying cells were labeled with pi ( red ) , and all nuclei were counterstained with hoechst 33342 ( blue ) for quantitative analysis ( c ) . grid distance= student 's t - test was performed , and p < .05 was considered statistically significant . both concentrations showed a significant increase in dying cells compared with controls , with the effect slightly more pronounced at 200 m . slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h ( a ) . after determination that an incubation of 72 h was sufficient , the experiment was repeated 3 times with 50 or 200 m tmz ( b ) . dying cells were labeled with pi ( red ) , and all nuclei were counterstained with hoechst 33342 ( blue ) for quantitative analysis ( c ) . grid distance= student 's t - test was performed , and p < .05 was considered statistically significant . both concentrations showed a significant increase in dying cells compared with controls , with the effect slightly more pronounced at 200 m . slices were exposed to either tmz or carbon ions in an sobp alone or in combination and fixed 48 h after irradiation . tmz treatment was started 24 h before irradiation , and a second dose was applied with the regular change of medium 48 h later . at the end of the treatment phase , induction of programmed cell death was determined using cleaved caspase 3 and h&e staining . all treatment regimens caused a decrease in cell density and nuclear alterations ( fig . therefore , instead of relating damaged cells to a total number of cell nuclei , the area coverage of caspase 3positive ( green ) and hoechst - positive ( blue ) pixels ( fig . 6e , caspase 3positive cells ; 6h , nuclear fragments ) was calculated as a measure of treatment - induced damage . after all treatments , the area of caspase 3positive pixels was significantly increased , whereas hoechst - positive pixels were decreased ( fig . a combination of both treatments did not show a synergistic or additive effect in the cases studied . irradiation with x - rays , however , also activated caspase 3 but did not cause significant cell loss ( fig . thus , effects of established treatment options on cell death can be studied in gbm slices . 6.combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . gbm slices were treated with tmz ( 200 m ) , x - ray irradiation ( 4 gy ) , sobp carbon ions ( 2 gy ) , or irradiation + tmz . tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time . slices were fixed 2 days after irradiation , and h&e staining was performed for neuropathological assessment ( a d ) . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . pictures of immunofluorescent stainings ( in rgb format ) were split into the three single channels of red , green , and blue , resulting in 3 gray - value pictures . the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting . in the green channel , the positive area represents the caspase 3positive cell population , and in the blue channel , the area covered by nuclei is displayed ( e j ) . carbon significantly reduced cell numbers ( g ) , whereas x - rays did not ( j ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . gbm slices were treated with tmz ( 200 m ) , x - ray irradiation ( 4 gy ) , sobp carbon ions ( 2 gy ) , or irradiation + tmz . tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time . slices were fixed 2 days after irradiation , and h&e staining was performed for neuropathological assessment ( a d ) . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . pictures of immunofluorescent stainings ( in rgb format ) were split into the three single channels of red , green , and blue , resulting in 3 gray - value pictures . the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting . in the green channel , the positive area represents the caspase 3positive cell population , and in the blue channel , the area covered by nuclei is displayed ( e j ) . carbon significantly reduced cell numbers ( g ) , whereas x - rays did not ( j ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . original magnification : 400 in a f . lack of promoter methylation of o - methylguanine - dna methyltransferase ( mgmt ) has been reported to significantly decrease patients ' susceptibility to tmz and thus survival , but there are also patients with nonmethylated promoter who benefit from tmz treatment . in fact , we identified one tumor in which tmz did not significantly induce cell death and we therefore requested the promoter methylation status , which is assessed by quantitative pcr and sequencing techniques of tumor material obtained from surgery . however , in line with the clinical observations that some patients with nonmethylated mgmt respond to tmz , we subsequently identified other tumors in which tmz significantly enhanced activation of caspase 3 to levels comparable to those of the methylated tumor ( fig . gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . this was independent of mgmt promoter methylation . from left to right : gbm 1011 with methylated mgmt promoter ( meth + ) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation . gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . this was independent of mgmt promoter methylation . from left to right : gbm 1011 with methylated mgmt promoter ( meth + ) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation . slices were at first cut with a vibratome and survived well , with histological preservation of the main features of the original tumor for at least 16 days ( fig . 1 ) . at later stages , cell density appeared to decline in some tumor slices , whereas cells in other slices survived longer ( fig . , however , were difficult or even impossible to cut due to their viscous texture , which may have resulted from altered collagen expression . using a tissue chopper resolved this problem with equally good histological preservation and maximal survival time . histological examination of cultured gbm slices and comparison with the original neuropathology used for diagnosis confirmed striking maintenance of the general and individual hallmarks of the tumor ( pleomorphic nuclei , palisading , invading vessels / neovascularization , and necrotic areas ) . as expected , gbm stained positive for gfap , nestin ( intermediate filament protein , gbm stem cell marker ) , and vimentin ( mesenchymal intermediate filament protein ) and differed strongly in their cell density and content of necrotic areas ( fig . 2 ) . slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute ( a ) slices at 1 day or at 3 days ( b ) , 6 days ( c ) , or 12 days original h&e neuropathology ( e and i ) and h&e - stained paraffin - embedded sections ( 8 m ; f h and j l ) prepared from slices after various culture periods . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . slices were fixed after 7 days in culture and processed for paraffin sectioning ( 8 m ) . characteristic marker proteins were visualized by antibody staining for gfap ( a ) , nestin ( b ) , vimentin ( c ) , and neurofilament ( d ) . slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute ( a ) slices at 1 day or at 3 days ( b ) , 6 days ( c ) , or 12 days ( d ) in vitro . original h&e neuropathology ( e and i ) and h&e - stained paraffin - embedded sections ( 8 m ; f h and j l ) prepared from slices after various culture periods . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . slices were fixed after 7 days in culture and processed for paraffin sectioning ( 8 m ) . characteristic marker proteins were visualized by antibody staining for gfap ( a ) , nestin ( b ) , vimentin ( c ) , and neurofilament ( d ) . if dna repair can not be conducted properly , cell proliferation is arrested at a cell cycle checkpoint and either is inactivated ( eg , in postmitotic cells ) or proceeds into programmed cell death . here , we tested the dose- and time - dependent decrease of the proliferation index after sobp carbon or x - irradiation . gbm slices were fixed 6 or 24 h after irradiation and processed for paraffin sectioning . 3a d ) , which marks cells in every state of the cell cycle except the g0 resting phase . the percentage of ki67-positive cells in relation to the total number of nuclei was calculated to express the proliferation index . carbon ions did not significantly diminish proliferation at 6 h , but after 24 h a reduction of 40% was found ( p = .018 ; fig . this is the first demonstration of specific effects of hi on human gbm tissue ex vivo . photons also showed the anticipated time - dependent reduction of proliferation ( here , 50% after 24 h ; fig . thus , gbm - derived slice cultures can be irradiated and the biological effects of photons and hi on tumor cells and mechanisms of resistance can be studied in this model . 3.proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions . slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later . proliferating cells were then visualized in paraffin - embedded sections ( 8 m ) using a ki67 antibody ( green ) combined with nuclear counterstaining ( hoechst 33342 ; blue ) for quantitative analysis . ( a ) , non - irradiated ; ( b ) , irradiated with 4 gy carbon ions ; ( c ) , non - irradiated ; ( d ) , irradiated with 4 gy x - rays ; scale bar = 50 m ) . the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . proliferation index of human gbm slice cultures irradiated with x - rays or carbon ions . slices were treated with either 4 gy of sobp carbon ions at gsi or 4 gy of x - rays and fixed 6 or 24 h later . proliferating cells were then visualized in paraffin - embedded sections ( 8 m ) using a ki67 antibody ( green ) combined with nuclear counterstaining ( hoechst 33342 ; blue ) for quantitative analysis . ( a ) , non - irradiated ; ( b ) , irradiated with 4 gy carbon ions ; ( c ) , non - irradiated ; ( d ) , irradiated with 4 gy x - rays ; scale bar = 50 m ) . the proliferative fraction in relation to total cell number was determined in at least 12 pictures per group by using imagej software . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . after induction of dna dsbs by ionizing radiation , repair proteins are rapidly recruited to the sites of damage . in mammalian cells , this involves a cascade of proteins that ultimately allows repair of the breaks in the form of rejoining the loose dna ends . when we established the irradiation setup of gbm slice cultures , we wanted to test whether the slices were evenly hit by the beam , and therefore we used h2ax as an early dsb marker for the visualization of dna damage . h2ax describes a phosphorylation of histone 2ax at serine 139 around the region of the dsb , which allows binding of further repair proteins , such as mdc1 and 53bp1 . once the repair process is completed , the repair proteins dissociate and h2ax is dephosphorylated by a distinct phosphatase complex . slices were irradiated with 4 gy carbon ions in an sobp to match therapeutic conditions , fixed after 1 h , and embedded in paraffin , and sections were stained with an antibody to h2ax . h2ax was mostly absent in controls but appeared in a punctuated nuclear pattern in all sections , confirming induction of dna damage throughout the irradiated tissues ( fig . 4 ) . slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later . paraffin sections ( 8 m ) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody ( green ) and nuclei with hoechst ( blue ) . although h2ax was rarely detected in non - irradiated controls ( a ) , exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax ( b ) . slices were irradiated with 4 gy of carbon ions in an sobp and fixed 1 h later . paraffin sections ( 8 m ) were assembled and dna dsbs were visualized by immunocytochemistry with a h2ax antibody ( green ) and nuclei with hoechst ( blue ) . although h2ax was rarely detected in non - irradiated controls ( a ) , exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax ( b ) . it is an alkylating agent that , in an aqueous solution at physiological ph , dissolves into its bioactive form mtic ( 5-(3-methyl-1-triazeno)imidazole-4-carboxamide ) , which is capable of penetrating the blood brain barrier . to test whether gbm tissue in culture responds to tmz treatment , slices were incubated with vehicle control or tmz ( 50 or 200 m ) . after 72 or 96 h , hoechst 33342 and pi were added to the cultures to visualize intact and dying nuclei using confocal live imaging . microimages were taken in which dying cells ( hoechst / pi double labeled ) were counted and their number related to the total number of nuclei ( hoechst single labeled ) , allowing calculation of a cell death rate . tmz - induced cell death was highly significant at 72 h , with little or no further increase until 96 h. this effect was more pronounced in slices treated with 200 m compared with 50 m of tmz ( fig . thus , chemotherapeutic effects can be mimicked in gbm slices and observed over time in situ . slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h ( a ) . after determination that an incubation of 72 h was sufficient , the experiment was repeated 3 times with 50 or 200 m tmz ( b ) . dying cells were labeled with pi ( red ) , and all nuclei were counterstained with hoechst 33342 ( blue ) for quantitative analysis ( c ) . grid distance= 50 m . student 's t - test was performed , and p < .05 was considered statistically significant . both concentrations showed a significant increase in dying cells compared with controls , with the effect slightly more pronounced at 200 m . slices were treated with either 50 or 200 m tmz and then incubated for 72 or 96 h ( a ) . after determination that an incubation of 72 h was sufficient , the experiment was repeated 3 times with 50 or 200 m tmz ( b ) . dying cells were labeled with pi ( red ) , and all nuclei were counterstained with hoechst 33342 ( blue ) for quantitative analysis ( c ) . grid distance= 50 m . student 's t - test was performed , and p < .05 was considered statistically significant . both concentrations showed a significant increase in dying cells compared with controls , with the effect slightly more pronounced at 200 m . slices were exposed to either tmz or carbon ions in an sobp alone or in combination and fixed 48 h after irradiation . tmz treatment was started 24 h before irradiation , and a second dose was applied with the regular change of medium 48 h later . at the end of the treatment phase , induction of programmed cell death was determined using cleaved caspase 3 and h&e staining . all treatment regimens caused a decrease in cell density and nuclear alterations ( fig . therefore , instead of relating damaged cells to a total number of cell nuclei , the area coverage of caspase 3positive ( green ) and hoechst - positive ( blue ) pixels ( fig . 6e , caspase 3positive cells ; 6h , nuclear fragments ) was calculated as a measure of treatment - induced damage . after all treatments , the area of caspase 3positive pixels was significantly increased , whereas hoechst - positive pixels were decreased ( fig . both irradiation and tmz alone induced cell death , but a combination of both treatments did not show a synergistic or additive effect in the cases studied . irradiation with x - rays , however , also activated caspase 3 but did not cause significant cell loss ( fig . thus , effects of established treatment options on cell death can be studied in gbm slices . 6.combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . gbm slices were treated with tmz ( 200 m ) , x - ray irradiation ( 4 gy ) , sobp carbon ions ( 2 gy ) , or irradiation + tmz . tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time . slices were fixed 2 days after irradiation , and h&e staining was performed for neuropathological assessment ( a d ) . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . pictures of immunofluorescent stainings ( in rgb format ) were split into the three single channels of red , green , and blue , resulting in 3 gray - value pictures . the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting . in the green channel , the positive area represents the caspase 3positive cell population , and in the blue channel , the area covered by nuclei is displayed ( e j ) . carbon significantly reduced cell numbers ( g ) , whereas x - rays did not ( j ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . gbm slices were treated with tmz ( 200 m ) , x - ray irradiation ( 4 gy ) , sobp carbon ions ( 2 gy ) , or irradiation + tmz . tmz treatment started 24 h before irradiation and was maintained throughout the entire incubation time . slices were fixed 2 days after irradiation , and h&e staining was performed for neuropathological assessment ( a d ) . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . pictures of immunofluorescent stainings ( in rgb format ) were split into the three single channels of red , green , and blue , resulting in 3 gray - value pictures . the area coverage is represented by white pixels in the respective channels and can be fine - tuned by threshold adjusting . in the green channel , the positive area represents the caspase 3positive cell population , and in the blue channel , the area covered by nuclei is displayed ( e j ) . carbon significantly reduced cell numbers ( g ) , whereas x - rays did not ( j ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . lack of promoter methylation of o - methylguanine - dna methyltransferase ( mgmt ) has been reported to significantly decrease patients ' susceptibility to tmz and thus survival , but there are also patients with nonmethylated promoter who benefit from tmz treatment . in fact , we identified one tumor in which tmz did not significantly induce cell death and we therefore requested the promoter methylation status , which is assessed by quantitative pcr and sequencing techniques of tumor material obtained from surgery . however , in line with the clinical observations that some patients with nonmethylated mgmt respond to tmz , we subsequently identified other tumors in which tmz significantly enhanced activation of caspase 3 to levels comparable to those of the methylated tumor ( fig . gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . this was independent of mgmt promoter methylation . from left to right : gbm 1011 with methylated mgmt promoter ( meth + ) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation . gbm slice cultures with different mgmt promoter methylation states were tested for their response to tmz treatment . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . this was independent of mgmt promoter methylation . from left to right : gbm 1011 with methylated mgmt promoter ( meth + ) and significant caspase 3 activation ; gbm 0411 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 1211 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0812 with nonmethylated mgmt promoter ( meth - ) and significant caspase 3 activation ; gbm 0611 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation ; gbm 0512 with nonmethylated mgmt promoter ( meth - ) and nonsignificant ( ns ) caspase 3 activation . organotypic slice cultures derived from early postnatal rodent brain are widely used in neuroscience due to their easy access for pharmacological intervention , electrophysiological studies , and live imaging . we have employed entorhinohippocampal preparations , which , due to the orientation of the trisynaptic pathway ( perpendicular to the longitudinal axis of the hippocampus ) , allow for maintenance of the major connectivity . in this study , we adjusted cutting and culturing methods to prepare slices from human gbm and demonstrated evidence for their suitability as a test system for novel therapies including irradiation with hi . we irradiated gbm slices with photons and carbon ions and in both instances found that radiation induced dna damage and strongly affected proliferation . carbon ion radiation also induced activation of caspase 3 , a potent inductor of programmed cell death ( figs . in contrast to photon radiation , which delivers energy all the way through the body ( eg , in an anterior - posterior direction ) , the depth of energy deposition of hi can be adjusted and limited to distinct areas of a few millimeters . in fact , much hope derives from successful intensity - modulated carbon therapy of chondrosarcomas of the skull base , a treatment first established at gsi . an accelerator specialized for medical applications has been constructed in heidelberg and opened for patients in 2009 . currently , clinical trials and work with cell lines are aimed at testing the effects of carbon ion radiation in tumors other than chondrosarcomas . our data support observations in gbm - derived cell lines and in a small group of patients , and our approach may lead to a more detailed understanding of the biological effects of hi and additional novel therapies . moreover , surviving tumor cells can be studied to understand their mode of resistance . we also applied tmz to gbm slices and analyzed the effect on cell survival using pi staining and live imaging , as well as labeling of activated caspase 3 in paraffin - embedded sections . tmz is an alkylating agent widely used to treat gbm that , in combination with radiation therapy , helps prolong patients ' survival time . methylation was significantly more frequent in patients who survived longer than 36 months after surgery ( p < .05 ) . statistically , methylation status apparently affects susceptibility to tmz , but some patients with nonmethylated status also seem to benefit from tmz . of note , determining methylation status is not trivial , as only a few of the 109 potential sites have been tested . a recent survey among 1053 members of the neuro - oncology community in the united states found that only a small percentage ( 10.9% ) of clinicians regard mgmt status as always or almost always thus , the next challenge will be to relate tmz - induced cell death rates obtained in slices to ( progression - free ) survival times ; we will address this issue with tumor - derived samples during the next 2 years to test the predictive value of this assay . it may be trivial to state that only in vitro systems allow different therapeutic options to be tested for an individual patient . our data demonstrate that tumor - derived gbm - slice cultures in principle are suitable for that task as a step on the way to more personalized therapies while also helping unravel basic mechanisms of tumor resistance . this work was supported by the messer foundation , the kassel foundation , and the dr .

backgroundglioblastoma multiforme is the most common lethal brain tumor in human adults , with no major therapeutic breakthroughs in recent decades . research is based mostly on human tumor cell lines deprived of their organotypic environment or inserted into immune - deficient animals required for graft survival . here , we describe how glioblastoma specimens obtained from surgical biopsy material can be sectioned and transferred into cultures within minutes.methodsslices were kept in 6-well plates , allowing direct observation , application of temozolomide , and irradiation . at the end of experiments , slice cultures were processed for histological analysis including hematoxylin - eosin staining , detection of proliferation ( ki67 ) , apoptosis / cell death ( cleaved caspase 3 , propidium iodide ) , dna double - strand breaks ( h2ax ) , and neural subpopulations . first clinical trials employed irradiation with the heavy ion carbon for the treatment of glioblastoma patients , but the biological effects and most effective dose regimens remain to be established . therefore , we developed an approach to expose glioblastoma slice cultures to 12c and x-rays.resultswe found preservation of the individual histopathology over at least 16 days . treatments resulted in activation of caspase 3 , inhibition of proliferation , and cell loss . irradiation induced h2ax . in line with clinical observations , individual tumors differed significantly in their susceptibility to temozolomide ( 0.4%2.5% apoptosis and 1%15% cell loss).conclusionglioblastoma multiforme slice cultures provide a unique tool to explore susceptibility of individual tumors for specific therapies including heavy ions , thus potentially allowing more personalized treatments plus exploration of mechanisms of ( and strategies to overcome ) tumor resistance .

Materials and Methods Tissue Slice Preparation Analysis of Proliferation in Slices Treatment of Glioblastoma Tissue Slices With Temozolomide Irradiation of Glioblastoma Slice Cultures Results Slice Cultures From Glioblastoma: Histology and Survival Irradiation of Slices: Effect on Proliferation Exposure of Slices to Carbon Ions and Detection of DNA Damage Exposure of Glioblastoma Tissue Slices to Temozolomide: Detection of Cell Death Using Live Imaging Exposure of Slices to Carbon Ions and TMZ: Detection of Cell Death Using Activated Caspase 3 Staining TMZ Treatment of Slice Cultures From Tumors With Different MGMT Promoter Methylation Statuses Discussion Funding

slices were then transferred by the glass pipette with the wide opening onto membrane culture inserts ( millipore ) in 6-well plates at a maximum of 34 slices per insert depending on size . then , sections were washed in phosphate buffered saline ( pbs ) , permeabilized with 1.5% triton / pbs for 10 min , blocked with 10% normal goat serum in 1.5% triton / pbs for 1 h , and incubated overnight at 4c with primary antibodies against ki67 ( rabbit , 1:100 ; dcs ) , cleaved caspase 3 ( rabbit , 1:400 ; cell signaling ) , glial fibrillary acidic protein ( gfap ; dako , rabbit , 1:600 ; dako ) , nestin ( rabbit , 1:600 ; chemicon ) , vimentin ( mouse , 1:100 ; dako ) , neurofilament ( mouse , 1:100 ; dako ) , or h2ax ( mouse , 1:100 ; millipore ) . 3 , 8 , 4 , and 611102011carbon ion irradiation and tmz treatmentfigs . paraffin sections ( 8 m ) of slices were dewaxed as previously described here , and proliferating cells were stained with the ki67 antibody . control slices were incubated with the corresponding amount of dimethyl sulfoxide ( 0.2% v / v ) . one - way anova was used , and p < .05 was considered to be statistically significant . photon irradiation of slices was performed at the department for radiation therapy and radio - oncology , university of leipzig , with a 150-kv x - ray unit ( darpac 150-mc ) with an energy of 13.2 ma and a dose rate of 0.86 gy / min . hi irradiation with a carbon beam was performed at gsi ( gesellschaft fr schwerionenforschung ) , darmstadt , at the former patient irradiation site . before and after irradiation , slice cultures were kept in an incubator as previously described here and were removed for only about 15 min for transport and to place them on the irradiation belt . glioblastoma tissue not required for neuropathological diagnostic procedures was obtained after surgical resection at the department of neurosurgery ( leipzig or mainz ) . slice cultures were prepared using a vibratome ( leica vt 1000 ) or a tissue chopper ( mcilwain tc752 ) at a thickness of 350 m under sterile conditions . slices were then transferred by the glass pipette with the wide opening onto membrane culture inserts ( millipore ) in 6-well plates at a maximum of 34 slices per insert depending on size . the cultivation medium consisted of mem ( gibco ) , 25% hank 's balanced salt solution ( with ca and mg ; gibco ) , 25% n - hydroxysuccinimide ( gibco ) , 1% l - glutamine ( braun ) , 1% glucose ( stock solution 45% , final concentration 0.45% ; braun ) , and 1% penicillin / streptomycin ( sigma ) . then , sections were washed in phosphate buffered saline ( pbs ) , permeabilized with 1.5% triton / pbs for 10 min , blocked with 10% normal goat serum in 1.5% triton / pbs for 1 h , and incubated overnight at 4c with primary antibodies against ki67 ( rabbit , 1:100 ; dcs ) , cleaved caspase 3 ( rabbit , 1:400 ; cell signaling ) , glial fibrillary acidic protein ( gfap ; dako , rabbit , 1:600 ; dako ) , nestin ( rabbit , 1:600 ; chemicon ) , vimentin ( mouse , 1:100 ; dako ) , neurofilament ( mouse , 1:100 ; dako ) , or h2ax ( mouse , 1:100 ; millipore ) . paraffin sections ( 8 m ) of slices were dewaxed as previously described here , and proliferating cells were stained with the ki67 antibody . control slices were incubated with the corresponding amount of dimethyl sulfoxide ( 0.2% v / v ) . photon irradiation of slices was performed at the department for radiation therapy and radio - oncology , university of leipzig , with a 150-kv x - ray unit ( darpac 150-mc ) with an energy of 13.2 ma and a dose rate of 0.86 gy / min . hi irradiation with a carbon beam was performed at gsi ( gesellschaft fr schwerionenforschung ) , darmstadt , at the former patient irradiation site . before and after irradiation , slice cultures were kept in an incubator as previously described here and were removed for only about 15 min for transport and to place them on the irradiation belt . after irradiation , slices were fixed in 4% paraformaldehyde after one of several time points , washed in pbs , and further processed for paraffin embedding or cryosectioning . slices were at first cut with a vibratome and survived well , with histological preservation of the main features of the original tumor for at least 16 days ( fig . histological examination of cultured gbm slices and comparison with the original neuropathology used for diagnosis confirmed striking maintenance of the general and individual hallmarks of the tumor ( pleomorphic nuclei , palisading , invading vessels / neovascularization , and necrotic areas ) . as expected , gbm stained positive for gfap , nestin ( intermediate filament protein , gbm stem cell marker ) , and vimentin ( mesenchymal intermediate filament protein ) and differed strongly in their cell density and content of necrotic areas ( fig . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . characteristic marker proteins were visualized by antibody staining for gfap ( a ) , nestin ( b ) , vimentin ( c ) , and neurofilament ( d ) . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . characteristic marker proteins were visualized by antibody staining for gfap ( a ) , nestin ( b ) , vimentin ( c ) , and neurofilament ( d ) . here , we tested the dose- and time - dependent decrease of the proliferation index after sobp carbon or x - irradiation . 3a d ) , which marks cells in every state of the cell cycle except the g0 resting phase . this is the first demonstration of specific effects of hi on human gbm tissue ex vivo . photons also showed the anticipated time - dependent reduction of proliferation ( here , 50% after 24 h ; fig . thus , gbm - derived slice cultures can be irradiated and the biological effects of photons and hi on tumor cells and mechanisms of resistance can be studied in this model . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . when we established the irradiation setup of gbm slice cultures , we wanted to test whether the slices were evenly hit by the beam , and therefore we used h2ax as an early dsb marker for the visualization of dna damage . although h2ax was rarely detected in non - irradiated controls ( a ) , exposure to therapeutic heavy ions caused massive induction of phosphorylation of h2ax ( b ) . microimages were taken in which dying cells ( hoechst / pi double labeled ) were counted and their number related to the total number of nuclei ( hoechst single labeled ) , allowing calculation of a cell death rate . dying cells were labeled with pi ( red ) , and all nuclei were counterstained with hoechst 33342 ( blue ) for quantitative analysis ( c ) . both concentrations showed a significant increase in dying cells compared with controls , with the effect slightly more pronounced at 200 m . dying cells were labeled with pi ( red ) , and all nuclei were counterstained with hoechst 33342 ( blue ) for quantitative analysis ( c ) . at the end of the treatment phase , induction of programmed cell death was determined using cleaved caspase 3 and h&e staining . irradiation with x - rays , however , also activated caspase 3 but did not cause significant cell loss ( fig . thus , effects of established treatment options on cell death can be studied in gbm slices . 6.combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . in fact , we identified one tumor in which tmz did not significantly induce cell death and we therefore requested the promoter methylation status , which is assessed by quantitative pcr and sequencing techniques of tumor material obtained from surgery . however , in line with the clinical observations that some patients with nonmethylated mgmt respond to tmz , we subsequently identified other tumors in which tmz significantly enhanced activation of caspase 3 to levels comparable to those of the methylated tumor ( fig . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . slices were at first cut with a vibratome and survived well , with histological preservation of the main features of the original tumor for at least 16 days ( fig . histological examination of cultured gbm slices and comparison with the original neuropathology used for diagnosis confirmed striking maintenance of the general and individual hallmarks of the tumor ( pleomorphic nuclei , palisading , invading vessels / neovascularization , and necrotic areas ) . as expected , gbm stained positive for gfap , nestin ( intermediate filament protein , gbm stem cell marker ) , and vimentin ( mesenchymal intermediate filament protein ) and differed strongly in their cell density and content of necrotic areas ( fig . slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute ( a ) slices at 1 day or at 3 days ( b ) , 6 days ( c ) , or 12 days original h&e neuropathology ( e and i ) and h&e - stained paraffin - embedded sections ( 8 m ; f h and j l ) prepared from slices after various culture periods . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . slices were cultured on membrane inserts in six - well plates with no signs of degeneration in acute ( a ) slices at 1 day or at 3 days ( b ) , 6 days ( c ) , or 12 days ( d ) in vitro . two different tumors ( e h and i note that typical features of individual tumors were maintained at least from 1 to 16 days ( f g ) and 1 to 13 days ( j k ) in vitro ; massive cell loss was observed after 20 days in vitro ( h and l ) . here , we tested the dose- and time - dependent decrease of the proliferation index after sobp carbon or x - irradiation . this is the first demonstration of specific effects of hi on human gbm tissue ex vivo . photons also showed the anticipated time - dependent reduction of proliferation ( here , 50% after 24 h ; fig . thus , gbm - derived slice cultures can be irradiated and the biological effects of photons and hi on tumor cells and mechanisms of resistance can be studied in this model . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . after 6 h , the effect on proliferation was not yet significant after both irradiation types , but after 24 h both treatments resulted in a significant decrease of ki67-positive cells ( e and f ) . when we established the irradiation setup of gbm slice cultures , we wanted to test whether the slices were evenly hit by the beam , and therefore we used h2ax as an early dsb marker for the visualization of dna damage . microimages were taken in which dying cells ( hoechst / pi double labeled ) were counted and their number related to the total number of nuclei ( hoechst single labeled ) , allowing calculation of a cell death rate . tmz - induced cell death was highly significant at 72 h , with little or no further increase until 96 h. this effect was more pronounced in slices treated with 200 m compared with 50 m of tmz ( fig . tmz treatment was started 24 h before irradiation , and a second dose was applied with the regular change of medium 48 h later . at the end of the treatment phase , induction of programmed cell death was determined using cleaved caspase 3 and h&e staining . therefore , instead of relating damaged cells to a total number of cell nuclei , the area coverage of caspase 3positive ( green ) and hoechst - positive ( blue ) pixels ( fig . irradiation with x - rays , however , also activated caspase 3 but did not cause significant cell loss ( fig . thus , effects of established treatment options on cell death can be studied in gbm slices . 6.combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . both treatments result in a significant increase in cell death and morphological alterations compared with the vehicle - treated control . combined treatment of tmz and irradiation with carbon ions and x - rays on human gbm slice cultures . in addition , activated caspase 3 was labeled ( green in e ) , and fragmented nuclei were visualized using hoechst 33342 ( blue in e and h ) . in fact , we identified one tumor in which tmz did not significantly induce cell death and we therefore requested the promoter methylation status , which is assessed by quantitative pcr and sequencing techniques of tumor material obtained from surgery . however , in line with the clinical observations that some patients with nonmethylated mgmt respond to tmz , we subsequently identified other tumors in which tmz significantly enhanced activation of caspase 3 to levels comparable to those of the methylated tumor ( fig . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . after tmz treatment , caspase 3 activation was detected in some specimens , whereas others seemed to be resistant . in a gbm specimen with methylated promoter sequence ( a ) , tmz treatment resulted in significant induction of caspase 3 cleavage ( gbm 1011 ) , whereas caspase 3 activation was not significantly induced ( b ) in gbm with an unmethylated promoter sequence ( gbm 0611 ) . immunocytochemistry is shown for the methylated ( c , right ) and unmethylated ( c , left ) tumor for cleaved caspase 3 ( green ) and nuclei ( hoechst , blue ) . organotypic slice cultures derived from early postnatal rodent brain are widely used in neuroscience due to their easy access for pharmacological intervention , electrophysiological studies , and live imaging . in this study , we adjusted cutting and culturing methods to prepare slices from human gbm and demonstrated evidence for their suitability as a test system for novel therapies including irradiation with hi . carbon ion radiation also induced activation of caspase 3 , a potent inductor of programmed cell death ( figs . currently , clinical trials and work with cell lines are aimed at testing the effects of carbon ion radiation in tumors other than chondrosarcomas . our data support observations in gbm - derived cell lines and in a small group of patients , and our approach may lead to a more detailed understanding of the biological effects of hi and additional novel therapies . statistically , methylation status apparently affects susceptibility to tmz , but some patients with nonmethylated status also seem to benefit from tmz . our data demonstrate that tumor - derived gbm - slice cultures in principle are suitable for that task as a step on the way to more personalized therapies while also helping unravel basic mechanisms of tumor resistance .

food security has become a major challenge given the projected need to increase world food supply by about 70% by 2050 ( anon . , 2009 ) . considering the limitations on expanding crop - growing areas , a significant increase in crop productivity will be required to achieve this target ( parry et al . , 2011 ; reynolds et al . , 2011 ) . wheat production is highly sensitive to climatic and environmental variations ( porter and semenov , 2005 ) . global warming is characterised by shifts in weather patterns and increase in frequency and magnitude of extreme events ( lobell et al . , 2012 ; semenov and shewry , 2011 ; sillmann and roeckner , 2008 ) . increasing temperature and incidence of drought associated with global warming are posing serious threats to food security ( lobell et al . , 2013 ) . climate change , therefore , represents a considerable challenge in achieving the 70%-increase target in world food production . however , the intrinsic uncertainty of climate change predictions poses a challenge to plant breeders and crop scientists who have limited time and resources and must select the most appropriate traits for improvement ( foulkes et al . , 2011 ; semenov and halford , 2009 ; zheng et al . , 2012 ) . modelling provides a rational framework to design and test in silico new wheat ideotypes optimised for target environments and future climatic conditions ( hammer et al . , 2006 , 2010 ; semenov and halford , 2009 ; semenov and shewry , 2011 ; sylvester - bradley et al . , 2012 ; eco - physiological process - based crop models are commonly used in basic and applied research in the plant sciences and in natural resource management ( hammer et al . , 2002 ; passioura , 1996 ; , 2011 ; sinclair and seligman , 1996 ; white et al . , 2011 ) . they provide the best - available framework for integrating our understanding of complex plant processes and their responses to climate and environment . such models are playing an increasing role in guiding the direction of fundamental research by providing quantitative predictions and highlighting gaps in our knowledge ( hammer et al . , 2006 ; hammer et al . , 2010 ; semenov and halford , 2009 ; semenov and shewry , 2011 ; tardieu , 2003 ) . the objective of our study was to assess wheat yield potential under climate change in europe and identify challenges which must be overcome to achieve high wheat yields in the future . firstly , we used the sirius wheat model to optimise wheat ideotypes for future climate scenarios ( jamieson and semenov , 2000 ; lawless et al . wheat ideotype was defined as a set of sirius cultivar parameters . by changing cultivar parameters , we change wheat growth and development in response to climatic and environment variations and can select ideotypes with better performance under future climates and environments . sirius is a well validated model and was able to simulate accurately wheat growth and grain yield in a wide range of environments , including europe , usa , new zealand and australia , and for experiments reproducing conditions of climate change , e.g. free - air carbon dioxide enrichment ( face ) experiments ( ewert et al . , 2002 ; he et al . , 2012 ; jamieson et al . , 2000 ; it remains a challenge for such models to capture the yield response of wheat to extreme events , particularly when they coincide with sensitive growth stages ( craufurd et al . crop models need an overhaul to incorporate such responses to extreme weather events ( rtter et al . , 2011 ) . for example , it has been established that wheat yield is particularly sensitive to abiotic stresses during microsporogenesis , anther dehiscence and fertilization because of effects on grain set ( as reviewed by barnabas et al . 2013 ) ; and just after fertilization because of effects on grain size ( gooding et al . , 2003 ) . to facilitate model development additional data from carefully designed experiments will be required . the second approach presented here is , therefore , to describe the response of wheat to heat and drought stress as imposed at booting and anthesis , using pot - grown plants and controlled environment facilities . we selected ten sites for our study representing wheat growing regions in europe ( table 1 ) . wheat ideotypes were described by nine model parameters used in the sirius wheat model to describe wheat cultivars and considered as most promising for improvement of yield potential under climate change ( table 2 ) . we used an evolutionary algorithm to optimize ideotypes for future climatic conditions as predicted by the hadcm3 global climate model . the ranges were based on parameters calibrated by sirius for modern cultivars allowing for variations reported in the literature for existing wheat germplasm ( he et al . , 2012 ; semenov et al . , we assume that a 10% increase in light conversion efficiency could be achieved in the future . using a model of canopy photosynthesis , ( tambussi et al . , 2007 ) showed that the value of parameter ( rubisco specificity factor that represents the discrimination between co2 and o2 ) found in current c3 crops exceeds the level that would be optimal for the present co2 concentration ( [ co2 ] ) , but would be optimal for [ co2 ] of about 220 ppm , the average over the last 400,000 years . the simulation results showed that up to 10% more carbon could be assimilated , if was optimal for the current [ co2 ] level . in sirius , radiation use efficiency ( rue ) is proportional to [ co2 ] with an increase of 30% for doubling in [ co2 ] compared with the baseline of 338 ppm , which is in agreement with the recent meta - analysis of field - scale experiments on the effects of [ co2 ] on crops ( vanuytrecht et al . , 2012 ) . a similar response was used by other wheat simulation models , e.g. ceres ( jamieson et al . , 2000 ) and epic ( tubiello et al . , 2000 ) . three cultivar parameters are directly related to phenological development of wheat , i.e. phylochron ph , daylength response pp and duration of grain filling gf ( table 2 ) . modifying the duration and timing of crop growth cycle in relation to seasonal variations of solar radiation and water availability may have significant effects on yield ( akkaya et al . an optimal flowering time has been the single most important factor to maximise yield in dry environments ( richards , 1991 ) . the phyllochron ph is the thermal time required for the appearance of successive leaves , and is a major driver of phenological development ( jamieson et al . , 1995 , 2007 , 1998a ) . details of the response of final leaf number to daylength pp could be found in brooking et al . by modifying phyllochron ph and daylength response pp we alter the rate of crop development and , therefore , the date of flowering and maturity . increasing the duration of the grain filling period gf has been suggested as a possible trait for increasing grain yield in wheat ( evans and fischer , 1999 ) . in sirius , gf is defined as a cultivar - specific amount of thermal time which needs to be accumulated to complete grain filling ( jamieson et al . , 1998b ) . during grain filling , assimilates for the grain are available from two sources : new biomass produced from intercepted radiation and water - soluble carbohydrates stored mostly in the stem before anthesis . in sirius , the labile carbohydrate pool is calculated as a fixed 25% of biomass at anthesis , and is translocated to the grain during grain filling . increasing gf will increase the amount of radiation intercepted by the crop and , consequently , grain yield . however , in the model , water - soluble carbohydrates accumulated before anthesis are transferred into the grain at a rate inversely proportional to gf . therefore , any increase of gf will also reduce the rate of biomass remobilisation . under stress conditions , when grain growth could be terminated as a results of leaves dying early due to water or heat stress , grain yield could decrease not only because of the reduction in intercepted radiation but also because of the reduction in translocation of the labile carbohydrate pool to the grain ( brooks et al . two cultivar parameters to be optimised are related to canopy , i.e. maximum area of flag leaf layer a , and duration of leaf senescence s. by varying the maximum area of the flag leaf layer , we change the rate of canopy expansion and the maximum achievable leaf area index ( lai ) . this in turn will change the pattern of light interception and transpiration and , therefore , will affect crop growth and final grain yield . one of the strategies to increase grain yield is to extend duration of leaf senescence and maintain green leaf area longer after anthesis , the so called stay - green trait ( austin , 1999 ; silva et al . , 2000 ; triboi and triboi - blondel , 2002 ) . both daily biomass production ( photosynthesis ) and leaf senescence depend on the drought stress factor sf calculated daily as the ratio of actual to potential evapotranspiration . production of new daily biomass decreases proportionally to the drought biomass reduction factor wsa defined as wsa = sf . by varying , wsa can change significantly , particularly , for values of sf < 0.4 . in sirius , leaf senescence requires a cultivar - specific amount of thermal time which could be accelerated by nitrogen shortage to sustain grain filling or by water or temperature stresses . in the presence of drought stress , the rate of leaf senescence increases , because the daily increment of thermal time is modified by the drought leaf senescence factor . increasing tolerance to drought stress ( reducing wss ) will make leaves stay green longer under water stress and potentially increase grain yield . in sirius , the soil is represented by 5 cm layers and only a proportion of available soil water can be extracted from each layer by the plant on any single day . by default , plants can extract up to 10% of available soil water from the top layer at any single day and only ru ( % ) from the bottom layer at the maximum root depth . a faster water uptake reduces current stress experienced by the plant in anticipation of additional water coming in the form of precipitation or irrigation later in the season . in dry environments with a likely drought at the end of the growing season , a slower water uptake ( lower values for ru ) may achieve , on average , higher yields ( manschadi et al . , 2006 ) . an evolutionary search algorithm was incorporated in sirius 2010 , which allows optimisation of cultivar parameters for the best performance of wheat ideotypes in a target environment . sirius employs an evolutionary algorithm with self - adaptation ( ea - sa ) which is shown to be applicable for solving complex optimisation problems in a high - dimensional parameter space ( back , 1998 ; beyer , 1995 ; meyer - nieberg and beyer , 2007 ; schwefel and rudolph , 1995 ) . ea - sa was used in the past by the authors for calibration of cultivar parameters ( stratonovitch and semenov , 2010 ) . in the current study , ea - sa optimised cultivar parameters by randomly perturbing ( mutating ) their values and comparing ideotypes ' performance under climate change . at every step , 16 candidates ( new wheat ideotypes ) were generated from a parent by perturbing the parent 's cultivar parameters . for each of 16 new candidates , 100-year mean yield was calculated for a future climate scenario . the candidate with the highest 100-year mean yield was selected as a parent for the next step . general conditions of convergence of ea - sa are given in ( semenov and terkel , 2003 ) . the main advantage of ea - sa , compared with genetic algorithms , is that they do not require tuning control parameters during the search , where predefined heuristic rules are unavailable or difficult to formulate in a high - dimensional space with a complex optimisation function ( back , 1998 ; beyer , 1995 ; semenov and terkel , 1985 ) . in our study , we optimised wheat ideotypes at 10 european sites with contrasting climates , which represent wheat growing areas in europe ( table 1 ) . local - scale climate scenarios , named as 2050(a1b ) , were based on climate projections from the hadcm3 global climate model for the a1b emission scenario for 2050 ( meehl et al . , 2007 ) . one hundred years of site - specific daily weather were generated at each site by the lars - wg stochastic weather generator ( semenov et al . , 2010 ) . to eliminate the effect of site - specific soils from the analysis , a single soil , hafren , with available water capacity of 177 mm was used for all locations . the sowing dates and cultivars are given in table 1 and represent typical cultivars and sowing dates for selected sites . ideotypes with the coefficient of variation ( cv ) of yield exceeding 15% were excluded from the selection process . the yield increase in the past 50 years was largely a result of increase in harvest index ( hi ) . there are several estimations of theoretical maximum hi for wheat : ( austin et al . , 1980 ) estimated this value as 0.62 and more recent analysis ( foulkes et al . , 2011 ) suggested using 0.64 . during optimization the stopping rule for optimisation was : ( 1 ) no further improvement was possible ( the search found a local optimum , or ea - sa prematurely converged ) , or ( 2 ) the 95-percentile of yield ( y ) exceeds 20 t ha . all simulations were assumed to be water - limited , but no n limitation was considered . ea - sa is a local search algorithm which converges to one of the local maxima in a multi - dimension parameter space . to avoid convergence to a local maximum and to explore fully the parameter space parents. for each of 10 sites , we used 20 parents randomly scattered in the parameter space except for one parent which has the same initial cultivar parameters as a wheat cultivar currently grown in that region . for each of 20 parents , ea - sa converged to one of the local maxima or found a wheat ideotype with the the optimisation function computes 100-year mean yield with additional constraints on yield cv and hi . this is a complex function which will have significantly different responses ( sensitivity ) to variations in cultivar parameters . ea - sa will converge quickly to an optimal value of the most sensitive cultivar parameter ( or several parameters ) at the bottom of the valley , leaving other parameters in a state not fully optimised . this phenomenon is known as premature convergence ( back et al . , 2000 ) . to overcome premature convergence , we adopted the following procedure . when we observed convergence of a parameter ( or several parameters ) to a single value for most of 20 parents , we assumed that the optimal value for this parameter was found . we assigned this optimal value to a parameter and repeated optimisation for the remaining parameters . after one or two iterations ( depending on a site ) duration of grain filling gf , maximum area of flag leaf a and the stay - green parameter s converged to near - maximum values of 900 , 0.01 and 1.5 , respectively . ideotypes with a longer duration of grain filling gf can potentially produce higher grain yield if green leaf area is maintained during grain filling . ideotypes with maximum values of a and s intercept more solar radiation during the growing season because of earlier establishment of canopy at the beginning of the season and later senescence of leaves at the end of the season . after parameters gf , a and s were fixed to their optimal values , convergence was observed for two more parameters , phyllochron ph and daylength response pp ( fig . 1 ) . both of these parameters control wheat phenology including flowering date and were responsible for shifting grain filling to the most favourable part of the season , maximising intercepted solar radiation and minimising the effect of water limitation on grain yield . parameters related to water - stress , i.e. maximum acceleration of leaf senescence wss and response of photosynthesis to water stress wsa , showed convergence only at those sites where water - limitation could have significant effect on grain yield , e.g. in sl . root water uptake ru did not converge at any of the european sites , because there is no an optimal strategy of extracting soil available water during the growing season . 1 shows normalised values of cultivar parameters for the best ideotype at each of 10 sites optimised for the 2050(a1b ) climate scenario . for each site , we selected the best performing ideotype out of 20 candidates and compared it with the current wheat cultivar for the 2050(a1b ) climate scenario . maturity date for all optimized ideotypes was later than for current wheat cultivars by about 19 days on average with maximum of 34 days later at tr ( fig . the grain filling period for all ideotypes was also longer by about two weeks on average with a maximum of 3 weeks at ma . due to a longer grain filling period 2c , d shows simulated mean grain yields with the 95-percentiles for current cultivars ( 2c ) and ideotypes ( 2d ) . grain yields for ideotypes were 78% higher on average with a maximum of 109% yield increase at cf . because of longer grain filling , mean harvest index ( hi ) across all sites for ideotypes was 0.56 , which was on average 15% higher than hi simulated for current wheat cultivar ( fig . 2c , d ) . 95-percentiles of hi have not exceeded 0.60 , which is below a theoretical maximum of 0.620.64 suggested in austin et al . ( 2011 ) . by tailoring phenology to future weather patterns and improving at some sites cultivar parameters responsible for drought tolerance , i.e. wsa and wss , ideotypes were able to reduce the overall effects of water stress on grain yield ( fig . , we computed a drought stress index , dsi , which is defined as a relative proportion of the yield lost due to water stress , i.e. dsi = ( yp ywl)/yp , where ywl and yp are water - limited and potential grain yields . 3 presents 95-percentiles of dsi ( dsi95 ) for the current cultivars ( 3a ) and future ideotypes ( 3b ) . dsi95 is a yield loss due to water stress , which could be expected to occur once every 20 years on average . dsi95 was on average 52% lower for ideotypes compared with current wheat cultivars . however , soil water deficit at anthesis was on average higher for ideotypes , except for 3 sites ( ed , rr and ma ) ( fig . the preceding analysis does not account for effects of drought and excessive heat at growth stages with particular sensitivity to stress . the most sensitive stages for grain set are associated with the processes of microsporogenesis , anther dehiscence and fertilization ( as reviewed by barnabas et al . , 2008 ; craufurd et al . , 2013 ) , and just after fertilization for grain size ( gooding et al . , 2003 ) . the model presented here assumes that if the crop is tolerant to drought and excessive heat around anthesis , it establishes sufficient grain numbers of adequate potential size to accommodate biomass produced during grain filling . however , the grain number can be substantially reduced if wheat is exposed to a short period of stress around meiosis , sometimes assumed to be concurrent with booting ( saini and aspinall , 1982 ; westgate et al . , 1996 ) , and during flowering ( wheeler et al . , 1996 ; ferris et al . , 1998 ) . reductions in grain numbers or potential grain size limits the capacity of grains to store newly produced biomass . the omission of these effects contributes to an apparent inconsistency in fig . 2 , i.e. current wheat cultivars in warmer , lower latitudes flower and mature earlier ( fig . 2a ) , but the prediction for higher yielding ideotypes in a future warmer climate is that flowering and maturation should occur later ( fig . 2b ) . the earlier maturation of wheat in warmer areas is thought , in part , to be necessary to avoid heat stress events at booting and anthesis ( worland et al . , 1998 ) . more needs to be known , therefore , about the response of wheat yield to stress events at meiosis and anthesis to improve accuracy of crop models , and to identify potential for improving drought and heat stress tolerance in wheat . 48 are results from three successive complete factorial replicated pot experiments ( table 3 ) conducted at the plant environment laboratory , university of reading , uk ( 51 27 n latitude , 00 56 w longitude ) , from 2010 to 2012 , to compare responses of wheat for susceptibility to heat and drought stress during booting and anthesis . further details are available ( alghabari , 2013 ; alghabari et al . , 2014 ) , but in summary , factors included genotype ( elite and near - isogenic lines of winter wheat varying for reduced height ( rht ) alleles ) , high temperature stress ( ranging from 20 c to 40 c ) , timing of stress ( booting or anthesis ) and irrigation ( withholding water during heat stress or irrigating to field capacity ( fc ) ) . all experiments used plastic pots ( 180 mm diameter ; 4 l volume ) containing 2:1:2:0.5 of vermiculite : sand : gravel : compost mixed with osmocote slow release granules ( 2 kg m ) containing a ratio of 15 : 11 : 13:2 of n : p2o5 : k2o : mgo . weight of growing media per pot was 2.60 kg and 3.05 kg at 0 and 100% fc respectively . heat stress treatments comprised transferring pots to matched 1.37 1.47 m saxil growth cabinets at 15:30 h gmt for three 16 h day ( 700 mm photon m s ; 70 2% relative humidity ; 350360 mol co2 mol air ) , 8 h night cycles ( 8 c below day temperature ) before returning to the original , completely randomized , position outside . the approach was similar to that of saini and aspinall ( 1982 ) in that stress was applied for three days in an attempt to detect effects of tolerance , rather than escape due to variations in growth stage within spikes ( lukac et al . , 2012 ) . stems in a pot were scored and tagged for their precise growth stage ( gs , zadoks et al . , 1974 ) when the pot was transferred . ears tagged at different gs when transferred to the cabinets were harvested and assessed from each pot separately . numbers of ears and spikelets were counted before the ears were threshed , cleaned and counted by hand . fitted logistic responses ( y = c/1+e ) described declining grain yield and grains per spikelet with increasing t , and t5 was that fitted to give a 5% reduction in grain set 4 ) reduced grain yield , principally by reducing grain numbers per spikelet ( fig . 4d ) . partial compensation through increased mean grain weight was slight and variable ( fig . critical temperatures ( t5 ) at booting for grains per spikelet are fitted ( fig . 4 ) at 32.4 c and 24.5 c for irrigated and non - irrigated situations respectively . at anthesis , it appears that grain set is less susceptible to drought than at booting ( fig . 5d ; saini and aspinall , 1981 ) : critical temperatures fitted at 31.7 c for the irrigated , and 29.9 c for the non - irrigated condition . a threshold of around 30 c is broadly consistent for the critical temperature of grain set at anthesis across a number of environments and curve fitting procedures . in an experiment on the combined effects of co2 and temperature on grain yield , mitchell et al . ( 1993 ) observed that a temperature of 27 c or higher applied mid - way through anthesis could result in a high number of sterile grains and considerable yield losses . ( 1996 ) used a temperature gradient tunnel system on field - grown wheat to demonstrate that temperatures of 30 c or higher shortly prior to anthesis was significantly associated with reduced grain number and , subsequently , yield of cv . in contrast to effects at booting , drought and heat stress during flowering can reduce mean grain weight as well as grain numbers ( fig . tashiro and wardlaw , 1989 ) , plants were transferred into controlled rooms with high temperatures 7 days after the first anthers appeared , showing that a temperature of 27 c and above could substantially reduce the maximum grain size of several australian wheat cultivars , resulting in yield losses . it appears that the transition from a stress mainly influencing grain numbers to one which mostly affects grain size , occurs over a very narrow range of growth stages ( fig . 6 ) . the earliest flowers on ears assessed as having just completed anthesis ( gs 69 ) may have been fertilized four or five days earlier ( lukac , personal communication ) , and hence beyond the vulnerable growth stage ( saini and aspinall , 1982 ; stone and nicolas , 1995a , b , c ) for grain set . hence , at gs 69 it is still possible to have 1.5 grains per spikelet at temperatures as high as 40 c under irrigated conditions . such grains are , however , significantly reduced in final mean grain weight ( fig . there is also a large effect of drought shortly after anthesis on final mean grain weight even when subsequent water availability is high before the end of grain growth ( gooding et al . , 2003 ) . to assess the risk of heat stress around flowering for the 2050 ( a1b ) climate scenario , we computed the probability of maximum temperature to exceed 30 c at anthesis ( which could affect the grain number ) and the probability of maximum temperature to exceed 30 c 5 days after anthesis ( which could affect the grain size ) . both probabilities are presented at fig . 3 for the current cultivars ( 3c ) and ideotypes ( 3d ) . semenov and shewry ( 2011 ) demonstrated in a modelling study that the risk of heat stress around flowering is predicted to increase in future for modern wheat cultivars in europe and advocated to focus breeding efforts on developing heat tolerant wheat varieties to avoid a decrease in wheat yield . the risk of heat stress around anthesis was relatively high for ideotypes , between 0.1 and 0.3 , ( fig . this means that in order to achieve the high yield potential predicted for optimized ideotypes , the heat tolerance trait needs to be incorporated in ideotypes and breeding for heat tolerance will remain a priority . drought , with or without heat , can increase spike and floret concentrations of abscisic acid ( aba ) , which can be related closely with poor grain set ( westgate et al . , 1996 ; weldearegay et al . , additionally , heat and drought can reduce photosynthesis , and the subsequent dilution of sucrose in the ear can be associated with floret abortion ( as reviewed by barnabas et al . ( furthermore , temperatures above 30 c during meiosis can interfere with division and lead to abnormal pollen development ( saini et al . , 1984 ) . heat shock proteins ( hsps ) appear to offer some protection for mature and developing pollen grains against heat stress , when hsps are induced by high temperatures . however , young et al . ( 2001 ) suggest that quantities of hsps , so induced , are often insufficient to offer full protection ; and that the degree of insufficiency correlates with poor thermotolerance of pollen and reduced grain set . ( 2011 ) as potential breeding and pre - breeding targets for heat stress tolerance . what is clear is that tolerance to heat stress is greatly modified by water availability . higher temperatures increase water deficits whilst drought reduces capacity for evaporative cooling , and hence lessens possible protection from high air temperatures ( reynolds and trethowan , 2007 ; craufurd et al . , 2013 ; steinmeyer et al . , this latter point is likely to modify the difference between dsi and swd for future wheat ideotypes illustrated in fig . greater water availability to the above ground crop may be achieved by selecting lines with greater investment in roots at depth compared to the surface layers ( ford et al . , 2006 ; wasson et al . , a further challenge for relating the results from the controlled environment work to effects likely in the field is the difficulty in simulating the responses to the more gradual development of a drought down through the soil profile that would likely occur in the field , compared to the more dramatic on / off availability of water imposed within the confines of a pot . despite the suggestions that heat stress tolerance could be improved , it is somewhat disappointing that wheats grown in southern europe are not necessarily more tolerant of stresses than wheats grown in the more temperate conditions of north western europe ( fig . currently much of the adaptation to regions that are prone to summer heat and drought is through escape , e.g. alleles such as those conferring photoperiod insensitivity , and agronomic practices such as sowing date are used to ensure booting and flowering occur before heat and drought is excessive ( worland et al . , 1998 ; the plethora of genes and alleles influencing developmental rate ( vrn-1 , vernalisation ; ppd-1 , photoperiodism ; eps , earliness per se ) , their interaction , and varying sensitivities at different growth stages ( snape et al . , 2001 ) has probably meant that adaptation through escape has been much more easy to achieve than adaptation through tolerance . furthermore , relatively minor variations in growth stage amongst genotypes can lead to large variations in responses to stress ( fig . further , wheat plants have long been known to flower throughout the day , from at least 5:00 h in the morning ( percival , 1921 ) , so differences between genotypes in responses to stress are likely to arise through alterations in the diurnal distribution of flowering . one aspect of escape that has received little attention to date is the extent to which duration of meiosis or flowering within and/or between ears might be related to yield stability , e.g. extending the period over which a crop flowered would mean that a damaging spike in temperature would disrupt the fertilization of a smaller proportion of florets ( lukac et al . , 2012 ) . other modelling studies have demonstrated the importance of flowering duration for estimating the impact of brief periods of high temperature on crop yield ( challinor et al . , 2005 ) but more detailed investigation and parameter estimation would be required to assess this in a highly determinate and synchronised crop such as wheat . a key factor to increase wheat yield potential is an extended duration of grain filling resulting in increased hi . this can only be possible if both capacities of the sink and the source are increased . the source capacity can be increased if the plant would be able to maintain healthy green area index until the end of grain filling ( stay - green ) . in water - limited environments such as sl , improvement in drought tolerance which delays the sink capacity can be increased if the number of fertile florets at anthesis and , as a result , the number of grains at maturity increased . the floret survival rate in most wheat cultivars varies between 25% and 40% ; so , in principal , there is a large potential for improvement ( gonzalez et al . , 2011 ) . ( 2013 ) studied the dynamics of floret development and its consequence on grain number and final yield in several semi - dwarf durum wheat cultivars in response to nitrogen ( n ) treatments and water availability . the study confirmed that increasing n availability resulted in an increased number of grains through increasing both the number of fertile florets and the percentage of them setting grains . it was shown that the fate of floret primordia in the intermediate positions of the spikelet ( f3f5 ) was related to the increase in assimilate supply during spike growth affected by n fertilization . we did not consider n limitation in our simulation , assuming plentiful supply of n. however , post - anthesis n uptake and redistribution could be a serious constraint in achieving greater yield potential . grain demand for n during grain filling is satisfied from three sources ( jamieson and semenov , 2000 ) . the first is excess of n in the stem including n released by natural leaf senescence . should these combined sources be insufficient , then n is remobilised from leaves reducing their photosynthetic capacity and accelerating leaf senescence ( killing leaves ) . as a result , grain filling duration can be shortened and grain yield potential can be reduced . one of the strategies to prevent this from happening is to increase the capacity to store n in non - photosynthetic organs , such as internodes , that allows the translocation of n to grains without reducing wheat photosynthetic capacity ( bancal , 2009 ; bertheloot et al . , 2008 ; dreccer et al . , 1998 ; martre et al . , 2007 ) . another strategy would be to improve n uptake from the soil in the post - anthesis period . however , the ability of roots to take up n could decline during grain filling ( andersson et al . , 2004 ; martre et al . 1995 ) . moreover , if the end of grain filling coincides with low water availability ( a typical situation in sl ) , then soil n available for uptake could be substantially reduced due to water shortage ( semenov et al . , 2007 ) . a significant requirement is for a better understanding of how higher temperatures and drought stresses , particularly during the booting and flowering periods constrain sink size , either through reducing grain numbers and/or potential grain weight ( lizana and calderini , 2013 ) . breeding for improved stress tolerance , particularly during meiosis and anthesis , remains a significant challenge . it would appear that much of the current adaptation of wheat to hotter and drier environments has involved tolerance. some escape strategies such as extending the duration , or modifying the diurnal pattern of susceptible stages may provide some improved stability of yield against the challenge of increased frequency of extreme events . additionally improved water availability through , for example , more efficient root systems and architectures may provide some protection against excessive temperatures . however , maintaining or increasing resource capture and partitioning in ways predicted to be optimal by sirius for a climate change scenario requires the development of heat tolerant varieties .

increasing cereal yield is needed to meet the projected increased demand for world food supply of about 70% by 2050 . sirius , a process - based model for wheat , was used to estimate yield potential for wheat ideotypes optimized for future climatic projections for ten wheat growing areas of europe . it was predicted that the detrimental effect of drought stress on yield would be decreased due to enhanced tailoring of phenology to future weather patterns , and due to genetic improvements in the response of photosynthesis and green leaf duration to water shortage . yield advances could be made through extending maturation and thereby improve resource capture and partitioning . however the model predicted an increase in frequency of heat stress at meiosis and anthesis . controlled environment experiments quantify the effects of heat and drought at booting and flowering on grain numbers and potential grain size . a current adaptation of wheat to areas of europe with hotter and drier summers is a quicker maturation which helps to escape from excessive stress , but results in lower yields . to increase yield potential and to respond to climate change , increased tolerance to heat and drought stress should remain priorities for the genetic improvement of wheat .

Introduction Assessing yield potential of future wheat ideotypes The impact of heat and drought stress on grain set and potential grain size Concluding remarks

food security has become a major challenge given the projected need to increase world food supply by about 70% by 2050 ( anon . considering the limitations on expanding crop - growing areas , a significant increase in crop productivity will be required to achieve this target ( parry et al . global warming is characterised by shifts in weather patterns and increase in frequency and magnitude of extreme events ( lobell et al . climate change , therefore , represents a considerable challenge in achieving the 70%-increase target in world food production . however , the intrinsic uncertainty of climate change predictions poses a challenge to plant breeders and crop scientists who have limited time and resources and must select the most appropriate traits for improvement ( foulkes et al . modelling provides a rational framework to design and test in silico new wheat ideotypes optimised for target environments and future climatic conditions ( hammer et al . , 2012 ; eco - physiological process - based crop models are commonly used in basic and applied research in the plant sciences and in natural resource management ( hammer et al . the objective of our study was to assess wheat yield potential under climate change in europe and identify challenges which must be overcome to achieve high wheat yields in the future . firstly , we used the sirius wheat model to optimise wheat ideotypes for future climate scenarios ( jamieson and semenov , 2000 ; lawless et al . sirius is a well validated model and was able to simulate accurately wheat growth and grain yield in a wide range of environments , including europe , usa , new zealand and australia , and for experiments reproducing conditions of climate change , e.g. , 2000 ; it remains a challenge for such models to capture the yield response of wheat to extreme events , particularly when they coincide with sensitive growth stages ( craufurd et al . for example , it has been established that wheat yield is particularly sensitive to abiotic stresses during microsporogenesis , anther dehiscence and fertilization because of effects on grain set ( as reviewed by barnabas et al . 2013 ) ; and just after fertilization because of effects on grain size ( gooding et al . the second approach presented here is , therefore , to describe the response of wheat to heat and drought stress as imposed at booting and anthesis , using pot - grown plants and controlled environment facilities . we selected ten sites for our study representing wheat growing regions in europe ( table 1 ) . wheat ideotypes were described by nine model parameters used in the sirius wheat model to describe wheat cultivars and considered as most promising for improvement of yield potential under climate change ( table 2 ) . we used an evolutionary algorithm to optimize ideotypes for future climatic conditions as predicted by the hadcm3 global climate model . , we assume that a 10% increase in light conversion efficiency could be achieved in the future . , 2007 ) showed that the value of parameter ( rubisco specificity factor that represents the discrimination between co2 and o2 ) found in current c3 crops exceeds the level that would be optimal for the present co2 concentration ( [ co2 ] ) , but would be optimal for [ co2 ] of about 220 ppm , the average over the last 400,000 years . the simulation results showed that up to 10% more carbon could be assimilated , if was optimal for the current [ co2 ] level . in sirius , radiation use efficiency ( rue ) is proportional to [ co2 ] with an increase of 30% for doubling in [ co2 ] compared with the baseline of 338 ppm , which is in agreement with the recent meta - analysis of field - scale experiments on the effects of [ co2 ] on crops ( vanuytrecht et al . three cultivar parameters are directly related to phenological development of wheat , i.e. modifying the duration and timing of crop growth cycle in relation to seasonal variations of solar radiation and water availability may have significant effects on yield ( akkaya et al . the phyllochron ph is the thermal time required for the appearance of successive leaves , and is a major driver of phenological development ( jamieson et al . details of the response of final leaf number to daylength pp could be found in brooking et al . during grain filling , assimilates for the grain are available from two sources : new biomass produced from intercepted radiation and water - soluble carbohydrates stored mostly in the stem before anthesis . in sirius , the labile carbohydrate pool is calculated as a fixed 25% of biomass at anthesis , and is translocated to the grain during grain filling . however , in the model , water - soluble carbohydrates accumulated before anthesis are transferred into the grain at a rate inversely proportional to gf . under stress conditions , when grain growth could be terminated as a results of leaves dying early due to water or heat stress , grain yield could decrease not only because of the reduction in intercepted radiation but also because of the reduction in translocation of the labile carbohydrate pool to the grain ( brooks et al . maximum area of flag leaf layer a , and duration of leaf senescence s. by varying the maximum area of the flag leaf layer , we change the rate of canopy expansion and the maximum achievable leaf area index ( lai ) . one of the strategies to increase grain yield is to extend duration of leaf senescence and maintain green leaf area longer after anthesis , the so called stay - green trait ( austin , 1999 ; silva et al . both daily biomass production ( photosynthesis ) and leaf senescence depend on the drought stress factor sf calculated daily as the ratio of actual to potential evapotranspiration . in sirius , leaf senescence requires a cultivar - specific amount of thermal time which could be accelerated by nitrogen shortage to sustain grain filling or by water or temperature stresses . in the presence of drought stress , the rate of leaf senescence increases , because the daily increment of thermal time is modified by the drought leaf senescence factor . increasing tolerance to drought stress ( reducing wss ) will make leaves stay green longer under water stress and potentially increase grain yield . in dry environments with a likely drought at the end of the growing season , a slower water uptake ( lower values for ru ) may achieve , on average , higher yields ( manschadi et al . an evolutionary search algorithm was incorporated in sirius 2010 , which allows optimisation of cultivar parameters for the best performance of wheat ideotypes in a target environment . ea - sa was used in the past by the authors for calibration of cultivar parameters ( stratonovitch and semenov , 2010 ) . in the current study , ea - sa optimised cultivar parameters by randomly perturbing ( mutating ) their values and comparing ideotypes ' performance under climate change . the candidate with the highest 100-year mean yield was selected as a parent for the next step . in our study , we optimised wheat ideotypes at 10 european sites with contrasting climates , which represent wheat growing areas in europe ( table 1 ) . local - scale climate scenarios , named as 2050(a1b ) , were based on climate projections from the hadcm3 global climate model for the a1b emission scenario for 2050 ( meehl et al . to eliminate the effect of site - specific soils from the analysis , a single soil , hafren , with available water capacity of 177 mm was used for all locations . the yield increase in the past 50 years was largely a result of increase in harvest index ( hi ) . all simulations were assumed to be water - limited , but no n limitation was considered . ea - sa is a local search algorithm which converges to one of the local maxima in a multi - dimension parameter space . to avoid convergence to a local maximum and to explore fully the parameter space parents. for each of 10 sites , we used 20 parents randomly scattered in the parameter space except for one parent which has the same initial cultivar parameters as a wheat cultivar currently grown in that region . for each of 20 parents , ea - sa converged to one of the local maxima or found a wheat ideotype with the the optimisation function computes 100-year mean yield with additional constraints on yield cv and hi . this is a complex function which will have significantly different responses ( sensitivity ) to variations in cultivar parameters . we assigned this optimal value to a parameter and repeated optimisation for the remaining parameters . ideotypes with a longer duration of grain filling gf can potentially produce higher grain yield if green leaf area is maintained during grain filling . after parameters gf , a and s were fixed to their optimal values , convergence was observed for two more parameters , phyllochron ph and daylength response pp ( fig . both of these parameters control wheat phenology including flowering date and were responsible for shifting grain filling to the most favourable part of the season , maximising intercepted solar radiation and minimising the effect of water limitation on grain yield . parameters related to water - stress , i.e. maximum acceleration of leaf senescence wss and response of photosynthesis to water stress wsa , showed convergence only at those sites where water - limitation could have significant effect on grain yield , e.g. 1 shows normalised values of cultivar parameters for the best ideotype at each of 10 sites optimised for the 2050(a1b ) climate scenario . for each site , we selected the best performing ideotype out of 20 candidates and compared it with the current wheat cultivar for the 2050(a1b ) climate scenario . by tailoring phenology to future weather patterns and improving at some sites cultivar parameters responsible for drought tolerance , i.e. wsa and wss , ideotypes were able to reduce the overall effects of water stress on grain yield ( fig . , we computed a drought stress index , dsi , which is defined as a relative proportion of the yield lost due to water stress , i.e. dsi = ( yp ywl)/yp , where ywl and yp are water - limited and potential grain yields . dsi95 is a yield loss due to water stress , which could be expected to occur once every 20 years on average . the preceding analysis does not account for effects of drought and excessive heat at growth stages with particular sensitivity to stress . , 2013 ) , and just after fertilization for grain size ( gooding et al . the model presented here assumes that if the crop is tolerant to drought and excessive heat around anthesis , it establishes sufficient grain numbers of adequate potential size to accommodate biomass produced during grain filling . , 1996 ) , and during flowering ( wheeler et al . reductions in grain numbers or potential grain size limits the capacity of grains to store newly produced biomass . the earlier maturation of wheat in warmer areas is thought , in part , to be necessary to avoid heat stress events at booting and anthesis ( worland et al . more needs to be known , therefore , about the response of wheat yield to stress events at meiosis and anthesis to improve accuracy of crop models , and to identify potential for improving drought and heat stress tolerance in wheat . 48 are results from three successive complete factorial replicated pot experiments ( table 3 ) conducted at the plant environment laboratory , university of reading , uk ( 51 27 n latitude , 00 56 w longitude ) , from 2010 to 2012 , to compare responses of wheat for susceptibility to heat and drought stress during booting and anthesis . , 2014 ) , but in summary , factors included genotype ( elite and near - isogenic lines of winter wheat varying for reduced height ( rht ) alleles ) , high temperature stress ( ranging from 20 c to 40 c ) , timing of stress ( booting or anthesis ) and irrigation ( withholding water during heat stress or irrigating to field capacity ( fc ) ) . the approach was similar to that of saini and aspinall ( 1982 ) in that stress was applied for three days in an attempt to detect effects of tolerance , rather than escape due to variations in growth stage within spikes ( lukac et al . fitted logistic responses ( y = c/1+e ) described declining grain yield and grains per spikelet with increasing t , and t5 was that fitted to give a 5% reduction in grain set 4 ) reduced grain yield , principally by reducing grain numbers per spikelet ( fig . critical temperatures ( t5 ) at booting for grains per spikelet are fitted ( fig . at anthesis , it appears that grain set is less susceptible to drought than at booting ( fig . 5d ; saini and aspinall , 1981 ) : critical temperatures fitted at 31.7 c for the irrigated , and 29.9 c for the non - irrigated condition . a threshold of around 30 c is broadly consistent for the critical temperature of grain set at anthesis across a number of environments and curve fitting procedures . in an experiment on the combined effects of co2 and temperature on grain yield , mitchell et al . ( 1996 ) used a temperature gradient tunnel system on field - grown wheat to demonstrate that temperatures of 30 c or higher shortly prior to anthesis was significantly associated with reduced grain number and , subsequently , yield of cv . in contrast to effects at booting , drought and heat stress during flowering can reduce mean grain weight as well as grain numbers ( fig . it appears that the transition from a stress mainly influencing grain numbers to one which mostly affects grain size , occurs over a very narrow range of growth stages ( fig . the earliest flowers on ears assessed as having just completed anthesis ( gs 69 ) may have been fertilized four or five days earlier ( lukac , personal communication ) , and hence beyond the vulnerable growth stage ( saini and aspinall , 1982 ; stone and nicolas , 1995a , b , c ) for grain set . there is also a large effect of drought shortly after anthesis on final mean grain weight even when subsequent water availability is high before the end of grain growth ( gooding et al . to assess the risk of heat stress around flowering for the 2050 ( a1b ) climate scenario , we computed the probability of maximum temperature to exceed 30 c at anthesis ( which could affect the grain number ) and the probability of maximum temperature to exceed 30 c 5 days after anthesis ( which could affect the grain size ) . 3 for the current cultivars ( 3c ) and ideotypes ( 3d ) . semenov and shewry ( 2011 ) demonstrated in a modelling study that the risk of heat stress around flowering is predicted to increase in future for modern wheat cultivars in europe and advocated to focus breeding efforts on developing heat tolerant wheat varieties to avoid a decrease in wheat yield . the risk of heat stress around anthesis was relatively high for ideotypes , between 0.1 and 0.3 , ( fig . this means that in order to achieve the high yield potential predicted for optimized ideotypes , the heat tolerance trait needs to be incorporated in ideotypes and breeding for heat tolerance will remain a priority . , additionally , heat and drought can reduce photosynthesis , and the subsequent dilution of sucrose in the ear can be associated with floret abortion ( as reviewed by barnabas et al . heat shock proteins ( hsps ) appear to offer some protection for mature and developing pollen grains against heat stress , when hsps are induced by high temperatures . ( 2001 ) suggest that quantities of hsps , so induced , are often insufficient to offer full protection ; and that the degree of insufficiency correlates with poor thermotolerance of pollen and reduced grain set . ( 2011 ) as potential breeding and pre - breeding targets for heat stress tolerance . what is clear is that tolerance to heat stress is greatly modified by water availability . , this latter point is likely to modify the difference between dsi and swd for future wheat ideotypes illustrated in fig . , a further challenge for relating the results from the controlled environment work to effects likely in the field is the difficulty in simulating the responses to the more gradual development of a drought down through the soil profile that would likely occur in the field , compared to the more dramatic on / off availability of water imposed within the confines of a pot . despite the suggestions that heat stress tolerance could be improved , it is somewhat disappointing that wheats grown in southern europe are not necessarily more tolerant of stresses than wheats grown in the more temperate conditions of north western europe ( fig . currently much of the adaptation to regions that are prone to summer heat and drought is through escape , e.g. alleles such as those conferring photoperiod insensitivity , and agronomic practices such as sowing date are used to ensure booting and flowering occur before heat and drought is excessive ( worland et al . further , wheat plants have long been known to flower throughout the day , from at least 5:00 h in the morning ( percival , 1921 ) , so differences between genotypes in responses to stress are likely to arise through alterations in the diurnal distribution of flowering . , 2005 ) but more detailed investigation and parameter estimation would be required to assess this in a highly determinate and synchronised crop such as wheat . a key factor to increase wheat yield potential is an extended duration of grain filling resulting in increased hi . the source capacity can be increased if the plant would be able to maintain healthy green area index until the end of grain filling ( stay - green ) . the floret survival rate in most wheat cultivars varies between 25% and 40% ; so , in principal , there is a large potential for improvement ( gonzalez et al . ( 2013 ) studied the dynamics of floret development and its consequence on grain number and final yield in several semi - dwarf durum wheat cultivars in response to nitrogen ( n ) treatments and water availability . it was shown that the fate of floret primordia in the intermediate positions of the spikelet ( f3f5 ) was related to the increase in assimilate supply during spike growth affected by n fertilization . we did not consider n limitation in our simulation , assuming plentiful supply of n. however , post - anthesis n uptake and redistribution could be a serious constraint in achieving greater yield potential . grain demand for n during grain filling is satisfied from three sources ( jamieson and semenov , 2000 ) . as a result , grain filling duration can be shortened and grain yield potential can be reduced . one of the strategies to prevent this from happening is to increase the capacity to store n in non - photosynthetic organs , such as internodes , that allows the translocation of n to grains without reducing wheat photosynthetic capacity ( bancal , 2009 ; bertheloot et al . another strategy would be to improve n uptake from the soil in the post - anthesis period . moreover , if the end of grain filling coincides with low water availability ( a typical situation in sl ) , then soil n available for uptake could be substantially reduced due to water shortage ( semenov et al . a significant requirement is for a better understanding of how higher temperatures and drought stresses , particularly during the booting and flowering periods constrain sink size , either through reducing grain numbers and/or potential grain weight ( lizana and calderini , 2013 ) . breeding for improved stress tolerance , particularly during meiosis and anthesis , remains a significant challenge . it would appear that much of the current adaptation of wheat to hotter and drier environments has involved tolerance. some escape strategies such as extending the duration , or modifying the diurnal pattern of susceptible stages may provide some improved stability of yield against the challenge of increased frequency of extreme events . however , maintaining or increasing resource capture and partitioning in ways predicted to be optimal by sirius for a climate change scenario requires the development of heat tolerant varieties .

the superior cervical ganglion ( scg ) is the center of sympathetic innervation of all head and neck organs . it provides sympathetic input to numerous key organs and structures such as the salivary glands , pineal gland , thyroid , carotid body , choroid plexus , brain and cranial muscle vasculature , eyes , and lacrimal gland , thus playing a vital role in a number of neuroendocrine , visceral , or vision - related processes ( cardinali et al . studies showed that scg is involved in circadian rhythm regulation ( lingappa and zigmond 2013 ) , thyroid function modulation ( young et al . 2005 ) , tear production ( ding et al . 2003 ; tangkrisanavinont 1984 ) , skin vasoconstriction ( gibbins et al . 1998 ) , and cerebral arterial supply regulation ( arbab et al . 1986 ; cassaglia et al . 1983 ) , a complex neurochemical phenotype of its preganglionic neurons and fibers has yet to be completed . in animal models studied so far , sympathetic preganglionic neurons ( spn ) supplying the scg were found in cervico - thoracic neuromeres , which were confined to the following nuclei : ( 1 ) nucleus intermediolateralis pars principalis ( imlpp ) , ( 2 ) nucleus intermediolateralis pars funicularis ( imlpf ) , ( 3 ) nucleus intercalatus spinalis ( ic ) , and ( 4 ) nucleus intercalatus spinalis pars paraependymalis ( icpe ) ( dalsgaard and elfvin 1979 ; petras and faden 1978 ; yau et al . 1991 ) ; however , until now there is no data clarifying the origins of porcine scg innervation . studies conducted in laboratory animals show that the chemical phenotype of these neurons is very diverse and complex , underscoring the importance of the ganglion in autonomic regulation . acetylcholine ( ach ) is the main spn neurotransmitter ; however , studies show that its actions upon scg activity are largely supported by nitric oxide ( no ) present in preganglionic neurons alongside ach ( okamura et al . it has been shown that no modulates cyclic gmp synthesis in cholinergic neurons , affecting ach release from these neurons and thus regulating overall ach activity ( prast et al . furthermore , data obtained from human and small animal laboratory species reveal that aside from ach and no , a number of other neuromodulatory substances such as serotonin ( jensen et al . 1995 ) , leu - enkephalin ( klimaschewski et al . 1995 ) , pituitary adenylate cyclase - activating polypeptide ( pacap ) ( beaudet et al . 1998 ) , neuropeptide ( npy ) , calcitonin gene - related peptide ( cgrp ) ( yamamoto et al . 1989 ) , substance p ( sp ) ( klimaschewski et al . 1995 ; llewellyn - smith et al . 1996 ) , and vasoactive intestinal peptide ( vip ) ( sasek et al . 1991 ) are present in the scg - innervating preganglionic neurons , likely contributing to the scg neuroendocrine function . here in the present study , we investigated the distribution and neurochemical coding of scg supplying neurons in the pig , an animal model physiologically and anatomically similar to humans . in our previous study , we showed a complete 2d reconstruction of porcine scg and provided neurochemical characteristics of scg neurons supplying the porcine parotid gland ( wojtkiewicz et al . in this project , we have advanced in our research , paving the way to the source of scg innervation and providing a detailed overview of scg preganglionic neuron distribution and their complex neurochemical phenotype . six 8-week - old female pigs of the large white polish breed , weighing ca . the animals were housed in accordance with the principles of laboratory animal care ( nih publication no . 86 - 23 , revised 1985 ) and approved by the ethical commission of veterinary medicine faculty at the university of warmia and mazury , olsztyn , poland ( resolution no . the animals were preanesthetized with propionyl promazine ( 0.4 mg / kg b / w , i.m . ) and deeply anesthetized with sodium barbital ( 25 mg / kg b / w ; i.v . ) . the left scg , exposed via midline neck incision , was injected with retrograde tracer , fast blue ( fb , ems , grivory , deutschland , gmbh , postfach ) at multiple sites along the scg . injections were given with a hamilton microsyringe at a total volume of 20 l 5 % dye solution ( 1 l dye solution per injection ) . after a 3-week survival time , animals were re - anesthetized and sacrificed by an overdose of pentobarbital . following sacrifice , animals were perfused transcardially with heparinized physiological saline in 0.1 phosphate buffer ( pb , ph 7.4 ) and fixative solution containing 4 % paraformaldehyde 0.1 m pb ( ph 7.4 ) . immediately after perfusion , spinal cord c5th8 segments were exposed by laminectomy and dissected . the segments were immersed in the same fixative for 20 min and stored in 18 % phosphate - buffered sucrose solution for further processing . the spinal cord segments were sectioned transversally ( n = 3 ) and longitudinally ( n = 3 ) on the cryostat at 10 m in thickness , mounted on glass slides , and stored at 70 c . briefly , 10-m - thick cryostat sections were air - dried at rt for 45 min and rinsed ( 3 15 min ) with phosphate - buffered saline ( pbs , ph 7.4 ) . afterwards , the samples were incubated with a blocking mixture containing 1 % triton x-100 ( sigma - aldrich , usa ) , 0.1 % bovine serum albumin ( sigma - aldrich , usa ) , 0.05 % thimerosal ( sigma - aldrich , usa ) , 0.01 % nan3 , and 10 % of normal goat serum ( mp biomedicals , usa ) in 0.01 m phosphate - buffered saline for 1 h at rt , rinsed in pbs ( 3 15 min ) , and incubated overnight with the following primary antibodies : mouse anti - nitric oxide synthase ( nos ) , co - stained with an array of rabbit antisera against dynorphin a ( dyn a ) , leu-5 enkephalin ( lenk ) , neuropeptide y ( npy ) , -neoendorphin ( neo ) , calbindin d-28 k ( cb - d28k ) , calretinin ( crt ) , galanin ( gal ) , vasoactive intestinal peptide ( vip ) , peptide histidine - isoleucine ( phi-27 ) , vesicular acetylcholine transporter ( vacht ) , substance p ( sp ) , calcitonin gene - related peptide ( cgrp ) , pituitary adenylate cyclase - activating peptide-27 ( pacap-27 ) , somatostatin ( som ) , choline acetyltransferase ( chat ) , serotonin ( 5-ht ) , cholecystokinin ( cck ) , cocaine- and amphetamine - regulated transcript ( cart ) , and gaba ( for dilutions and secondary antibodies , see table 1 ) . sections were then incubated with a mixture of appropriate fluorescein isothiocyanate ( fitc)-conjugated secondary antisera and biotinylated goat anti - rabbit antibodies ( 1 h ) . after staining , the sections were mounted with carbonate - buffered glycerol ( ph 8.6 ) and cover slipped . the specificity of primary antisera was tested as follows : sections were incubated with antibody , preabsorbed with synthetic antigen ( 10 g of antigen / ml diluted antiserum ) ; the primary antibody was omitted from the incubation ; or normal rabbit or mouse serum was substituted for the primary antibody.table 1detailed list of primary and secondary antibodies used in the studyantiserareagentcodehost speciesdilutionsupplierprimary antibodies chatab5052rabbit1:10,000chemicon int . inc , us ; www.biomol.com som8330 - 0154rabbit1:10,000biogenesis inc , www.biogenesis.co.uk vip11428rabbit1:10,000mp biomedicals ; www.mpbio.com galrin7153rabbit1:10,000bachem ag ; www.bachem.com cb - d28kcb-38rabbit1:10,000swant , s ; www.swant.com crt7699/4rabbit1:20,000swant , s ; www.swant.com nosn2280mouse1:2,000sigma ; http://www.sigmaaldrich.com lenkea 1149rabbit1:10,000bioreagents inc , uk ; www.bioreagents.com dynas-4019rabbit1 : 10,000bachem ag ; www.bachem.com neos-3149rabbit1:10,000bachem ag ; www.bachem.com vachth - v006rabbit1:10,000phoenix , ; www.phoenixpeptide.com pacapihc 8922rabbit1:20,000bachem ag ; www.bachem.com phis-3130rabbit1:10,000bachem ag ; www.bachem.com cgrp11189rabbit1:10,000mp biomedicals ; www.mpbio.com sp8450 - 0505rabbit1:10,000biogenesis inc , www.biogenesis.co.uk 5hts5545rabbit1:5,000sigma , us ; www.sigma-aldrich.com secondary antibodiesdonkey anti - mouse igg ( h + l ) conjugated with fitc1:800715 - 095 - 151 ; jackson ir lab , us ; www.jacksonimmuno.com cy3-conjugated f(ab)2 donkey anti - rabbit igg ( h + l ) -1:9,000711 - 166 - 152 , jackson ir lab , us ; www.jacksonimmuno.com detailed list of primary and secondary antibodies used in the study sections were examined under an olympus bx51 fluorescence microscope equipped with a barrier filter for fb . microphotographs were acquired with a ccd camera connected to a pc equipped with analysis image analysis software ( ver . 3.2 ; soft imaging system gmbh , mnster , germany ) . to determine the relative number of fb - positive cells , neurons were counted in every 16th section at 20 objective . data was pooled from animals and analyzed with graphpad prism 5 software ( graphpad software , la jolla , ca , usa ) . nerve fiber distribution was assessed under 40 objective by subjective observation ( two independent researchers ) as previously described ( gonkowski et al . 2013 ) and depending on the density of labeling , described as not found or very few , small , moderate , and large number of nerve fibers . following fb injections of left scg , fb - positive neurons were found on the ipsilateral side of the spinal cord . a total of 13,013 neurons , combined from longitudinal and transverse sections , were counted . the sympathetic preganglionic neurons ( spn ) projecting to the porcine scg were found in c8 to th6 neuromeres , with the vast majority of them located in th1th3 segments ( 94 4.4 % ) , ranging from 27.6 3.0 % ( th1 ) to 34.5 4.0 % ( th2 ) and 31.6 6.3 % ( th3 ) , respectively . fb neurons were localized predominantly in imlpp ( 85.9 2.4 % ; 4,313 cells ) and imlpf ( 9.0 1.5 % ; 451 cells ) and about 5 % of the cells were seen in icpe ( 0.5 0.2 % ; 23 cells ) . chat- , nos- , vacht- , crt- , cb - d28k- , som- , pacap- , and cck - immunoreactive fb neurons constituted approximately 88 , 72 , 7 , 3.5 , 3 , 3 , 1 , and 0.6 % of all fb neurons in the whole region , respectively . the largest population of fb+/nos + neurons was positive for chat ( 62.8 4.9 % ) , followed by a much smaller number of vacht ( 5.3 2.7 % ) -positive neurons . a few of the fb - positive nitrergic neurons also contained cb-28 k ( 2.2 0.4 ) , som ( 2.0 0.7 % ) , and pacap ( 1.0 0.5 % ) and a very limited number of fb+/nos + neurons were positive to cck ( 0.6 0.4 % ) and crt ( 0.6 0.4 % ) ( table 2 ; figs 1 and 2 ) . none of the fb - positive perikarya was immunopositive to lenk , sp , phi , npy , vip , gal , cgrp , 5ht , and -neo . retrogradely labelled spn were surrounded by a very dense network of opioidergic ( lenk- , dyn a- , or -neo - ir ) , gaba- , pacap- , cart- , or vacht - ir nerve terminals , a moderately dense network of sp- , som- , calb- , crt- , or th - ir nerve fibers , and a scarce network of cck- , vip- , phi- , npy- , 5ht- , gal- , or cgrp - ir axons ( figs . 1 and 2).table 2percentages of retrogradely labelled cells in sympathetic preganglionic neurons ( spn ; in neuromers th1th3 ) projecting to the porcine scgsubstancefb / nos / p fb / nos / p fb / nos / p fb / nos / p chat62.8 4.910.8 1.216.2 3.210.6 1.4vacht5.3 2.765.9 5.32.1 1.126.1 5.7cb-28 k2.2 0.464.3 4.50.8 0.532.6 4.9som2.0 0.767.5 1.30.8 0.229.7 1.5pacap1.0 0.566.0 3.90.2 0.232.7 3.4cck0.6 0.467.1 3.5032.4 3.6crt0.6 0.265.1 5.72.9 1.231.3 5.3p substance ( chat , vacht , pacap .. ) . all pictures are composites of merged images taken separately from the red , green , and/or blue fluorescent channels . a double - labelled nos+/pacap + neurons in the th3 neuromere , a compilation of three photos . b , c three different populations of nos / chat - positive neurons in th3 neuromere ; fb / nos neurons positive for chat ( two double arrows ) , fb+/nos + negative for chat ( three long arrows ) , and double - labeled fb / chat negative for nos ( two small arrows ) . d , e single fb + neurons scattered between a small population of fb / nos / vacht + ( triple staining , two double arrows ) and fb+/vacht + neurons ( small arrow ) . f a population of double fb / nos / pacap ( two single arrows ) and fb / nos / pacap + ( small arrow ) neurons observed in th3 neuromere . g , j the fb / nos / cb-28 k ( one double arrow ) and fb / nos / cb-28 k ( single arrow ) neurons observed in th3 neuromere ( merged image ) . enlarged images of the triple - stained neuron , split channels : h blue k the fb / nos / crt ( single arrows ) and l fb / noscrt ( small arrow ) neurons observed in th3 neuromere . scale bar image 1120 m ; images 2 , 4 , 6 , 11 , and 12100 m ; images 3 and 550 m ; image 725 m ; images 8 , 9 , and 1020 mfig . 2 a j representative images of scg - projecting fb+/nos + neurons ( spn ) and surrounding fibers observed in th3 neuromere . all images are composites of merged images taken separately from the red , green , and blue fluorescent channels . retrogradely labelled spn were surrounded by a lenk- , b dyn a- , c -neo - ir , d 5ht- , e cgrp- , f sp- , g npy- , h phi- , i som- , j gal - ir axons ; scale bar 50 m percentages of retrogradely labelled cells in sympathetic preganglionic neurons ( spn ; in neuromers th1th3 ) projecting to the porcine scg p substance ( chat , vacht , pacap .. ) . data expressed as mean standard deviation ( sd ) representative images of scg - projecting neurons located in the spinal cord iml column . all pictures are composites of merged images taken separately from the red , green , and/or blue fluorescent channels . a double - labelled nos+/pacap + neurons in the th3 neuromere , a compilation of three photos . b , c three different populations of nos / chat - positive neurons in th3 neuromere ; fb / nos neurons positive for chat ( two double arrows ) , fb+/nos + negative for chat ( three long arrows ) , and double - labeled fb / chat negative for nos ( two small arrows ) . d , e single fb + neurons scattered between a small population of fb / nos / vacht + ( triple staining , two double arrows ) and fb+/vacht + neurons ( small arrow ) . f a population of double fb / nos / pacap ( two single arrows ) and fb / nos / pacap + ( small arrow ) neurons observed in th3 neuromere . g , j the fb / nos / cb-28 k ( one double arrow ) and fb / nos / cb-28 k ( single arrow ) neurons observed in th3 neuromere ( merged image ) . k the fb / nos / crt ( single arrows ) and l fb / noscrt ( small arrow ) neurons observed in th3 neuromere . scale bar image 1120 m ; images 2 , 4 , 6 , 11 , and 12100 m ; images 3 and 550 m ; image 725 m ; images 8 , 9 , and 1020 m a j representative images of scg - projecting fb+/nos + neurons ( spn ) and surrounding fibers observed in th3 neuromere . all images are composites of merged images taken separately from the red , green , and blue fluorescent channels . retrogradely labelled spn were surrounded by a lenk- , b dyn a- , c -neo - ir , d 5ht- , e cgrp- , f sp- , g npy- , h phi- , i som- , j gal - ir axons ; scale bar 50 m our results show that the majority of all neurons supplying scg is localized between first through third spinal cord thoracic segments ( th13 ) and originates predominantly from nucleus intermediolateralis pars principalis and pars funicularis with only a small percentage found elsewhere , that is , in nucleus intercalatus spinalis pars paraependymali . these results are consistent with data obtained from other species ( dalsgaard and elfvin 1979 ; petras and faden 1978 ; yau et al . 1991 ) and provide further evidence establishing the predominant importance of intermediolateral nuclei in scg innervation . the observation bears clinical relevance as intermediolateral column injury apparently disrupts scg function , subsequently resulting in various vascular , respiratory , and cardiac dysfunctions ( grigorean et al . it has been shown that sympathetic iml morphological abnormalities in th13 segments observed in patients with familiar dysautonomia lead to scg shrinkage and activity suppression , resulting in numerous autonomic dysfunctions such as difficulties in swallowing , paresthesia , frequent vomiting , labile hypertension , propensity to lung infections , and corneal problems ( pearson and pytel 1978 ) ; these findings further support the notion of central role of scg in the autonomic coordination of major vital organs . further , our study revealed that the neurochemical composition of neurons supplying scg is complex , underscoring the importance of this ganglion as a neuroendocrine center . first , we found that the majority of scg preganglionic neurons is positive to both chat and nos , showing a much higher level of colocalization than previously observed ( blottner and baumgarten 1992 ; calka et al . chat , an acetylcholine marker , previously reported in the intermediolateral ( iml ) nuclei of several species ( caka et al . 2008 ; ichikawa and shimizu 1998 ; markham and vaughn 1990 ) is an important player in numerous neuromodulatory functions ( oda 1999 ) . it has been found that disturbance in neuronal chat levels contributes to the mechanisms underlying development of certain neurodegenerative diseases such as alzheimer s or huntington s disorder , schizophrenia , or amyotrophic lateral sclerosis ( oda 1999 ) . nos , an indicator of nitric oxide presence in the neuron , was shown in the iml nuclei of a number of small and large laboratory animals as well as in humans ( calka et al . it has been shown that nos is involved in the regulation of several metabolic pathways such as soluble guanylyl cyclase or adp - ribosyltransferase activation ( blottner and baumgarten 1992 ) and , most importantly , modulates acetylcholine neurotransmission in central nervous system neurons affecting their activity ( prast and philippu 2001 ) . to date , only a few studies have indicated that in the iml column , nos and chat co - exist in the same neurons ( burnett et al . it is plausible to assume that both nos and chat play a vital role in the scg preganglionic neurons , complementing each other s functions and synergistically modulating neuronal activity of autonomic nervous system cells . interestingly , vacht , an acetylcholine transporter and another marker of cholinergic neurons , has been found in only 7 % of all scg supplying neurons . this discrepancy between chat and vacht distribution in cholinergic neurons might be explained by the differential genetic expression and diverse functional patterns of these two molecules at the molecular level ( weihe and eiden 2000 ) . chat and vacht share the same genetic locus ( eiden 1998 ) ; however , it has been established that their expression levels might substantially differ and , depending on neuronal region and developmental stage , do not always correlate ( schutz et al . 2001 ) , as observed in our study . furthermore , aside from chat , nos , and vacht , here for the first time we show the presence of a number of other neurochemicals such as crt , cb - d28k , som , pacap , and cck in iml neurons innervating the scg , indicating that these neurons largely contribute to and play an important role in maintaining the role of the scg as a neuroendocrine center . crt has been identified in iml preganglionic sympathetic neurons of the rat ( murphy et al . 2003 ; ren and ruda 1994 ) , mouse ( ninomiya et al . 1993 ) , cat ( edwards et al . 1996 ) , and lizard ( gecko ) ( morona et al . it belongs to the family of calcium - binding proteins and is involved in a wide array of processes from intracellular calcium signaling through molecular protein targeting to long - time potentiation modification ( schwaller 2014 ) . it is also thought to be involved in neuroprotection , neuronal development , and homeostasis ( barinka and druga 2010 ; schwaller 2014 ) . in the sympathetic nervous system , crt is presumed to function as a regulator of sympathetic ganglion activity and as a potential contributor to age - related changes of target organ functions ( corns et al . cb , another member of the calcium - binding protein family , has been reported in the sympathetic preganglionic neurons of the rat s iml column ( grkovic and anderson 1997 ) . in the sympathetic nervous system , cb has been speculated to contribute to the secretory functions of salivary glands and fatty brown tissues ( grkovic and anderson 1997 ) , play a role in the development of the enteric nervous system ( hagl et al . 2013 ) , and contribute to the pathogenesis of proliferative enteropathy ( wojtkiewicz et al . recent studies indicate that cb may also play a role in excitatory activity of gaba and glutamate neurons projecting to iml sympathetic preganglionic neurons ( llewellyn - smith et al . cck , first described as an intestinal hormone ( chandra and liddle 2007 ) , reported previously in rat , human , and guinea pig sympathetic neurons of iml nuclei ( chiba and masuko 1987 ; chung et al . 1989 ; schroder 1983 ) , has been shown to participate in a number of gastrointestinal processes , affecting the activity of numerous digestive system organs , i.e. , stomach , gall bladder , pancreas , and intestines ( chandra and liddle 2007 ) . studies have shown that changes in cck expression or a reduction in a > number of cck receptors contributes to various gastrointestinal and metabolic diseases such as diabetes mellitus , gall stone disease and irritable bowel syndrome ( chandra and liddle 2007 ) . som , observed previously in the iml column of the guinea pig spinal cord ( chiba and masuko 1989 ) , has been shown to modulate inflammatory response between neuronal and mast cells in the intestine ( van op den bosch et al . 2009 ) and likely acts alongside cck in digestion and food intake ( schmidt et al . it has been demonstrated that in the sympathetic nervous system som affects vasodepressor response ( rioux et al . 1981 ) and gastrin secretion ( olesen et al . 1984 ) and that it plays a role in insulin release ( lechin et al . 2013 ) . it might be speculated that in the scg , som plays a regulatory role , affecting the function of scg target organs . finally , pacap , previously found in scg neurons of the iml column in the rat ( beaudet et al . 1998 ; klimaschewski et al . 1996 ) , is critically important in an array of autonomic nervous system - regulated activities , from metabolic and cardiovascular regulation through hormone secretion , stress response to appetite suppression , and food intake regulation ( merriam et al . 2013 ; roy et al . it has been shown that in the sympathetic nervous system , pacap contributes to the neuronal development of the sympathetic neuroblast ( lu , et al . , 1998 ) and modulates sympathetic neuron function via ip3 , cyclic amp / pka , and mapk / erk regulatory pathways ( girard et al . 2004 ; may et al . furthermore , pacap contributes to npy and catecholamine expression in scg neurons by regulating the mrna levels of these neurotransmitters , thus affecting the function of this ganglion overall ( braas and may 1999 ; may and braas 1995 ) . in addition to numerous neurochemicals present in scg - projecting neurons , we found a dense network of neurochemically complex fibers surrounding the neurons and containing a plethora of neuropeptides such as lenk , dyn a , -neo , gaba , pacap , cart , vacht , sp , som , calb , crt , th , cck , vip , phi , npy , 5ht , gal , or cgrp that likely modulate the activity of scg preganglionic neurons , thus regulating overall scg function . in conclusion , this is the first evidence of such a neurochemical complexity of iml preganglionic sympathetic neurons supplying mammalian scg . it might be speculated that such complexity is indispensable to scg activity and , as such , likely contributes to the central role of scg in numerous crucial physiological and metabolic functions . the findings of our studies underscore the importance of scg in the autonomic nervous system and lay the foundation for future translational and clinical studies .

the superior cervical ganglion ( scg ) is a center of sympathetic innervation of all head and neck organs . scg sympathetic preganglionic neurons ( spn ) were found in the nucleus intermediolateralis pars principalis ( imlpp ) , the nucleus intermediolateralis pars funicularis ( imlpf ) , the nucleus intercalatus spinalis ( ic ) , and the nucleus intercalatus spinalis pars paraependymalis ( icpe ) . despite its importance , little is known of scg innervation and chemical coding in the laboratory pig , a model that is physiologically and anatomically representative of humans . here in our study , we established the distribution and chemical coding of fast blue ( fb ) retrogradely labelled spn innervating porcine scg . after unilateral injection of fb retrograde tracer into the left scg , labeled neurons were found solely on the ipsilateral side with approximately 98 % located in th1th3 segments and predominantly distributed in the imlpp and imlpf . neurochemical analysis revealed that approximately 80 % of spn were positive both to choline acetyltransferase ( chat ) and nitric oxide synthase ( nos ) and were surrounded by a plethora of opioidergic and peptiergic nerve terminals . the results of our study provide a detailed description of the porcine preganglionic neuroarchitecture of neurons controlling the scg , setting the stage for further studies concerning spn plasticity under experimental / pathological conditions .

Introduction Materials and Methods Results Discussion

the superior cervical ganglion ( scg ) is the center of sympathetic innervation of all head and neck organs . 1998 ) , and cerebral arterial supply regulation ( arbab et al . 1983 ) , a complex neurochemical phenotype of its preganglionic neurons and fibers has yet to be completed . in animal models studied so far , sympathetic preganglionic neurons ( spn ) supplying the scg were found in cervico - thoracic neuromeres , which were confined to the following nuclei : ( 1 ) nucleus intermediolateralis pars principalis ( imlpp ) , ( 2 ) nucleus intermediolateralis pars funicularis ( imlpf ) , ( 3 ) nucleus intercalatus spinalis ( ic ) , and ( 4 ) nucleus intercalatus spinalis pars paraependymalis ( icpe ) ( dalsgaard and elfvin 1979 ; petras and faden 1978 ; yau et al . 1991 ) ; however , until now there is no data clarifying the origins of porcine scg innervation . acetylcholine ( ach ) is the main spn neurotransmitter ; however , studies show that its actions upon scg activity are largely supported by nitric oxide ( no ) present in preganglionic neurons alongside ach ( okamura et al . 1996 ) , and vasoactive intestinal peptide ( vip ) ( sasek et al . 1991 ) are present in the scg - innervating preganglionic neurons , likely contributing to the scg neuroendocrine function . here in the present study , we investigated the distribution and neurochemical coding of scg supplying neurons in the pig , an animal model physiologically and anatomically similar to humans . in our previous study , we showed a complete 2d reconstruction of porcine scg and provided neurochemical characteristics of scg neurons supplying the porcine parotid gland ( wojtkiewicz et al . in this project , we have advanced in our research , paving the way to the source of scg innervation and providing a detailed overview of scg preganglionic neuron distribution and their complex neurochemical phenotype . the left scg , exposed via midline neck incision , was injected with retrograde tracer , fast blue ( fb , ems , grivory , deutschland , gmbh , postfach ) at multiple sites along the scg . the segments were immersed in the same fixative for 20 min and stored in 18 % phosphate - buffered sucrose solution for further processing . the spinal cord segments were sectioned transversally ( n = 3 ) and longitudinally ( n = 3 ) on the cryostat at 10 m in thickness , mounted on glass slides , and stored at 70 c . afterwards , the samples were incubated with a blocking mixture containing 1 % triton x-100 ( sigma - aldrich , usa ) , 0.1 % bovine serum albumin ( sigma - aldrich , usa ) , 0.05 % thimerosal ( sigma - aldrich , usa ) , 0.01 % nan3 , and 10 % of normal goat serum ( mp biomedicals , usa ) in 0.01 m phosphate - buffered saline for 1 h at rt , rinsed in pbs ( 3 15 min ) , and incubated overnight with the following primary antibodies : mouse anti - nitric oxide synthase ( nos ) , co - stained with an array of rabbit antisera against dynorphin a ( dyn a ) , leu-5 enkephalin ( lenk ) , neuropeptide y ( npy ) , -neoendorphin ( neo ) , calbindin d-28 k ( cb - d28k ) , calretinin ( crt ) , galanin ( gal ) , vasoactive intestinal peptide ( vip ) , peptide histidine - isoleucine ( phi-27 ) , vesicular acetylcholine transporter ( vacht ) , substance p ( sp ) , calcitonin gene - related peptide ( cgrp ) , pituitary adenylate cyclase - activating peptide-27 ( pacap-27 ) , somatostatin ( som ) , choline acetyltransferase ( chat ) , serotonin ( 5-ht ) , cholecystokinin ( cck ) , cocaine- and amphetamine - regulated transcript ( cart ) , and gaba ( for dilutions and secondary antibodies , see table 1 ) . after staining , the sections were mounted with carbonate - buffered glycerol ( ph 8.6 ) and cover slipped . inc , us ; www.biomol.com som8330 - 0154rabbit1:10,000biogenesis inc , www.biogenesis.co.uk vip11428rabbit1:10,000mp biomedicals ; www.mpbio.com galrin7153rabbit1:10,000bachem ag ; www.bachem.com cb - d28kcb-38rabbit1:10,000swant , s ; www.swant.com crt7699/4rabbit1:20,000swant , s ; www.swant.com nosn2280mouse1:2,000sigma ; http://www.sigmaaldrich.com lenkea 1149rabbit1:10,000bioreagents inc , uk ; www.bioreagents.com dynas-4019rabbit1 : 10,000bachem ag ; www.bachem.com neos-3149rabbit1:10,000bachem ag ; www.bachem.com vachth - v006rabbit1:10,000phoenix , ; www.phoenixpeptide.com pacapihc 8922rabbit1:20,000bachem ag ; www.bachem.com phis-3130rabbit1:10,000bachem ag ; www.bachem.com cgrp11189rabbit1:10,000mp biomedicals ; www.mpbio.com sp8450 - 0505rabbit1:10,000biogenesis inc , www.biogenesis.co.uk 5hts5545rabbit1:5,000sigma , us ; www.sigma-aldrich.com secondary antibodiesdonkey anti - mouse igg ( h + l ) conjugated with fitc1:800715 - 095 - 151 ; jackson ir lab , us ; www.jacksonimmuno.com cy3-conjugated f(ab)2 donkey anti - rabbit igg ( h + l ) -1:9,000711 - 166 - 152 , jackson ir lab , us ; www.jacksonimmuno.com detailed list of primary and secondary antibodies used in the study sections were examined under an olympus bx51 fluorescence microscope equipped with a barrier filter for fb . to determine the relative number of fb - positive cells , neurons were counted in every 16th section at 20 objective . 2013 ) and depending on the density of labeling , described as not found or very few , small , moderate , and large number of nerve fibers . following fb injections of left scg , fb - positive neurons were found on the ipsilateral side of the spinal cord . the sympathetic preganglionic neurons ( spn ) projecting to the porcine scg were found in c8 to th6 neuromeres , with the vast majority of them located in th1th3 segments ( 94 4.4 % ) , ranging from 27.6 3.0 % ( th1 ) to 34.5 4.0 % ( th2 ) and 31.6 6.3 % ( th3 ) , respectively . fb neurons were localized predominantly in imlpp ( 85.9 2.4 % ; 4,313 cells ) and imlpf ( 9.0 1.5 % ; 451 cells ) and about 5 % of the cells were seen in icpe ( 0.5 0.2 % ; 23 cells ) . chat- , nos- , vacht- , crt- , cb - d28k- , som- , pacap- , and cck - immunoreactive fb neurons constituted approximately 88 , 72 , 7 , 3.5 , 3 , 3 , 1 , and 0.6 % of all fb neurons in the whole region , respectively . the largest population of fb+/nos + neurons was positive for chat ( 62.8 4.9 % ) , followed by a much smaller number of vacht ( 5.3 2.7 % ) -positive neurons . a few of the fb - positive nitrergic neurons also contained cb-28 k ( 2.2 0.4 ) , som ( 2.0 0.7 % ) , and pacap ( 1.0 0.5 % ) and a very limited number of fb+/nos + neurons were positive to cck ( 0.6 0.4 % ) and crt ( 0.6 0.4 % ) ( table 2 ; figs 1 and 2 ) . none of the fb - positive perikarya was immunopositive to lenk , sp , phi , npy , vip , gal , cgrp , 5ht , and -neo . retrogradely labelled spn were surrounded by a very dense network of opioidergic ( lenk- , dyn a- , or -neo - ir ) , gaba- , pacap- , cart- , or vacht - ir nerve terminals , a moderately dense network of sp- , som- , calb- , crt- , or th - ir nerve fibers , and a scarce network of cck- , vip- , phi- , npy- , 5ht- , gal- , or cgrp - ir axons ( figs . 1 and 2).table 2percentages of retrogradely labelled cells in sympathetic preganglionic neurons ( spn ; in neuromers th1th3 ) projecting to the porcine scgsubstancefb / nos / p fb / nos / p fb / nos / p fb / nos / p chat62.8 4.910.8 1.216.2 3.210.6 1.4vacht5.3 2.765.9 5.32.1 1.126.1 5.7cb-28 k2.2 0.464.3 4.50.8 0.532.6 4.9som2.0 0.767.5 1.30.8 0.229.7 1.5pacap1.0 0.566.0 3.90.2 0.232.7 3.4cck0.6 0.467.1 3.5032.4 3.6crt0.6 0.265.1 5.72.9 1.231.3 5.3p substance ( chat , vacht , pacap .. ) . a double - labelled nos+/pacap + neurons in the th3 neuromere , a compilation of three photos . b , c three different populations of nos / chat - positive neurons in th3 neuromere ; fb / nos neurons positive for chat ( two double arrows ) , fb+/nos + negative for chat ( three long arrows ) , and double - labeled fb / chat negative for nos ( two small arrows ) . d , e single fb + neurons scattered between a small population of fb / nos / vacht + ( triple staining , two double arrows ) and fb+/vacht + neurons ( small arrow ) . enlarged images of the triple - stained neuron , split channels : h blue k the fb / nos / crt ( single arrows ) and l fb / noscrt ( small arrow ) neurons observed in th3 neuromere . 2 a j representative images of scg - projecting fb+/nos + neurons ( spn ) and surrounding fibers observed in th3 neuromere . retrogradely labelled spn were surrounded by a lenk- , b dyn a- , c -neo - ir , d 5ht- , e cgrp- , f sp- , g npy- , h phi- , i som- , j gal - ir axons ; scale bar 50 m percentages of retrogradely labelled cells in sympathetic preganglionic neurons ( spn ; in neuromers th1th3 ) projecting to the porcine scg p substance ( chat , vacht , pacap .. ) . data expressed as mean standard deviation ( sd ) representative images of scg - projecting neurons located in the spinal cord iml column . a double - labelled nos+/pacap + neurons in the th3 neuromere , a compilation of three photos . b , c three different populations of nos / chat - positive neurons in th3 neuromere ; fb / nos neurons positive for chat ( two double arrows ) , fb+/nos + negative for chat ( three long arrows ) , and double - labeled fb / chat negative for nos ( two small arrows ) . d , e single fb + neurons scattered between a small population of fb / nos / vacht + ( triple staining , two double arrows ) and fb+/vacht + neurons ( small arrow ) . scale bar image 1120 m ; images 2 , 4 , 6 , 11 , and 12100 m ; images 3 and 550 m ; image 725 m ; images 8 , 9 , and 1020 m a j representative images of scg - projecting fb+/nos + neurons ( spn ) and surrounding fibers observed in th3 neuromere . retrogradely labelled spn were surrounded by a lenk- , b dyn a- , c -neo - ir , d 5ht- , e cgrp- , f sp- , g npy- , h phi- , i som- , j gal - ir axons ; scale bar 50 m our results show that the majority of all neurons supplying scg is localized between first through third spinal cord thoracic segments ( th13 ) and originates predominantly from nucleus intermediolateralis pars principalis and pars funicularis with only a small percentage found elsewhere , that is , in nucleus intercalatus spinalis pars paraependymali . 1991 ) and provide further evidence establishing the predominant importance of intermediolateral nuclei in scg innervation . it has been shown that sympathetic iml morphological abnormalities in th13 segments observed in patients with familiar dysautonomia lead to scg shrinkage and activity suppression , resulting in numerous autonomic dysfunctions such as difficulties in swallowing , paresthesia , frequent vomiting , labile hypertension , propensity to lung infections , and corneal problems ( pearson and pytel 1978 ) ; these findings further support the notion of central role of scg in the autonomic coordination of major vital organs . further , our study revealed that the neurochemical composition of neurons supplying scg is complex , underscoring the importance of this ganglion as a neuroendocrine center . first , we found that the majority of scg preganglionic neurons is positive to both chat and nos , showing a much higher level of colocalization than previously observed ( blottner and baumgarten 1992 ; calka et al . nos , an indicator of nitric oxide presence in the neuron , was shown in the iml nuclei of a number of small and large laboratory animals as well as in humans ( calka et al . it has been shown that nos is involved in the regulation of several metabolic pathways such as soluble guanylyl cyclase or adp - ribosyltransferase activation ( blottner and baumgarten 1992 ) and , most importantly , modulates acetylcholine neurotransmission in central nervous system neurons affecting their activity ( prast and philippu 2001 ) . to date , only a few studies have indicated that in the iml column , nos and chat co - exist in the same neurons ( burnett et al . it is plausible to assume that both nos and chat play a vital role in the scg preganglionic neurons , complementing each other s functions and synergistically modulating neuronal activity of autonomic nervous system cells . interestingly , vacht , an acetylcholine transporter and another marker of cholinergic neurons , has been found in only 7 % of all scg supplying neurons . 2001 ) , as observed in our study . furthermore , aside from chat , nos , and vacht , here for the first time we show the presence of a number of other neurochemicals such as crt , cb - d28k , som , pacap , and cck in iml neurons innervating the scg , indicating that these neurons largely contribute to and play an important role in maintaining the role of the scg as a neuroendocrine center . 1996 ) , and lizard ( gecko ) ( morona et al . in the sympathetic nervous system , crt is presumed to function as a regulator of sympathetic ganglion activity and as a potential contributor to age - related changes of target organ functions ( corns et al . cb , another member of the calcium - binding protein family , has been reported in the sympathetic preganglionic neurons of the rat s iml column ( grkovic and anderson 1997 ) . in the sympathetic nervous system , cb has been speculated to contribute to the secretory functions of salivary glands and fatty brown tissues ( grkovic and anderson 1997 ) , play a role in the development of the enteric nervous system ( hagl et al . 2013 ) , and contribute to the pathogenesis of proliferative enteropathy ( wojtkiewicz et al . recent studies indicate that cb may also play a role in excitatory activity of gaba and glutamate neurons projecting to iml sympathetic preganglionic neurons ( llewellyn - smith et al . cck , first described as an intestinal hormone ( chandra and liddle 2007 ) , reported previously in rat , human , and guinea pig sympathetic neurons of iml nuclei ( chiba and masuko 1987 ; chung et al . som , observed previously in the iml column of the guinea pig spinal cord ( chiba and masuko 1989 ) , has been shown to modulate inflammatory response between neuronal and mast cells in the intestine ( van op den bosch et al . it might be speculated that in the scg , som plays a regulatory role , affecting the function of scg target organs . finally , pacap , previously found in scg neurons of the iml column in the rat ( beaudet et al . 1996 ) , is critically important in an array of autonomic nervous system - regulated activities , from metabolic and cardiovascular regulation through hormone secretion , stress response to appetite suppression , and food intake regulation ( merriam et al . it has been shown that in the sympathetic nervous system , pacap contributes to the neuronal development of the sympathetic neuroblast ( lu , et al . , 1998 ) and modulates sympathetic neuron function via ip3 , cyclic amp / pka , and mapk / erk regulatory pathways ( girard et al . in addition to numerous neurochemicals present in scg - projecting neurons , we found a dense network of neurochemically complex fibers surrounding the neurons and containing a plethora of neuropeptides such as lenk , dyn a , -neo , gaba , pacap , cart , vacht , sp , som , calb , crt , th , cck , vip , phi , npy , 5ht , gal , or cgrp that likely modulate the activity of scg preganglionic neurons , thus regulating overall scg function . the findings of our studies underscore the importance of scg in the autonomic nervous system and lay the foundation for future translational and clinical studies .

the biological heterogeneity of breast cancer ( bc ) has been defined by various efforts in recent years , taking into account the distinct molecular and histopathological features of these tumours . luminal - type and basal - like bcs have been shown to be completely different diseases at the molecular level , as well as in terms of the course of the disease , patient prognosis and survival.16 while the luminal subtype , characterised by the expression of estrogen and/or progesterone receptors ( er / pr ) , shows well - known characteristics of adenocarcinoma , basal - like phenotypes exhibit a wider and more continuous spectrum of genomic evolution and have been linked to biological features of other malignancies.3 with recent results from clinical trials targeting well - known cancer - promoting pathways , this review is seeking to elucidate and summarise current new therapeutic aspects in metastatic bc ( mbc ) and shed light on translational aspects within this entity . articles from peer - reviewed journals as well as published abstracts were searched for using ncbis pubmed as well as esmo , asco , aacr and sabcs online library databases as of 22 march 2016 . her2 , luminal breast cancer , triple - negative , translational , hormone , metastases , brain , bone and names of medications as well as gene and protein symbols of therapeutic targets dealt with in this manuscript . targeted therapy in mbc consists of approaches where well - established or novel pathways are being targeted with the aim of prolonged disease control.79 besides the er , targeting her2 is today regarded as the best established targeted treatment approach in mbc . her2 is a transmembrane growth factor receptor of the erbb family ; her2 protein overexpression and/or her2/neu gene amplification result in an aggressive bc phenotype with high recurrence rates and poor outcome.10 of note , before the availability of targeted treatment options , median overall survival ( os ) in her2-positive mbc was low at around 20 months.11 addition of trastuzumab , a humanised monoclonal antibody targeting the extracellular domain of her2 , to chemotherapy significantly prolonged progression - free survival ( pfs ) and os over chemotherapy alone.11 12 still , secondary resistance to trastuzumab will eventually evolve and patients initially responding to her2-targeted therapy will usually progress within 18 months,13 indicating the need for further alternative treatment approaches . in the phase iii trial cleopatra , the classic first - line treatment standard of docetaxel plus trastuzumab was compared with a triple therapy of docetaxel , trastuzumab plus pertuzumab , a humanised monoclonal antibody targeting the dimerisation domain of her2 , thereby preventing receptor homodimerisation and heterodimerisation and consequently activation of her2 signalling.14 at a median follow - up of 50 months , median os in the pertuzumab group was 56.5 months.15 this number indicates the impressive outcome improvements achievable in her2-positive mbc with today 's therapeutic options . when the antibody binds to her2 , the cell internalises the antigen - antibody complex ; consequently , trastuzumab is degraded in the lysosome and dm1 is set free within the cancer cell . tdm1 was shown to be superior to lapatinib , a small molecule tyrosine kinase inhibitor ( tki ) of her2 and epidermal growth factor receptor ( egfr ) , plus capecitabine in terms of activity as well as tolerability in the phase iii trial emilia with pfs 9.6 vs 6.4 months ( hr 0.65 ; 95% ci 0.55 to 0.77).16 most patients received tdm1 as second - line therapy but 16% of patients had progressed on or within 6 months after the end of adjuvant trastuzumab ; this lead to the approval of tdm1 as first - line treatment standard in earlier relapse . another phase iii study , th3resa , randomised pretreated patients to tdm1 or treatment by investigator 's choice . since approximately 80% of patients in the control arm received trastuzumab - based therapy , th3resa is considered a comparison of tdm1 to trastuzumab treatment in multiple lines . in this study , tdm1 improved pfs from 3.3 to 6.2 months ( hr 0.53 ; 95% ci 0.42 to 0.66).17 in summary , these results suggest that despite considerable costs , tdm1 is indeed a valuable novel treatment option . besides , other antibody - drug conjugates targeting her2 are currently being tested in clinical trials and already showed favourable safety profiles , such as mm-302 . owing to the use of small amounts of its active agent doxorubicin , it caused only minor haematological toxicity when used as a monotherapy or in combination with trastuzumab , as well as with trastuzumab and cyclophosphamide in a phase i study . it is currently being evaluated in the randomised phase ii hermione trial in patients with anthracycline nave her2-positive locally advanced or mbc previously treated with trastuzumab , pertuzumab and tdm1.18 lapatinib was the second her2-targeted drug to become available after trastuzumab . this first - generation , reversible tki inhibits the tyrosine - kinase domains of her2 as well as egfr . in a prospective randomised phase iii trial , addition of lapatinib to capecitabine improved pfs over chemotherapy alone in pretreated patients ( median pfs 8.4 vs 4.4 months ; hr 0.47 ; 95% ci 0.32 to 0.68 ; p<0.001 ) while os was not changed.19 this was the first phase iii study to demonstrate that continuing anti - her2 therapy in combination with chemotherapy in patients who had virtually all received prior trastuzumab was superior to chemotherapy alone . owing to relevant side effects , however , lapatinib plus capecitabine was rarely used as second - line treatment . often physicians continued their patients on trastuzumab in multiple treatment lines after a switch of the cytotoxic combination partner ; indeed , several phase ii trials suggested that this was a feasible treatment approach.20 21 before second - generation antibodies became available , this strategy was so common that the only prospective randomised phase iii trial investigating trastuzumab treatment in multiple lines had to be closed early due to slow accrual after the inclusion of only 156 patients.22 similar to the lapatinib study , continuation of anti - her2 treatment resulted in longer pfs ( 8.2 vs 5.6 months ; hr 0.69 ; 95% ci 0.48 to 0.97 ; p=0.0338 ) while once again no difference in terms of os was observed . the concept of vertical dual blockade of her2 with a combination of trastuzumab plus lapatinib has recently led to renewed interest in her2 tkis . in a heavily pretreated population , the combination of trastuzumab with lapatinib improved os over lapatinib alone ( 14 vs 9.5 months ; hr 0.74 ; 95% ci 0.57 to 0.97 ; p=0.026).23 of note , this effect was more pronounced in the her2-positive / hormone receptor - negative subgroup , eventually leading to european medicines agency approval of vertical dual blockade in this subset . further , recent studies have focused on irreversible second - generation tkis such as neratinib and afatinib with potentially improved activity . afatinib yielded a partial response rate of 11% and stable disease rate of 37% in a phase ii trial of 41 patients progressing on prior trastuzumab treatment ( bibw 2992 trial).24 median pfs was 15.1 weeks and a median os of 61.0 weeks was reported . these results led to the initiation of the phase iii lux - breast 1 trial comparing afatinib plus vinorelbine to trastuzumab plus vinorelbine in patients with her2-positive mbc progressing on prior trastuzumab - based treatment . a similar outcome in terms of pfs was observed in both groups ; os , however , was shorter in the afatinib arm.25 this effect was most likely caused by higher rates of dose reductions and treatment discontinuations in patients receiving afatinib . indeed , toxicity issues especially diarrhoea remain a concern with first - generation and second - generation tkis inhibiting egfr and her2 , but may be less so with her2-specific third - generation tkis such as ont-380.26 the lack of benefit of afatinib over standard treatment options was further substantiated by results of a randomised phase ii trial of afatinib versus afatinib plus vinorelbine versus treatment by investigators ' choice in patients with progressive brain metastases after trastuzumab - based and/or lapatinib - based therapy ( lux - breast 3 trial ) . in this study including 121 patients , both regimen containing afatinib did not yield gains in terms of clinical benefit rate at 12 weeks ( which was defined as primary study end point ) as well as pfs or os . further , afatinib - containing treatments were less well tolerated.27 another irreversible tki , neratinib , has shown promising objective response rates ( orr ) of up to 56% as single agent in patients with her2-overexpressing advanced bc in an open - label phase ii trial ; pfs in this study was 39.6 weeks.28 however , the benefit was more substantial in patients without prior trastuzumab treatment compared with those with prior treatment ( orr 24% , pfs 22.3 weeks ) , suggesting that trastuzumab resistance may not be fully overcome by neratinib . when neratinib was directly compared with the former second - line standard of lapatinib plus capecitabine in a randomised phase ii trial , it failed to show benefit in terms of pfs and os ( pfs 4.5 months on neratinib vs 6.8 months ; not significant ; and os 19.7 months on neratinib vs 23.6 months ; not significant).29 while this trial could not establish the superiority of irreversible tkis over lapatinib plus capecitabine , single - agent activity of neratinib was confirmed ; still , the exact future place of neratinib in the continuum of anti - her2 therapy awaits further clarification . a phase i basket study of 60 patients with her2-overexpressing carcinomas was able to shed light on a potential future avenue for the development of neratinib ; in this trial , neratinib was combined with temsirolimus in order to achieve inhibition of her2-signalling by the additional abolishment of one of its major downstream effector kinases , mammalian target of rapamycin ( mtor ) . this combination produced responses in 2 of the 15 patients ( 13.3% ) with mbc resistant to trastuzumab ; on the downside , this dual blockade led to relevant treatment - related toxicity : diarrhoea was observed in 93% of all patients and also held responsible for 4 of the 10 dose - limiting toxicities ( dlts ) ; dose - limiting metabolic alterations were observed as well.30 a subsequent phase i / ii trial of neratinib plus temsirolimus in her2-positive mbc31 reported a partial response rate of 30% for patients treated at both the maximum tolerable dose ( mtd ) and dose escalation ( de ) cohorts ( median duration of response 3.0 and 7.4 months , respectively ) . while concerns regarding tolerability remain , these findings again strongly supported a model of ongoing her2 pathway addiction even in mbc resistant to trastuzumab . of note , neratinib may further play a role in maintenance therapy after adjuvant chemotherapy and trastuzumab treatment . in a randomised phase iii trial in the aforementioned setting , neratinib led to a significant 2.3% increase in disease - free survival when compared with placebo ( 93.9% vs 91.6% ; hr 0.67 ; p=0.009 ) . again , a high rate of grade 3 diarrhoea ( 40% ) was observed.32 the question whether the addition of mtor inhibitors to anti - her2 treatment might reverse resistance against her2-targeted therapy was also stressed in the bolero-3 trial.33 34 patients with advanced taxane - pretreated bc who had advanced on prior trastuzumab therapy were treated with vinorelbine plus trastuzumab with or without everolimus . the primary end point ( pfs ) was met , indicating a moderate prolongation of median pfs from 5.78 months ( 95% ci 5.49 to 6.90 ) to 7.00 months ( 95% ci 6.74 to 8.18 ) with the addition of everolimus ( hr 0.78 ; 95% ci 0.65 to 0.95 ; p=0.0067 ) . grade 3/4 side effects observed more commonly in the everolimus group included neutropenia ( 73% ) , leucopenia ( 38% ) , anaemia ( 19% ) , febrile neutropenia ( 16% ) , stomatitis ( 13% ) and fatigue ( 12% ) . serious adverse events were reported in 42% of patients in the everolimus arm as compared with 20% in the placebo group . on the basis of these results , the authors stated that the clinical benefit observed needs to be balanced in the context of the toxicity profile in an mbc population . owing to this fact , mtor inhibitors have not yet found their way into the clinical routine in her2-positive mbc . finally , novel antibodies may also help in optimising outcome in patients with her2-positive mbc . margetuximab is a chimeric monoclonal antibody with optimised fc fraction in order to increase antibody - dependent cell - mediated cytotoxicity activity . in a phase i trial , 52 patients with different her2-positive malignancies without any further standard therapy available were included.35 in the bc subset consisting of 19 participants , a reduction in tumour size was observed in 57.9% of patients . treatment was generally well tolerated , with grade i / ii fever and nausea as well as diarrhoea and fatigue being the main toxicities . heterodimers of her2 and her3 are known to be the most potent inducers of her2 signalling.36 furthermore , upregulation of her3 was also described as a relevant mechanism of resistance to treatment with trastuzumab and lapatinib.37 38 recently , it was shown that co - expression of her2 and her3 was associated with shorter os in patients with her2-positive mbc treated with trastuzumab in multiple lines.39 therefore , it appears reasonable to examine strategies of targeting her3 in combination with her2 . in a phase i trial , patritumab was added to trastuzumab plus paclitaxel in 18 patients with her2-positive mbc who had received at least one prior treatment line for mbc ; this fully human monoclonal antibody targets her3 , blocks receptor ligand interaction and receptor activation and induces her3 downregulation . tolerability of this approach was excellent and no dlts were observed with diarrhoea being the main side effect ; the authors reported a relatively high response rate of 38.9% , suggesting that this approach should be investigated further in future clinical trials.40 luminal bc is defined by the expression of the er and progesterone receptor ; the vast majority of all bc cases belong to this subtype . hormone - dependent cancer growth allows for targeted therapy with antihormonal agents . in this context , surgical oophorectomy , first described by beatson,41 is regarded as the first systemic anticancer therapy and the prototype of the concept of biologically targeted treatment . the former adjuvant treatment standard of tamoxifen administered for 5 years reduces recurrence risk by half and bc mortality by one - third.42 in the metastatic setting , sequential administration of different non - cross resistant classes of antihormonal therapy allows for delaying initiation of cytotoxic chemotherapy.43 still , even in highly hormone expressing tumours , resistance to endocrine therapy will eventually arise . several factors add to secondary resistance : ccnd1 amplification , esr1 mutation and activation of growth factor signalling pathways.4447 the latter results in er activation even in the absence of estrogen via the activation of signalling molecules downstream of receptor tyrosine kinases ( rtks ) ; phosphatidyl - inositol-3-kinase ( pi3k ) and mtor have been identified as central downstream molecules in this crosstalk leading to the testing of mtor inhibitors such as everolimus in combination with endocrine therapy in clinical trials . in the prospective randomised placebo - controlled trial bolero-2 , postmenopausal patients with er - positive advanced bc progressing on or after prior therapy with a non - steroidal aromatase inhibitor ( ai ) were randomised to receive everolimus or placebo in combination with the steroidal ai exemestane . bolero-2 was able to show a clear - cut benefit in terms of pfs ( 7.4 vs 3.2 months ; hr 0.34 ; 95% ci 0.36 to 0.53)48 for patients in the everolimus group . this supported findings from the randomised phase ii trial tamrad49 and the horizon50 trials ; on the downside , a significant os advantage could not be shown ( bolero-2 : os hr 0.89 ; 31.0 vs 26.6 months , p=0.14 ) and a relevant increase in toxicity was observed as well . pik3ca , a gene coding for the catalytic p110 subunit of pi3k , is among the most commonly mutated genes in bc51 ; mutations have been linked to endocrine resistance by activating the aforementioned crosstalk between er and rtks , thus suggesting to serve as a predictive marker for response of drugs targeting the pi3k / mtor pathway . indeed , activity of mtor inhibitors might be restricted to patients harbouring alterations in the pi3k / akt / mtor pathway , such as pi3kca mutations or pten deletions.52 while this notion is supported by preclinical data53 biological subprotocols of bolero-2 and tamrad suggested that , in contrast to expectation , patients without activating alterations in the pi3k / mtor pathway derived the greatest benefit from treatment.54 55 the quest for predictive markers for novel targeted therapies in luminal bc is therefore ongoing and interest in this field has recently focused on pi3k - inhibitors . while it appears reasonable to assume that pi3k - inhibitors may be most active in tumours harbouring activating pik3ca mutations , no such clinical correlation was proven henceforth . while the addition of bkm120 ( buparlisib , a pan - class pi3k inhibitor ) increased activity of endocrine therapy over endocrine therapy alone in the belle-2 trial , this effect was overall modest ( hr 0.78 , pfs 6.9 vs 5.0 months , p<0.001 ) and considerable toxicity was observed as well . of note , in a prospective substudy within belle-2 , it was shown that pik3ca mutations in cell - free tumour dna ( ctdna ) predicted further activity of buparlisib in this subset of patients ( hr 0.56 , pfs 7.0 vs 3.2 months , p<0.001).56 one hypothesis suggests that pik3ca mutations may predict especially for activity of -specific inhibitors of the p110 catalytic subunit of class i pi3k , but clinical proof of this concept is still awaited . if these data can be verified , however , a predictive biomarker beyond the er will be available in luminal bc for the first time . cyclin - dependent kinase 4 and 6 ( cdk 4/6 ) inhibitors are a novel and promising treatment approach as dysregulation of cell cycle control may cause resistance to antihormonal treatment as well . palbociclib , an irreversible inhibitor of cdk 4/6 , when added to letrozole , has been shown to prolong progression - free interval over letrozole alone to a significant and clinically relevant extent . in the prospective , randomised phase ii trial paloma-1/trio-18,57 median pfs at 29.6 months median follow - up was prolonged from 10.2 to 20.1 months ( hr 0.488 ; 95% ci 0.319 to 0.748 ; one - sided p=0.0004 ) with the addition of palbociclib while the tolerability profile was generally favourable . the main toxicity consisted of neutropenia and was easily manageable with dose delays and modifications . patients were enrolled into two separate sequential cohorts : cohort 1 ( n=66 ) included unselected patients with er - positive , her2-negative tumours ; cohort 2 ( n=99 ) enrolled patients with luminal cancers and ccdn1 amplification or p16 loss . disappointingly , however , results demonstrated that no reliable response prediction is possible on the basis of these markers . recently , the favourable results of paloma-1 were duplicated in the prospective randomised phase iii trial paloma-3 where palbociclib was added to the pure antiestrogen fulvestrant in pretreated patients . again , addition of palbociclib resulted in a clinically meaningful improvement of median pfs from 3.8 to 9.2 months ( hr 0.422 ; 95% ci 0.318 to 0.560 ; p<0.000001 ) ; of note , this study included premenopausal women as well and results were consistent in this population . no new safety signals were observed and main toxicity again consisted of neutropenia ( 78.8% vs 3.5% ) , leucopenia ( 45.5% vs 4.1% ) , and fatigue ( 38.0% vs 26.7% ) . still , febrile neutropenia was rare and comparable in between both groups with 0.6% each ; furthermore , the discontinuation rate due to adverse events was identical as well ( 2.0% vs 1.7%).58 several other trials of cdk 4/6 inhibitors in bc are currently ongoing . their results might add to the notion that a hormone - independent treatment modality is able to prolong the interval to development of hormone refractory disease , as also seen in other malignancies , such as prostate cancer.59 the role of immunotherapy and immune checkpoint modulators in luminal bc has not been fully characterised , although safety and comparably good response rates have been reported after multiple lines of therapy for patients suffering from programmed death ligand 1 ( pd - l1)-overexpressing tumours in the keynote-028 trial.60 further investigation in this field is greatly needed to objectify biological activity and to find biomarkers aside from pd - l1 that would point to responsiveness towards immune checkpoint inhibition in this setting . as indicated by the negative definition of being a bc subtype without expression of the er and progesterone receptor as well as by the lack of her2 protein overexpression or her2/neu gene amplification , tnbc is the disease subtype with the least clinically relevant attributes . this lack of potential targets is clinically reflected by the poor prognosis of patients with tnbc . chemotherapy remains the mainstay of tnbc therapy and patients with pathological complete remission to neoadjuvant chemotherapy are known to have a favourable prognosis despite the inherent aggressiveness of this disease subtype.61 patients with a less chemotherapy - sensitive disease , on the other hand , have a markedly poor prognosis with high recurrence rates and mortality . this warrants the search for novel targets in tnbc . around 10% of all bc cases can be attributed to brca-1/2 germline mutations with considerably higher rates of brca-1 mutations in women with tnbc.62 further , women with early - onset bc are at a higher risk of carrying brca-1/2 mutations , with brca-1 being the more frequently altered gene.63 64 owing to consecutive defects in genetic repair mechanisms such as homologous repair , brca - deficient cells must resort to other error - prone options of double - strand brake repair such as non - homologous end - joining ( nhej ) and base excision repair ( ber).65 both repair mechanisms depend on parp ( poly-(adp - ribose)-polymerases ) as molecular scaffolds for repairing double - strand breaks , making parp inhibitors either alone or in combination with dna - damaging agents a promising treatment option , inducing synthetic lethality due to loss of parp and brca-1/2 functions.66 a single - arm multicentre phase ii trial of olaparib as a single agent was conducted in patients with different malignancies harbouring brca-1/2 germline mutations ; 62 patients with bc who had received at least three prior treatment lines for mbc were included . in this subset , there was an overall response rate of 12.9% ( 95% ci 5.7% to 23.9% ) with an additional 47% ( 95% ci 34.0% to 59.9% ) of patients experiencing disease stabilisation for a minimum of 8 weeks.67 on the other hand , activity of olaparib in an unselected tnbc population was disappointing.68 several trials of parp inhibitors in bc are currently ongoing ; of special interest , the randomised phase iii trial olympia ( nct02032823 ) focuses on the potential role of olaparib as adjuvant therapy in patients with high - risk her2-negative primary bc harbouring brca germline mutations . in this context , it needs to be mentioned that , in contrast to germline brca mutations , the predictive role of the more abundant somatic brca mutations in bc remains elusive.69 the quest for novel therapeutic approaches has brought to light various potential targets including rtks such as met and fgf . met and its ligand hepatocyte growth factor are considered especially promising targets , being overexpressed in up to 45% of bcs and associated with poor clinical outcome , increased propensity to metastatic spread , increased tumour cell proliferation , high grading and a triple - negative phenotype . of note , the met - inhibitor cabozantinib has recently shown activity in humanised patient - derived xenograft models of tnbc.70 furthermore , phase i clinical trials have also suggested promising activity and relative safety of met inhibitors such as cabozantinib and foretinib , and results of later - phase clinical trials are therefore awaited with great interest.71 72 on the other side , targeting the extracellular domain of met by means of the monoclonal humanised antibody onartuzumab has currently shown no benefit when added to paclitaxel / bevacizumab ; in this context , it needs to be stated that most patients included in this study ( 88% ) had only weak met expression as defined by immunohistochemistry,73 raising the question whether this study was indeed conducted in the right population . fgf was shown to play a significant role in up to 10% of patients with bc,74 facilitating epithelial to mesenchymal transition and hence metastatic spread . subsequently , clinical trials of tkis targeting selected ( azd4547 ) or multiple subforms of fibroblast growth factor receptor ( fgfr ) ( dovitinib,75 lucitanib76 or nintedanib77 ) were initiated ; of note , the latter showed promising activity when combined with paclitaxel in a neoadjuvant setting . the rationale for the approach of fgfr - targeted therapy is to delay mesenchymal dedifferentiation and hence resistance to chemotherapy and targeted therapy that had originally been tailored for epithelial tumours . although activity was also seen in luminal tumours,74 75 recent trials using fgfr inhibitors were focusing on the triple - negative setting because of inherent toxicity and missing alternative treatments in tnbc . as with other malignancies , immune checkpoint modulators are currently being investigated in tnbc , although clinical development started with considerable delay as compared with melanoma or lung cancer . tnbc is characterised by high mutational load , which again is apparently associated with high immunogenicity.78 this makes tnbc an attractive field for developing immunotherapy in bc . first data on immunotherapy with immune checkpoint modulators in tnbc were presented at the 2014 san antonio breast cancer symposium.79 thirty - two patients with pretreated mbc received pembrolizumab ( mk-3475 ) , a humanised igg4 monoclonal antibody targeting pd-1 within the bc cohort of the phase 1b trial keynote-012 . of note , approximately 45% had already received at least three prior treatment lines for metastatic disease . while the overall response rate was relatively low at 18.5% , it is worth mentioning that responses were observed even in heavily pretreated participants and some patients seemed to experience prolonged disease stabilisation . another phase i trial tested mdpl3280a , an anti - pd - l1 antibody , in 54 patients with tnbc.80 again , 85% of all patients had at least four prior systemic therapy lines . in 21 evaluable patients , the authors observed a response rate of 19% , with two complete responses and two partial responses ; although it is too early to draw any firm conclusions , it must be mentioned that these four patients had high expression of pretreatment pd - l1 . recently , the phase ib javelin study reported first results from the bc cohort.81 the anti - pd - l1 antibody avelumab ( msb0010718c ) yielded an overall relative reponse ( rr ) of 4.8% in 168 unselected patients with pretreated mbc . in patients with tnbc ( 34.5% ) , rr was somewhat higher ( 8.6% ) but responses were seen across all subtypes with a median duration of response of 28.7 weeks . when analysing patients with tumours harbouring 10% pd - l1 expression , a response rate of 33% in the overall population was observed ; rr in patients with tnbc was a striking 44% . in summary , these data suggest that immunotherapy is indeed a valuable addition to the therapeutic armamentarium in tnbc ( table 1 ) . selected drugs and their molecular targets undergoing clinical evaluation in mbc lapatinib ( her family ) neratinib ( her family ) afatinib ( her family ) temsirolimus ( mtorc1 ) palbociclib ( cdk 4/6 ) cdk 4/6 , cyclin - dependent kinase 4 and 6 ; fgfr , fibroblast growth factor receptor ; mbc , metastatic breast cancer ; mtorc1 , mammalian target of rapamycine complex 1 ; pd - l1 , programmed death ligand 1 . targeted therapy in mbc consists of approaches where well - established or novel pathways are being targeted with the aim of prolonged disease control.79 besides the er , targeting her2 is today regarded as the best established targeted treatment approach in mbc . her2 is a transmembrane growth factor receptor of the erbb family ; her2 protein overexpression and/or her2/neu gene amplification result in an aggressive bc phenotype with high recurrence rates and poor outcome.10 of note , before the availability of targeted treatment options , median overall survival ( os ) in her2-positive mbc was low at around 20 months.11 addition of trastuzumab , a humanised monoclonal antibody targeting the extracellular domain of her2 , to chemotherapy significantly prolonged progression - free survival ( pfs ) and os over chemotherapy alone.11 12 still , secondary resistance to trastuzumab will eventually evolve and patients initially responding to her2-targeted therapy will usually progress within 18 months,13 indicating the need for further alternative treatment approaches . in the phase iii trial cleopatra , the classic first - line treatment standard of docetaxel plus trastuzumab was compared with a triple therapy of docetaxel , trastuzumab plus pertuzumab , a humanised monoclonal antibody targeting the dimerisation domain of her2 , thereby preventing receptor homodimerisation and heterodimerisation and consequently activation of her2 signalling.14 at a median follow - up of 50 months , median os in the pertuzumab group was 56.5 months.15 this number indicates the impressive outcome improvements achievable in her2-positive mbc with today 's therapeutic options . when the antibody binds to her2 , the cell internalises the antigen - antibody complex ; consequently , trastuzumab is degraded in the lysosome and dm1 is set free within the cancer cell . tdm1 was shown to be superior to lapatinib , a small molecule tyrosine kinase inhibitor ( tki ) of her2 and epidermal growth factor receptor ( egfr ) , plus capecitabine in terms of activity as well as tolerability in the phase iii trial emilia with pfs 9.6 vs 6.4 months ( hr 0.65 ; 95% ci 0.55 to 0.77).16 most patients received tdm1 as second - line therapy but 16% of patients had progressed on or within 6 months after the end of adjuvant trastuzumab ; this lead to the approval of tdm1 as first - line treatment standard in earlier relapse . another phase iii study , th3resa , randomised pretreated patients to tdm1 or treatment by investigator 's choice . since approximately 80% of patients in the control arm received trastuzumab - based therapy , th3resa is considered a comparison of tdm1 to trastuzumab treatment in multiple lines . in this study , tdm1 improved pfs from 3.3 to 6.2 months ( hr 0.53 ; 95% ci 0.42 to 0.66).17 in summary , these results suggest that despite considerable costs , tdm1 is indeed a valuable novel treatment option . besides , other antibody - drug conjugates targeting her2 are currently being tested in clinical trials and already showed favourable safety profiles , such as mm-302 . owing to the use of small amounts of its active agent doxorubicin , it caused only minor haematological toxicity when used as a monotherapy or in combination with trastuzumab , as well as with trastuzumab and cyclophosphamide in a phase i study . it is currently being evaluated in the randomised phase ii hermione trial in patients with anthracycline nave her2-positive locally advanced or mbc previously treated with trastuzumab , pertuzumab and tdm1.18 lapatinib was the second her2-targeted drug to become available after trastuzumab . this first - generation , reversible tki inhibits the tyrosine - kinase domains of her2 as well as egfr . in a prospective randomised phase iii trial , addition of lapatinib to capecitabine improved pfs over chemotherapy alone in pretreated patients ( median pfs 8.4 vs 4.4 months ; hr 0.47 ; 95% ci 0.32 to 0.68 ; p<0.001 ) while os was not changed.19 this was the first phase iii study to demonstrate that continuing anti - her2 therapy in combination with chemotherapy in patients who had virtually all received prior trastuzumab was superior to chemotherapy alone . owing to relevant side effects , often physicians continued their patients on trastuzumab in multiple treatment lines after a switch of the cytotoxic combination partner ; indeed , several phase ii trials suggested that this was a feasible treatment approach.20 21 before second - generation antibodies became available , this strategy was so common that the only prospective randomised phase iii trial investigating trastuzumab treatment in multiple lines had to be closed early due to slow accrual after the inclusion of only 156 patients.22 similar to the lapatinib study , continuation of anti - her2 treatment resulted in longer pfs ( 8.2 vs 5.6 months ; hr 0.69 ; 95% ci 0.48 to 0.97 ; p=0.0338 ) while once again no difference in terms of os was observed . the concept of vertical dual blockade of her2 with a combination of trastuzumab plus lapatinib has recently led to renewed interest in her2 tkis . in a heavily pretreated population , the combination of trastuzumab with lapatinib improved os over lapatinib alone ( 14 vs 9.5 months ; hr 0.74 ; 95% ci 0.57 to 0.97 ; p=0.026).23 of note , this effect was more pronounced in the her2-positive / hormone receptor - negative subgroup , eventually leading to european medicines agency approval of vertical dual blockade in this subset . further , recent studies have focused on irreversible second - generation tkis such as neratinib and afatinib with potentially improved activity . afatinib yielded a partial response rate of 11% and stable disease rate of 37% in a phase ii trial of 41 patients progressing on prior trastuzumab treatment ( bibw 2992 trial).24 median pfs was 15.1 weeks and a median os of 61.0 weeks was reported . these results led to the initiation of the phase iii lux - breast 1 trial comparing afatinib plus vinorelbine to trastuzumab plus vinorelbine in patients with her2-positive mbc progressing on prior trastuzumab - based treatment . a similar outcome in terms of pfs was observed in both groups ; os , however , was shorter in the afatinib arm.25 this effect was most likely caused by higher rates of dose reductions and treatment discontinuations in patients receiving afatinib . indeed , toxicity issues especially diarrhoea remain a concern with first - generation and second - generation tkis inhibiting egfr and her2 , but may be less so with her2-specific third - generation tkis such as ont-380.26 the lack of benefit of afatinib over standard treatment options was further substantiated by results of a randomised phase ii trial of afatinib versus afatinib plus vinorelbine versus treatment by investigators ' choice in patients with progressive brain metastases after trastuzumab - based and/or lapatinib - based therapy ( lux - breast 3 trial ) . in this study including 121 patients , both regimen containing afatinib did not yield gains in terms of clinical benefit rate at 12 weeks ( which was defined as primary study end point ) as well as pfs or os . further , afatinib - containing treatments were less well tolerated.27 another irreversible tki , neratinib , has shown promising objective response rates ( orr ) of up to 56% as single agent in patients with her2-overexpressing advanced bc in an open - label phase ii trial ; pfs in this study was 39.6 weeks.28 however , the benefit was more substantial in patients without prior trastuzumab treatment compared with those with prior treatment ( orr 24% , pfs 22.3 weeks ) , suggesting that trastuzumab resistance may not be fully overcome by neratinib . when neratinib was directly compared with the former second - line standard of lapatinib plus capecitabine in a randomised phase ii trial , it failed to show benefit in terms of pfs and os ( pfs 4.5 months on neratinib vs 6.8 months ; not significant ; and os 19.7 months on neratinib vs 23.6 months ; not significant).29 while this trial could not establish the superiority of irreversible tkis over lapatinib plus capecitabine , single - agent activity of neratinib was confirmed ; still , the exact future place of neratinib in the continuum of anti - her2 therapy awaits further clarification . a phase i basket study of 60 patients with her2-overexpressing carcinomas was able to shed light on a potential future avenue for the development of neratinib ; in this trial , neratinib was combined with temsirolimus in order to achieve inhibition of her2-signalling by the additional abolishment of one of its major downstream effector kinases , mammalian target of rapamycin ( mtor ) . this combination produced responses in 2 of the 15 patients ( 13.3% ) with mbc resistant to trastuzumab ; on the downside , this dual blockade led to relevant treatment - related toxicity : diarrhoea was observed in 93% of all patients and also held responsible for 4 of the 10 dose - limiting toxicities ( dlts ) ; dose - limiting metabolic alterations were observed as well.30 a subsequent phase i / ii trial of neratinib plus temsirolimus in her2-positive mbc31 reported a partial response rate of 30% for patients treated at both the maximum tolerable dose ( mtd ) and dose escalation ( de ) cohorts ( median duration of response 3.0 and 7.4 months , respectively ) . while concerns regarding tolerability remain , these findings again strongly supported a model of ongoing her2 pathway addiction even in mbc resistant to trastuzumab . of note , neratinib may further play a role in maintenance therapy after adjuvant chemotherapy and trastuzumab treatment . in a randomised phase iii trial in the aforementioned setting , neratinib led to a significant 2.3% increase in disease - free survival when compared with placebo ( 93.9% vs 91.6% ; hr 0.67 ; p=0.009 ) . again , a high rate of grade 3 diarrhoea ( 40% ) was observed.32 the question whether the addition of mtor inhibitors to anti - her2 treatment might reverse resistance against her2-targeted therapy was also stressed in the bolero-3 trial.33 34 patients with advanced taxane - pretreated bc who had advanced on prior trastuzumab therapy were treated with vinorelbine plus trastuzumab with or without everolimus . the primary end point ( pfs ) was met , indicating a moderate prolongation of median pfs from 5.78 months ( 95% ci 5.49 to 6.90 ) to 7.00 months ( 95% ci 6.74 to 8.18 ) with the addition of everolimus ( hr 0.78 ; 95% ci 0.65 to 0.95 ; p=0.0067 ) . grade 3/4 side effects observed more commonly in the everolimus group included neutropenia ( 73% ) , leucopenia ( 38% ) , anaemia ( 19% ) , febrile neutropenia ( 16% ) , stomatitis ( 13% ) and fatigue ( 12% ) . serious adverse events were reported in 42% of patients in the everolimus arm as compared with 20% in the placebo group . on the basis of these results , the authors stated that the clinical benefit observed needs to be balanced in the context of the toxicity profile in an mbc population . owing to this fact , mtor inhibitors have not yet found their way into the clinical routine in her2-positive mbc . finally , novel antibodies may also help in optimising outcome in patients with her2-positive mbc . margetuximab is a chimeric monoclonal antibody with optimised fc fraction in order to increase antibody - dependent cell - mediated cytotoxicity activity . in a phase i trial , 52 patients with different her2-positive malignancies without any further standard therapy available were included.35 in the bc subset consisting of 19 participants , a reduction in tumour size was observed in 57.9% of patients . treatment was generally well tolerated , with grade i / ii fever and nausea as well as diarrhoea and fatigue being the main toxicities . heterodimers of her2 and her3 are known to be the most potent inducers of her2 signalling.36 furthermore , upregulation of her3 was also described as a relevant mechanism of resistance to treatment with trastuzumab and lapatinib.37 38 recently , it was shown that co - expression of her2 and her3 was associated with shorter os in patients with her2-positive mbc treated with trastuzumab in multiple lines.39 therefore , it appears reasonable to examine strategies of targeting her3 in combination with her2 . in a phase i trial , patritumab was added to trastuzumab plus paclitaxel in 18 patients with her2-positive mbc who had received at least one prior treatment line for mbc ; this fully human monoclonal antibody targets her3 , blocks receptor ligand interaction and receptor activation and induces her3 downregulation . tolerability of this approach was excellent and no dlts were observed with diarrhoea being the main side effect ; the authors reported a relatively high response rate of 38.9% , suggesting that this approach should be investigated further in future clinical trials.40 luminal bc is defined by the expression of the er and progesterone receptor ; the vast majority of all bc cases belong to this subtype . hormone - dependent cancer growth allows for targeted therapy with antihormonal agents . in this context , surgical oophorectomy , first described by beatson,41 is regarded as the first systemic anticancer therapy and the prototype of the concept of biologically targeted treatment . the former adjuvant treatment standard of tamoxifen administered for 5 years reduces recurrence risk by half and bc mortality by one - third.42 in the metastatic setting , sequential administration of different non - cross resistant classes of antihormonal therapy allows for delaying initiation of cytotoxic chemotherapy.43 still , even in highly hormone expressing tumours , resistance to endocrine therapy will eventually arise . several factors add to secondary resistance : ccnd1 amplification , esr1 mutation and activation of growth factor signalling pathways.4447 the latter results in er activation even in the absence of estrogen via the activation of signalling molecules downstream of receptor tyrosine kinases ( rtks ) ; phosphatidyl - inositol-3-kinase ( pi3k ) and mtor have been identified as central downstream molecules in this crosstalk leading to the testing of mtor inhibitors such as everolimus in combination with endocrine therapy in clinical trials . in the prospective randomised placebo - controlled trial bolero-2 , postmenopausal patients with er - positive advanced bc progressing on or after prior therapy with a non - steroidal aromatase inhibitor ( ai ) were randomised to receive everolimus or placebo in combination with the steroidal ai exemestane . bolero-2 was able to show a clear - cut benefit in terms of pfs ( 7.4 vs 3.2 months ; hr 0.34 ; 95% ci 0.36 to 0.53)48 for patients in the everolimus group . this supported findings from the randomised phase ii trial tamrad49 and the horizon50 trials ; on the downside , a significant os advantage could not be shown ( bolero-2 : os hr 0.89 ; 31.0 vs 26.6 months , p=0.14 ) and a relevant increase in toxicity was observed as well . pik3ca , a gene coding for the catalytic p110 subunit of pi3k , is among the most commonly mutated genes in bc51 ; mutations have been linked to endocrine resistance by activating the aforementioned crosstalk between er and rtks , thus suggesting to serve as a predictive marker for response of drugs targeting the pi3k / mtor pathway . indeed , activity of mtor inhibitors might be restricted to patients harbouring alterations in the pi3k / akt / mtor pathway , such as pi3kca mutations or pten deletions.52 while this notion is supported by preclinical data53 biological subprotocols of bolero-2 and tamrad suggested that , in contrast to expectation , patients without activating alterations in the pi3k / mtor pathway derived the greatest benefit from treatment.54 55 the quest for predictive markers for novel targeted therapies in luminal bc is therefore ongoing and interest in this field has recently focused on pi3k - inhibitors . while it appears reasonable to assume that pi3k - inhibitors may be most active in tumours harbouring activating pik3ca mutations , no such clinical correlation was proven henceforth . while the addition of bkm120 ( buparlisib , a pan - class pi3k inhibitor ) increased activity of endocrine therapy over endocrine therapy alone in the belle-2 trial , this effect was overall modest ( hr 0.78 , pfs 6.9 vs 5.0 months , p<0.001 ) and considerable toxicity was observed as well . of note , in a prospective substudy within belle-2 , it was shown that pik3ca mutations in cell - free tumour dna ( ctdna ) predicted further activity of buparlisib in this subset of patients ( hr 0.56 , pfs 7.0 vs 3.2 months , p<0.001).56 one hypothesis suggests that pik3ca mutations may predict especially for activity of -specific inhibitors of the p110 catalytic subunit of class i pi3k , but clinical proof of this concept is still awaited . if these data can be verified , however , a predictive biomarker beyond the er will be available in luminal bc for the first time . cyclin - dependent kinase 4 and 6 ( cdk 4/6 ) inhibitors are a novel and promising treatment approach as dysregulation of cell cycle control may cause resistance to antihormonal treatment as well . palbociclib , an irreversible inhibitor of cdk 4/6 , when added to letrozole , has been shown to prolong progression - free interval over letrozole alone to a significant and clinically relevant extent . in the prospective , randomised phase ii trial paloma-1/trio-18,57 median pfs at 29.6 months median follow - up was prolonged from 10.2 to 20.1 months ( hr 0.488 ; 95% ci 0.319 to 0.748 ; one - sided p=0.0004 ) with the addition of palbociclib while the tolerability profile was generally favourable . the main toxicity consisted of neutropenia and was easily manageable with dose delays and modifications . patients were enrolled into two separate sequential cohorts : cohort 1 ( n=66 ) included unselected patients with er - positive , her2-negative tumours ; cohort 2 ( n=99 ) enrolled patients with luminal cancers and ccdn1 amplification or p16 loss . disappointingly , however , results demonstrated that no reliable response prediction is possible on the basis of these markers . recently , the favourable results of paloma-1 were duplicated in the prospective randomised phase iii trial paloma-3 where palbociclib was added to the pure antiestrogen fulvestrant in pretreated patients . again , addition of palbociclib resulted in a clinically meaningful improvement of median pfs from 3.8 to 9.2 months ( hr 0.422 ; 95% ci 0.318 to 0.560 ; p<0.000001 ) ; of note , this study included premenopausal women as well and results were consistent in this population . no new safety signals were observed and main toxicity again consisted of neutropenia ( 78.8% vs 3.5% ) , leucopenia ( 45.5% vs 4.1% ) , and fatigue ( 38.0% vs 26.7% ) . still , febrile neutropenia was rare and comparable in between both groups with 0.6% each ; furthermore , the discontinuation rate due to adverse events was identical as well ( 2.0% vs 1.7%).58 several other trials of cdk 4/6 inhibitors in bc are currently ongoing . their results might add to the notion that a hormone - independent treatment modality is able to prolong the interval to development of hormone refractory disease , as also seen in other malignancies , such as prostate cancer.59 the role of immunotherapy and immune checkpoint modulators in luminal bc has not been fully characterised , although safety and comparably good response rates have been reported after multiple lines of therapy for patients suffering from programmed death ligand 1 ( pd - l1)-overexpressing tumours in the keynote-028 trial.60 further investigation in this field is greatly needed to objectify biological activity and to find biomarkers aside from pd - l1 that would point to responsiveness towards immune checkpoint inhibition in this setting . as indicated by the negative definition of being a bc subtype without expression of the er and progesterone receptor as well as by the lack of her2 protein overexpression or her2/neu gene amplification , tnbc is the disease subtype with the least clinically relevant attributes . this lack of potential targets is clinically reflected by the poor prognosis of patients with tnbc . chemotherapy remains the mainstay of tnbc therapy and patients with pathological complete remission to neoadjuvant chemotherapy are known to have a favourable prognosis despite the inherent aggressiveness of this disease subtype.61 patients with a less chemotherapy - sensitive disease , on the other hand , have a markedly poor prognosis with high recurrence rates and mortality . this warrants the search for novel targets in tnbc . around 10% of all bc cases can be attributed to brca-1/2 germline mutations with considerably higher rates of brca-1 mutations in women with tnbc.62 further , women with early - onset bc are at a higher risk of carrying brca-1/2 mutations , with brca-1 being the more frequently altered gene.63 64 owing to consecutive defects in genetic repair mechanisms such as homologous repair , brca - deficient cells must resort to other error - prone options of double - strand brake repair such as non - homologous end - joining ( nhej ) and base excision repair ( ber).65 both repair mechanisms depend on parp ( poly-(adp - ribose)-polymerases ) as molecular scaffolds for repairing double - strand breaks , making parp inhibitors either alone or in combination with dna - damaging agents a promising treatment option , inducing synthetic lethality due to loss of parp and brca-1/2 functions.66 a single - arm multicentre phase ii trial of olaparib as a single agent was conducted in patients with different malignancies harbouring brca-1/2 germline mutations ; 62 patients with bc who had received at least three prior treatment lines for mbc were included . in this subset , there was an overall response rate of 12.9% ( 95% ci 5.7% to 23.9% ) with an additional 47% ( 95% ci 34.0% to 59.9% ) of patients experiencing disease stabilisation for a minimum of 8 weeks.67 on the other hand , activity of olaparib in an unselected tnbc population was disappointing.68 several trials of parp inhibitors in bc are currently ongoing ; of special interest , the randomised phase iii trial olympia ( nct02032823 ) focuses on the potential role of olaparib as adjuvant therapy in patients with high - risk her2-negative primary bc harbouring brca germline mutations . in this context , it needs to be mentioned that , in contrast to germline brca mutations , the predictive role of the more abundant somatic brca mutations in bc remains elusive.69 the quest for novel therapeutic approaches has brought to light various potential targets including rtks such as met and fgf . met and its ligand hepatocyte growth factor are considered especially promising targets , being overexpressed in up to 45% of bcs and associated with poor clinical outcome , increased propensity to metastatic spread , increased tumour cell proliferation , high grading and a triple - negative phenotype . of note , the met - inhibitor cabozantinib has recently shown activity in humanised patient - derived xenograft models of tnbc.70 furthermore , phase i clinical trials have also suggested promising activity and relative safety of met inhibitors such as cabozantinib and foretinib , and results of later - phase clinical trials are therefore awaited with great interest.71 72 on the other side , targeting the extracellular domain of met by means of the monoclonal humanised antibody onartuzumab has currently shown no benefit when added to paclitaxel / bevacizumab ; in this context , it needs to be stated that most patients included in this study ( 88% ) had only weak met expression as defined by immunohistochemistry,73 raising the question whether this study was indeed conducted in the right population . fgf was shown to play a significant role in up to 10% of patients with bc,74 facilitating epithelial to mesenchymal transition and hence metastatic spread . subsequently , clinical trials of tkis targeting selected ( azd4547 ) or multiple subforms of fibroblast growth factor receptor ( fgfr ) ( dovitinib,75 lucitanib76 or nintedanib77 ) were initiated ; of note , the latter showed promising activity when combined with paclitaxel in a neoadjuvant setting . the rationale for the approach of fgfr - targeted therapy is to delay mesenchymal dedifferentiation and hence resistance to chemotherapy and targeted therapy that had originally been tailored for epithelial tumours . although activity was also seen in luminal tumours,74 75 recent trials using fgfr inhibitors were focusing on the triple - negative setting because of inherent toxicity and missing alternative treatments in tnbc . as with other malignancies , immune checkpoint modulators are currently being investigated in tnbc , although clinical development started with considerable delay as compared with melanoma or lung cancer . tnbc is characterised by high mutational load , which again is apparently associated with high immunogenicity.78 this makes tnbc an attractive field for developing immunotherapy in bc . first data on immunotherapy with immune checkpoint modulators in tnbc were presented at the 2014 san antonio breast cancer symposium.79 thirty - two patients with pretreated mbc received pembrolizumab ( mk-3475 ) , a humanised igg4 monoclonal antibody targeting pd-1 within the bc cohort of the phase 1b trial keynote-012 . of note , approximately 45% had already received at least three prior treatment lines for metastatic disease . while the overall response rate was relatively low at 18.5% , it is worth mentioning that responses were observed even in heavily pretreated participants and some patients seemed to experience prolonged disease stabilisation . another phase i trial tested mdpl3280a , an anti - pd - l1 antibody , in 54 patients with tnbc.80 again , 85% of all patients had at least four prior systemic therapy lines . in 21 evaluable patients , the authors observed a response rate of 19% , with two complete responses and two partial responses ; although it is too early to draw any firm conclusions , it must be mentioned that these four patients had high expression of pretreatment pd - l1 . recently , the phase ib javelin study reported first results from the bc cohort.81 the anti - pd - l1 antibody avelumab ( msb0010718c ) yielded an overall relative reponse ( rr ) of 4.8% in 168 unselected patients with pretreated mbc . in patients with tnbc ( 34.5% ) , rr was somewhat higher ( 8.6% ) but responses were seen across all subtypes with a median duration of response of 28.7 weeks . when analysing patients with tumours harbouring 10% pd - l1 expression , a response rate of 33% in the overall population was observed ; rr in patients with tnbc was a striking 44% . in summary , these data suggest that immunotherapy is indeed a valuable addition to the therapeutic armamentarium in tnbc ( table 1 ) . selected drugs and their molecular targets undergoing clinical evaluation in mbc lapatinib ( her family ) neratinib ( her family ) afatinib ( her family ) temsirolimus ( mtorc1 ) palbociclib ( cdk 4/6 ) cdk 4/6 , cyclin - dependent kinase 4 and 6 ; fgfr , fibroblast growth factor receptor ; mbc , metastatic breast cancer ; mtorc1 , mammalian target of rapamycine complex 1 ; pd - l1 , programmed death ligand 1 . following the discovery of crucial molecular signalling mechanisms , targeted drugs have been developed for mbc . although generally applicable as a concept , the identification of a true driver pathway , the identification of biomarkers for patient selection and , finally and most importantly , the therapeutic efficacy of a specific targeted approach has to be proven for each bc subtype and each setting separately . an abundance of drugs is in development which will challenge the scientific community to develop appropriate models for preclinical as well as clinical testing of these compounds but may eventually help in further improving patient outcomes .

biologically distinct subtypes of metastatic breast cancer ( mbc ) have been defined by multiple efforts in recent years , showing broad heterogeneity at the molecular level of disease . throughout this endeavour , oncogenic drivers within mbc were identified as potential therapeutic targets . with recent results from clinical trials targeting these well - known cancer - promoting pathways , this review is trying to elucidate as well as summarise current new therapeutic aspects in mbc and shed light on translational aspects within this entity .

Introduction Methods HER2-overexpressing advanced BC Luminal BC Triple-negative BC (TNBC) Conclusion

the biological heterogeneity of breast cancer ( bc ) has been defined by various efforts in recent years , taking into account the distinct molecular and histopathological features of these tumours . luminal - type and basal - like bcs have been shown to be completely different diseases at the molecular level , as well as in terms of the course of the disease , patient prognosis and survival.16 while the luminal subtype , characterised by the expression of estrogen and/or progesterone receptors ( er / pr ) , shows well - known characteristics of adenocarcinoma , basal - like phenotypes exhibit a wider and more continuous spectrum of genomic evolution and have been linked to biological features of other malignancies.3 with recent results from clinical trials targeting well - known cancer - promoting pathways , this review is seeking to elucidate and summarise current new therapeutic aspects in metastatic bc ( mbc ) and shed light on translational aspects within this entity . articles from peer - reviewed journals as well as published abstracts were searched for using ncbis pubmed as well as esmo , asco , aacr and sabcs online library databases as of 22 march 2016 . her2 , luminal breast cancer , triple - negative , translational , hormone , metastases , brain , bone and names of medications as well as gene and protein symbols of therapeutic targets dealt with in this manuscript . targeted therapy in mbc consists of approaches where well - established or novel pathways are being targeted with the aim of prolonged disease control.79 besides the er , targeting her2 is today regarded as the best established targeted treatment approach in mbc . tdm1 was shown to be superior to lapatinib , a small molecule tyrosine kinase inhibitor ( tki ) of her2 and epidermal growth factor receptor ( egfr ) , plus capecitabine in terms of activity as well as tolerability in the phase iii trial emilia with pfs 9.6 vs 6.4 months ( hr 0.65 ; 95% ci 0.55 to 0.77).16 most patients received tdm1 as second - line therapy but 16% of patients had progressed on or within 6 months after the end of adjuvant trastuzumab ; this lead to the approval of tdm1 as first - line treatment standard in earlier relapse . owing to the use of small amounts of its active agent doxorubicin , it caused only minor haematological toxicity when used as a monotherapy or in combination with trastuzumab , as well as with trastuzumab and cyclophosphamide in a phase i study . this first - generation , reversible tki inhibits the tyrosine - kinase domains of her2 as well as egfr . in this study including 121 patients , both regimen containing afatinib did not yield gains in terms of clinical benefit rate at 12 weeks ( which was defined as primary study end point ) as well as pfs or os . a phase i basket study of 60 patients with her2-overexpressing carcinomas was able to shed light on a potential future avenue for the development of neratinib ; in this trial , neratinib was combined with temsirolimus in order to achieve inhibition of her2-signalling by the additional abolishment of one of its major downstream effector kinases , mammalian target of rapamycin ( mtor ) . this combination produced responses in 2 of the 15 patients ( 13.3% ) with mbc resistant to trastuzumab ; on the downside , this dual blockade led to relevant treatment - related toxicity : diarrhoea was observed in 93% of all patients and also held responsible for 4 of the 10 dose - limiting toxicities ( dlts ) ; dose - limiting metabolic alterations were observed as well.30 a subsequent phase i / ii trial of neratinib plus temsirolimus in her2-positive mbc31 reported a partial response rate of 30% for patients treated at both the maximum tolerable dose ( mtd ) and dose escalation ( de ) cohorts ( median duration of response 3.0 and 7.4 months , respectively ) . while concerns regarding tolerability remain , these findings again strongly supported a model of ongoing her2 pathway addiction even in mbc resistant to trastuzumab . treatment was generally well tolerated , with grade i / ii fever and nausea as well as diarrhoea and fatigue being the main toxicities . tolerability of this approach was excellent and no dlts were observed with diarrhoea being the main side effect ; the authors reported a relatively high response rate of 38.9% , suggesting that this approach should be investigated further in future clinical trials.40 luminal bc is defined by the expression of the er and progesterone receptor ; the vast majority of all bc cases belong to this subtype . several factors add to secondary resistance : ccnd1 amplification , esr1 mutation and activation of growth factor signalling pathways.4447 the latter results in er activation even in the absence of estrogen via the activation of signalling molecules downstream of receptor tyrosine kinases ( rtks ) ; phosphatidyl - inositol-3-kinase ( pi3k ) and mtor have been identified as central downstream molecules in this crosstalk leading to the testing of mtor inhibitors such as everolimus in combination with endocrine therapy in clinical trials . this supported findings from the randomised phase ii trial tamrad49 and the horizon50 trials ; on the downside , a significant os advantage could not be shown ( bolero-2 : os hr 0.89 ; 31.0 vs 26.6 months , p=0.14 ) and a relevant increase in toxicity was observed as well . pik3ca , a gene coding for the catalytic p110 subunit of pi3k , is among the most commonly mutated genes in bc51 ; mutations have been linked to endocrine resistance by activating the aforementioned crosstalk between er and rtks , thus suggesting to serve as a predictive marker for response of drugs targeting the pi3k / mtor pathway . while the addition of bkm120 ( buparlisib , a pan - class pi3k inhibitor ) increased activity of endocrine therapy over endocrine therapy alone in the belle-2 trial , this effect was overall modest ( hr 0.78 , pfs 6.9 vs 5.0 months , p<0.001 ) and considerable toxicity was observed as well . again , addition of palbociclib resulted in a clinically meaningful improvement of median pfs from 3.8 to 9.2 months ( hr 0.422 ; 95% ci 0.318 to 0.560 ; p<0.000001 ) ; of note , this study included premenopausal women as well and results were consistent in this population . still , febrile neutropenia was rare and comparable in between both groups with 0.6% each ; furthermore , the discontinuation rate due to adverse events was identical as well ( 2.0% vs 1.7%).58 several other trials of cdk 4/6 inhibitors in bc are currently ongoing . their results might add to the notion that a hormone - independent treatment modality is able to prolong the interval to development of hormone refractory disease , as also seen in other malignancies , such as prostate cancer.59 the role of immunotherapy and immune checkpoint modulators in luminal bc has not been fully characterised , although safety and comparably good response rates have been reported after multiple lines of therapy for patients suffering from programmed death ligand 1 ( pd - l1)-overexpressing tumours in the keynote-028 trial.60 further investigation in this field is greatly needed to objectify biological activity and to find biomarkers aside from pd - l1 that would point to responsiveness towards immune checkpoint inhibition in this setting . as indicated by the negative definition of being a bc subtype without expression of the er and progesterone receptor as well as by the lack of her2 protein overexpression or her2/neu gene amplification , tnbc is the disease subtype with the least clinically relevant attributes . of note , the met - inhibitor cabozantinib has recently shown activity in humanised patient - derived xenograft models of tnbc.70 furthermore , phase i clinical trials have also suggested promising activity and relative safety of met inhibitors such as cabozantinib and foretinib , and results of later - phase clinical trials are therefore awaited with great interest.71 72 on the other side , targeting the extracellular domain of met by means of the monoclonal humanised antibody onartuzumab has currently shown no benefit when added to paclitaxel / bevacizumab ; in this context , it needs to be stated that most patients included in this study ( 88% ) had only weak met expression as defined by immunohistochemistry,73 raising the question whether this study was indeed conducted in the right population . subsequently , clinical trials of tkis targeting selected ( azd4547 ) or multiple subforms of fibroblast growth factor receptor ( fgfr ) ( dovitinib,75 lucitanib76 or nintedanib77 ) were initiated ; of note , the latter showed promising activity when combined with paclitaxel in a neoadjuvant setting . first data on immunotherapy with immune checkpoint modulators in tnbc were presented at the 2014 san antonio breast cancer symposium.79 thirty - two patients with pretreated mbc received pembrolizumab ( mk-3475 ) , a humanised igg4 monoclonal antibody targeting pd-1 within the bc cohort of the phase 1b trial keynote-012 . selected drugs and their molecular targets undergoing clinical evaluation in mbc lapatinib ( her family ) neratinib ( her family ) afatinib ( her family ) temsirolimus ( mtorc1 ) palbociclib ( cdk 4/6 ) cdk 4/6 , cyclin - dependent kinase 4 and 6 ; fgfr , fibroblast growth factor receptor ; mbc , metastatic breast cancer ; mtorc1 , mammalian target of rapamycine complex 1 ; pd - l1 , programmed death ligand 1 . targeted therapy in mbc consists of approaches where well - established or novel pathways are being targeted with the aim of prolonged disease control.79 besides the er , targeting her2 is today regarded as the best established targeted treatment approach in mbc . tdm1 was shown to be superior to lapatinib , a small molecule tyrosine kinase inhibitor ( tki ) of her2 and epidermal growth factor receptor ( egfr ) , plus capecitabine in terms of activity as well as tolerability in the phase iii trial emilia with pfs 9.6 vs 6.4 months ( hr 0.65 ; 95% ci 0.55 to 0.77).16 most patients received tdm1 as second - line therapy but 16% of patients had progressed on or within 6 months after the end of adjuvant trastuzumab ; this lead to the approval of tdm1 as first - line treatment standard in earlier relapse . owing to the use of small amounts of its active agent doxorubicin , it caused only minor haematological toxicity when used as a monotherapy or in combination with trastuzumab , as well as with trastuzumab and cyclophosphamide in a phase i study . this first - generation , reversible tki inhibits the tyrosine - kinase domains of her2 as well as egfr . in this study including 121 patients , both regimen containing afatinib did not yield gains in terms of clinical benefit rate at 12 weeks ( which was defined as primary study end point ) as well as pfs or os . a phase i basket study of 60 patients with her2-overexpressing carcinomas was able to shed light on a potential future avenue for the development of neratinib ; in this trial , neratinib was combined with temsirolimus in order to achieve inhibition of her2-signalling by the additional abolishment of one of its major downstream effector kinases , mammalian target of rapamycin ( mtor ) . this combination produced responses in 2 of the 15 patients ( 13.3% ) with mbc resistant to trastuzumab ; on the downside , this dual blockade led to relevant treatment - related toxicity : diarrhoea was observed in 93% of all patients and also held responsible for 4 of the 10 dose - limiting toxicities ( dlts ) ; dose - limiting metabolic alterations were observed as well.30 a subsequent phase i / ii trial of neratinib plus temsirolimus in her2-positive mbc31 reported a partial response rate of 30% for patients treated at both the maximum tolerable dose ( mtd ) and dose escalation ( de ) cohorts ( median duration of response 3.0 and 7.4 months , respectively ) . treatment was generally well tolerated , with grade i / ii fever and nausea as well as diarrhoea and fatigue being the main toxicities . tolerability of this approach was excellent and no dlts were observed with diarrhoea being the main side effect ; the authors reported a relatively high response rate of 38.9% , suggesting that this approach should be investigated further in future clinical trials.40 luminal bc is defined by the expression of the er and progesterone receptor ; the vast majority of all bc cases belong to this subtype . several factors add to secondary resistance : ccnd1 amplification , esr1 mutation and activation of growth factor signalling pathways.4447 the latter results in er activation even in the absence of estrogen via the activation of signalling molecules downstream of receptor tyrosine kinases ( rtks ) ; phosphatidyl - inositol-3-kinase ( pi3k ) and mtor have been identified as central downstream molecules in this crosstalk leading to the testing of mtor inhibitors such as everolimus in combination with endocrine therapy in clinical trials . while the addition of bkm120 ( buparlisib , a pan - class pi3k inhibitor ) increased activity of endocrine therapy over endocrine therapy alone in the belle-2 trial , this effect was overall modest ( hr 0.78 , pfs 6.9 vs 5.0 months , p<0.001 ) and considerable toxicity was observed as well . cyclin - dependent kinase 4 and 6 ( cdk 4/6 ) inhibitors are a novel and promising treatment approach as dysregulation of cell cycle control may cause resistance to antihormonal treatment as well . again , addition of palbociclib resulted in a clinically meaningful improvement of median pfs from 3.8 to 9.2 months ( hr 0.422 ; 95% ci 0.318 to 0.560 ; p<0.000001 ) ; of note , this study included premenopausal women as well and results were consistent in this population . their results might add to the notion that a hormone - independent treatment modality is able to prolong the interval to development of hormone refractory disease , as also seen in other malignancies , such as prostate cancer.59 the role of immunotherapy and immune checkpoint modulators in luminal bc has not been fully characterised , although safety and comparably good response rates have been reported after multiple lines of therapy for patients suffering from programmed death ligand 1 ( pd - l1)-overexpressing tumours in the keynote-028 trial.60 further investigation in this field is greatly needed to objectify biological activity and to find biomarkers aside from pd - l1 that would point to responsiveness towards immune checkpoint inhibition in this setting . as indicated by the negative definition of being a bc subtype without expression of the er and progesterone receptor as well as by the lack of her2 protein overexpression or her2/neu gene amplification , tnbc is the disease subtype with the least clinically relevant attributes . around 10% of all bc cases can be attributed to brca-1/2 germline mutations with considerably higher rates of brca-1 mutations in women with tnbc.62 further , women with early - onset bc are at a higher risk of carrying brca-1/2 mutations , with brca-1 being the more frequently altered gene.63 64 owing to consecutive defects in genetic repair mechanisms such as homologous repair , brca - deficient cells must resort to other error - prone options of double - strand brake repair such as non - homologous end - joining ( nhej ) and base excision repair ( ber).65 both repair mechanisms depend on parp ( poly-(adp - ribose)-polymerases ) as molecular scaffolds for repairing double - strand breaks , making parp inhibitors either alone or in combination with dna - damaging agents a promising treatment option , inducing synthetic lethality due to loss of parp and brca-1/2 functions.66 a single - arm multicentre phase ii trial of olaparib as a single agent was conducted in patients with different malignancies harbouring brca-1/2 germline mutations ; 62 patients with bc who had received at least three prior treatment lines for mbc were included . of note , the met - inhibitor cabozantinib has recently shown activity in humanised patient - derived xenograft models of tnbc.70 furthermore , phase i clinical trials have also suggested promising activity and relative safety of met inhibitors such as cabozantinib and foretinib , and results of later - phase clinical trials are therefore awaited with great interest.71 72 on the other side , targeting the extracellular domain of met by means of the monoclonal humanised antibody onartuzumab has currently shown no benefit when added to paclitaxel / bevacizumab ; in this context , it needs to be stated that most patients included in this study ( 88% ) had only weak met expression as defined by immunohistochemistry,73 raising the question whether this study was indeed conducted in the right population . subsequently , clinical trials of tkis targeting selected ( azd4547 ) or multiple subforms of fibroblast growth factor receptor ( fgfr ) ( dovitinib,75 lucitanib76 or nintedanib77 ) were initiated ; of note , the latter showed promising activity when combined with paclitaxel in a neoadjuvant setting . first data on immunotherapy with immune checkpoint modulators in tnbc were presented at the 2014 san antonio breast cancer symposium.79 thirty - two patients with pretreated mbc received pembrolizumab ( mk-3475 ) , a humanised igg4 monoclonal antibody targeting pd-1 within the bc cohort of the phase 1b trial keynote-012 . selected drugs and their molecular targets undergoing clinical evaluation in mbc lapatinib ( her family ) neratinib ( her family ) afatinib ( her family ) temsirolimus ( mtorc1 ) palbociclib ( cdk 4/6 ) cdk 4/6 , cyclin - dependent kinase 4 and 6 ; fgfr , fibroblast growth factor receptor ; mbc , metastatic breast cancer ; mtorc1 , mammalian target of rapamycine complex 1 ; pd - l1 , programmed death ligand 1 . following the discovery of crucial molecular signalling mechanisms , targeted drugs have been developed for mbc . an abundance of drugs is in development which will challenge the scientific community to develop appropriate models for preclinical as well as clinical testing of these compounds but may eventually help in further improving patient outcomes .

hereditary haemorrhagic telangiectasia ( hht ) is an autosomal dominant inherited disease characterised by vascular malformations , ranging from small telangiectases in skin and mucosal membranes to large visceral arteriovenous malformations ( avms ) predominantly localised in the lungs , brain and liver [ 13 ] . pulmonary arteriovenous malformations ( pavms ) are abnormally dilated vessels between pulmonary arteries and veins that cause a permanent extra - cardiac right - to - left shunt , which carries the risk of cerebral paradoxical embolisation of both thrombotic and septic origin . transcatheter embolisation of pavms can be safely performed , in order to prevent these potentially severe neurological complications , such as ischaemic stroke or cerebral abscess [ 3 , 4 ] . currently , there are no data regarding the potential haemodynamic changes occurring directly after pavm embolisation . therefore , the present study investigated the direct haemodynamic effects of pavm embolisation , using non - invasive finger pressure measurements . between 2008 and 2010 , we included 29 patients who underwent transcatheter embolisation of pavms in the st . a pavm was defined as a direct communication between a pulmonary artery and a pulmonary vein , bypassing the pulmonary capillary filter , and was diagnosed using transthoracic contrast echocardiography ( ttce ) and subsequent chest computed tomography ( ct ) . before and after pavm embolisation , the right - to - left shunt fraction was measured using the 100 % oxygen method as previously described . pavms with a feeding artery diameter of 23 mm or greater were found suitable for embolisation therapy . 1a c ) was performed under local anaesthesia ( lidocaine 1 % ) . percutaneous access was derived through the right femoral vein and a six french sheath was inserted . the interventional radiologist selected the pavm closure device , based on the diameter and anatomy of the pavm . the most preferred closure device was the amplatzer vascular plug ( aga medical , golden valley , mn , usa ) ( fig . plugs with a diameter of 412 mm were used . if pavm closure with a plug was not possible , detachable coils ( boston scientific , natick , ma ) the embolic material was implanted under fluoroscopic guidance , with a maximum contrast volume of 300 ml ( xenetix ; iobitrol , guerbet , villepinte , france ) . within 24 h after embolisation c embolisation of pulmonary arteriovenous malformation in the left lower lobe with a amplatzer vascular plug . d amplatzer vascular plug a pulmonary angiogram of pulmonary arteriovenous malformation in the left lower lobe . c embolisation of pulmonary arteriovenous malformation in the left lower lobe with a amplatzer vascular plug . d amplatzer vascular plug during the transcatheter embolisation of pavms , arterial pressure was measured on a finger of the left hand using a finometer device ( fms , finapres medical systems , amsterdam , the netherlands ) . the finometer measures blood pressure by a combination of the volume clamp method of penaz and the physiocal criteria developed by wesseling [ 810 ] . the hand was kept at heart level and a cuff was wrapped around the same arm for individual blood pressure calibration using the return - to - flow calibration [ 8 , 11 ] . because of potential distortion of the measurements at the time of pavm closure , parameters were recorded after stabilisation of the finger pressure signal during a blanking period of 1 min immediately before and after placement of the first and the last plug . during the pavm embolisation , finger pressure measurements with the associated event marks were monitored and digitally stored . review of these data was performed using beatscope software . five minute averages of systolic ( sbp ) , diastolic ( dbp ) and mean blood pressure ( mbp ) , heart rate ( hr ) , stroke volume ( sv ) , cardiac output ( co ) , total peripheral resistance ( tpr ) and delta pressure / delta time ( dp / dt ) were calculated after an electronic calibration procedure . sv and co ( co is the product of sv and hr ) were calculated from the finger pressure wave using the modelflow methodology . the cardiac index ( ci ) return - to - flow calibration , using the arm cuff , was used for calibration of the blood pressure . both absolute values and absolute and relative changes ( delta absolute and delta percent ) are presented . differences within groups were analysed performing paired samples t - tests . a significance level of p < 0.05 was considered significant . between 2008 and 2010 , we included 29 patients who underwent transcatheter embolisation of pavms in the st . a pavm was defined as a direct communication between a pulmonary artery and a pulmonary vein , bypassing the pulmonary capillary filter , and was diagnosed using transthoracic contrast echocardiography ( ttce ) and subsequent chest computed tomography ( ct ) . before and after pavm embolisation , the right - to - left shunt fraction was measured using the 100 % oxygen method as previously described . pavms with a feeding artery diameter of 23 mm or greater were found suitable for embolisation therapy . the procedure ( fig . 1a c ) was performed under local anaesthesia ( lidocaine 1 % ) . percutaneous access was derived through the right femoral vein and a six french sheath was inserted . the interventional radiologist selected the pavm closure device , based on the diameter and anatomy of the pavm . the most preferred closure device was the amplatzer vascular plug ( aga medical , golden valley , mn , usa ) ( fig . plugs with a diameter of 412 mm were used . if pavm closure with a plug was not possible , detachable coils ( boston scientific , natick , ma ) were used . the embolic material was implanted under fluoroscopic guidance , with a maximum contrast volume of 300 ml ( xenetix ; iobitrol , guerbet , villepinte , france ) . within 24 h after embolisation c embolisation of pulmonary arteriovenous malformation in the left lower lobe with a amplatzer vascular plug . d amplatzer vascular plug a pulmonary angiogram of pulmonary arteriovenous malformation in the left lower lobe . c embolisation of pulmonary arteriovenous malformation in the left lower lobe with a amplatzer vascular plug . during the transcatheter embolisation of pavms , arterial pressure was measured on a finger of the left hand using a finometer device ( fms , finapres medical systems , amsterdam , the netherlands ) . the finometer measures blood pressure by a combination of the volume clamp method of penaz and the physiocal criteria developed by wesseling [ 810 ] . the hand was kept at heart level and a cuff was wrapped around the same arm for individual blood pressure calibration using the return - to - flow calibration [ 8 , 11 ] . because of potential distortion of the measurements at the time of pavm closure , parameters were recorded after stabilisation of the finger pressure signal during a blanking period of 1 min immediately before and after placement of the first and the last plug . during the pavm embolisation , finger pressure measurements with the associated event marks were monitored and digitally stored . review of these data was performed using beatscope software . five minute averages of systolic ( sbp ) , diastolic ( dbp ) and mean blood pressure ( mbp ) , heart rate ( hr ) , stroke volume ( sv ) , cardiac output ( co ) , total peripheral resistance ( tpr ) and delta pressure / delta time ( dp / dt ) were calculated after an electronic calibration procedure . sv and co ( co is the product of sv and hr ) were calculated from the finger pressure wave using the modelflow methodology . the cardiac index ( ci ) return - to - flow calibration , using the arm cuff , was used for calibration of the blood pressure . both absolute values and absolute and relative changes ( delta absolute and delta percent ) are presented . the statistics were performed using spss version 17.0 for windows ( spss inc . , chicago , il , usa ) . differences within groups were analysed performing paired samples t - tests . a significance level of p < 0.05 was considered significant . a total of 29 hht patients ( 62 % female , mean age 39.2 15.3 years ) were included , in which 72 pavms were embolised ( mean per patient 2.5 ) . an amplatzer plug was used in 54 cases and a detachable coil in the remaining 18 cases . the baseline characteristics are presented in tables 1 and 2.table 1baseline characteristics of patientstotal29gendermale11 ( 37.9)female18 ( 62.1)age ( years)39.2 15.3bmi ( kg / m)24.0 5.0bsa ( m)1.9 0.2hhtdefinite28 ( 96.6)type 119 ( 65.5)type 22 ( 6.9)type unknown8 ( 27.6)sao2 ( % ) before procedure95.5 3.5after procedure98.4 2.3shunt fraction ( % ) before procedure13.6 8.3after procedure4.9 6.6all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformationtable 2baseline characteristics of embolisation proceduretreated pavms1 pavm9 ( 31.0)2 pavms9 ( 31.0)3 pavms2 ( 6.9)4 pavms8 ( 27.6)>4 pavms1 ( 3.4)closure deviceamplatzer plug54 ( 75.0)coil18 ( 25.0)plug diameter < 4 ( mm)1 ( 1.9)4 ( mm)13 ( 24.1)6 ( mm)13 ( 24.1)8 ( mm)9 ( 16.7)10 ( mm)10 ( 18.5)>10 ( mm)6 ( 11.1)not known2 ( 3.7)all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know baseline characteristics of patients all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformation baseline characteristics of embolisation procedure all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know directly after pavm embolisation the sv and co decreased significantly : 6.4 13.0 ml ( range 45.9 to 17.9 ml ; 11.9 % , p = 0.01 ) and 0.4 0.8 l / min ( range 3.0 to 1.58 l / min ; 9.5 % , p = 0.01 ) . as expected , the ci decreased as well ( range 1.5 to 0.9 l / min / m ; 9.5 % , p = 0.01 ) . dbp and mbp increased significantly by 5.2 10.3 mmhg ( range 9.0 to 32.9 mmhg ; 5.9 % , p = 0.01 ) for dbp and 5.7 12.1 mmhg ( range 12.7 to 32.6 mmhg ; 4.1 % , p = 0.02 ) for mbp , respectively . there was no significant change in sbp ( 4.0 16.3 mmhg ( range 25.5 to 38.5 mmhg ; 1.7 % , p = 0.20 ) . the dp / dt did not change significantly : 2.1 290.0 mmhg / s ( range 750.0 to 848.2 mmhg / s ; 0.2 % , p = 0.97 ) . there was a correlation between the delta dp / dt and the sbp ( pearson coefficient r = 0.73 , r = 0.53 , p < 0.0001 ) . hr and tpr increased , but this appeared to be non - significant : 1.6 7.4 beats / min ( range 17.4 to 21.2 beats / min ; 1.8 % , p = 0.24 ) and 0.1 0.5 woods units ( range 1.62 to 1.39 woods units ; 5.8 % , p = 0.16 ) . these data are summarised in table 3.table 3haemodynamic measurements before and after embolisation with absolute and relative changesbefore sdafter sddelta absolutedelta percentpsbp ( mmhg)144.9 25.3148.9 28.44.01.70.20dpb ( mmhg)85.5 11.590.7 16.35.25.90.01mbp ( mmhg)108.0 16.0113.7 19.95.74.10.02hr ( beats / min)78.8 14.680.4 13.91.61.80.24sv ( ml)70.9 20.964.5 19.36.411.90.01co ( l / min)5.5 1.65.1 1.40.49.50.01ci ( l / min / m)3.0 0.82.8 0.80.29.50.01tpr ( woods units)1.4 0.51.5 0.50.15.80.16dp / dt ( mmhg / s)1,233.7 463.81,235.8 481.72.10.20.97 sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second haemodynamic measurements before and after embolisation with absolute and relative changes sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second a total of 29 hht patients ( 62 % female , mean age 39.2 15.3 years ) were included , in which 72 pavms were embolised ( mean per patient 2.5 ) . an amplatzer plug was used in 54 cases and a detachable coil in the remaining 18 cases . the baseline characteristics are presented in tables 1 and 2.table 1baseline characteristics of patientstotal29gendermale11 ( 37.9)female18 ( 62.1)age ( years)39.2 15.3bmi ( kg / m)24.0 5.0bsa ( m)1.9 0.2hhtdefinite28 ( 96.6)type 119 ( 65.5)type 22 ( 6.9)type unknown8 ( 27.6)sao2 ( % ) before procedure95.5 3.5after procedure98.4 2.3shunt fraction ( % ) before procedure13.6 8.3after procedure4.9 6.6all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformationtable 2baseline characteristics of embolisation proceduretreated pavms1 pavm9 ( 31.0)2 pavms9 ( 31.0)3 pavms2 ( 6.9)4 pavms8 ( 27.6)>4 pavms1 ( 3.4)closure deviceamplatzer plug54 ( 75.0)coil18 ( 25.0)plug diameter < 4 ( mm)1 ( 1.9)4 ( mm)13 ( 24.1)6 ( mm)13 ( 24.1)8 ( mm)9 ( 16.7)10 ( mm)10 ( 18.5)>10 ( mm)6 ( 11.1)not known2 ( 3.7)all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know baseline characteristics of patients all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformation baseline characteristics of embolisation procedure all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know directly after pavm embolisation the sv and co decreased significantly : 6.4 13.0 ml ( range 45.9 to 17.9 ml ; 11.9 % , p = 0.01 ) and 0.4 0.8 l / min ( range 3.0 to 1.58 l / min ; 9.5 % , p = 0.01 ) . as expected , the ci decreased as well ( range 1.5 to 0.9 l / min / m ; 9.5 % , p = 0.01 ) . dbp and mbp increased significantly by 5.2 10.3 mmhg ( range 9.0 to 32.9 mmhg ; 5.9 % , p = 0.01 ) for dbp and 5.7 12.1 mmhg ( range 12.7 to 32.6 mmhg ; 4.1 % , p = 0.02 ) for mbp , respectively . there was no significant change in sbp ( 4.0 16.3 mmhg ( range 25.5 to 38.5 mmhg ; 1.7 % , p = 0.20 ) . the dp / dt did not change significantly : 2.1 290.0 mmhg / s ( range 750.0 to 848.2 mmhg / s ; 0.2 % , p = 0.97 ) . there was a correlation between the delta dp / dt and the sbp ( pearson coefficient r = 0.73 , r = 0.53 , p < 0.0001 ) . hr and tpr increased , but this appeared to be non - significant : 1.6 7.4 beats / min ( range 17.4 to 21.2 beats / min ; 1.8 % , p = 0.24 ) and 0.1 0.5 woods units ( range 1.62 to 1.39 woods units ; 5.8 % , p = 0.16 ) . these data are summarised in table 3.table 3haemodynamic measurements before and after embolisation with absolute and relative changesbefore sdafter sddelta absolutedelta percentpsbp ( mmhg)144.9 25.3148.9 28.44.01.70.20dpb ( mmhg)85.5 11.590.7 16.35.25.90.01mbp ( mmhg)108.0 16.0113.7 19.95.74.10.02hr ( beats / min)78.8 14.680.4 13.91.61.80.24sv ( ml)70.9 20.964.5 19.36.411.90.01co ( l / min)5.5 1.65.1 1.40.49.50.01ci ( l / min / m)3.0 0.82.8 0.80.29.50.01tpr ( woods units)1.4 0.51.5 0.50.15.80.16dp / dt ( mmhg / s)1,233.7 463.81,235.8 481.72.10.20.97 sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second haemodynamic measurements before and after embolisation with absolute and relative changes sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second to our knowledge , this is the first study describing the occurrence of significant haemodynamic changes directly after transcatheter embolisation of pavms . using the finometer and modelflow methodology , our study accurately recorded beat - to - beat non - invasive finger pressure measurements and thereby the immediate haemodynamic changes after pavm embolisation , amongst which a decrease in sv and co were most important . only one case report previously documented haemodynamic changes 4 months after pavm embolisation with a marked reduction in co of 5.1 l / min ( 41 % ) . this seems to be in line with the results in our current study in 29 hht patients who all underwent transcatheter embolisation of pavms . however , we found a less pronounced decrease in co , which can be explained by the smaller right - to - left shunts in our study population , with a mean shunt fraction before closure of 14 % versus 31 % described by andrivet et al . . it is possible that the co may further decrease over time as a result of additional thrombosis of the plug or coil in the pavm feeding artery . however , the long - term haemodynamic changes after pavm embolisation remain hard to predict , since new pavms may occur and existing pavms may grow , so this is still subject for larger studies in the future . the haemodynamic responses after pavm embolisation may differ between hht patients , which can be related to difference in number and size of pavms at baseline . in the present study , nine patients underwent embolisation of at least 4 pavms in one session , whereas less pavms were embolised in the remaining 20 patients . furthermore , a total of 16 pavms were treated with a large plug ( diameter 10 mm ) , whereas 14 pavms could be treated with smaller endovascular plugs ( diameter 4 mm ) . unfortunately , we could not find a significant association between plug size ( size of the pavm ) and the changes in haemodynamic parameters . furthermore , the haemodynamic response can be influenced by a different prevalence of underlying hepatic arteriovenous malformations ( havms ) in different hht subtypes [ 5 , 14 , 15 ] . only one patient had a history of havms , which might be an underestimation , since screening for havms was only performed when clinically or biochemically suspected . clinically significant havms seemed to be absent in the present study , as a hyperdynamic circulation with an abnormal high ci at baseline was not documented in the present study ( mean ci within the normal range of 3.0 0.8 l / min / m ) . the amount of microscopic pavms under the detection limit of chest ct may further influence the shunt percentage and different haemodynamic responses in hht patients . due to the decrease in pulmonary right - to - left shunt from 14 to 5 % after pavm embolisation , a consequent decrease in preload and sv can be expected ( mean change 12 % in the current study ) . as there was no change in hr , the mbp increased by 4 % after pavm embolisation , which is probably due to the non - significant increase in tpr ( 6 % ) , as the mbp is a product of tpr and co. as blood pressure is inversely related to indoor temperature , the fall in ambient temperature during the procedure may have caused further vasoconstriction and thereby an increase in blood pressure . in a previous study about the association between brachial pulse dp / dt and other haemodynamic parameters in a chronic haemodialysis population , a pearson coefficient of r = 0.6 ( r = 0.36 , p < 0.001 ) was reported for the correlation between the delta blood pressure and the delta dp / dt . this is in line with the results found in our study ( r = 0.73 , r = 0.53 , p < 0.0001 ) and demonstrates that the dp / dt is responsible for more than 50 % of variance in the blood pressure . because of the significant increase in mbp and decrease in co , we also expected a significant increase in tpr . a possible explanation might be an increase in central venous pressure due to the embolisation . unfortunately , no measurements of the right atrium pressure were performed during the embolisation procedure . a potential clinical implication of our findings might be associated with the presence of pulmonary hypertension ( ph ) in hht [ 18 , 19 ] . ph can occur both as gene - related pulmonary arterial hypertension and as a response to high output due to havms . as pavms are abnormally dilated vessels between pulmonary arteries and veins they provide low resistance pathways for the pulmonary blood flow and one may therefore expect an elevation in pulmonary artery pressure ( pap ) after transcatheter closure . it was suggested that this might be caused by other haemodynamic changes , for example recruitment of the pulmonary vasculature or a decrease in co , although this has been previously suggested in only one case report . we now present the first study that confirms the decrease in co after embolisation in a larger population of hht patients , which may indeed provide a potential explanation for the absent increase in pap after embolisation . furthermore , pavm - related hypoxaemia can induce pulmonary vasoconstriction with a concomitant increase in pulmonary vascular resistance ( pvr ) . in our study there was indeed an increase in saturation after embolisation of pavms ( table 1 ) with probably an decrease in pulmonary vasoconstriction and pvr . first , our study is limited by its small sample , which may have influenced the results . second , the modelflow model does not seem to be accurate in measuring absolute values of sv , co and tpr . the differences between the uncalibrated model and invasive determinations ( measured with the thermodilution method ) in individual patients are usually small , but can be substantial and unreliable in some [ 8 , 20 ] . however , the modelflow methodology is an accurate model to compare changes in haemodynamics within one patient and the british hypertension society has recommended the finometer for measurements in the clinical set - up as well as for research purposes [ 8 , 21 ] . first , our study is limited by its small sample , which may have influenced the results . second , the modelflow model does not seem to be accurate in measuring absolute values of sv , co and tpr . the differences between the uncalibrated model and invasive determinations ( measured with the thermodilution method ) in individual patients are usually small , but can be substantial and unreliable in some [ 8 , 20 ] . however , the modelflow methodology is an accurate model to compare changes in haemodynamics within one patient and the british hypertension society has recommended the finometer for measurements in the clinical set - up as well as for research purposes [ 8 , 21 ] . the present study shows that significant haemodynamic changes occur directly after embolisation of pulmonary arteriovenous malformations , amongst which a decrease in stroke volume and cardiac output are most important . this may especially provide additional insights into the haemodynamic responses after pavm embolisation in hht patients prone to ph .

backgroundtranscatheter embolisation is widely used to close pulmonary arteriovenous malformations ( pavms ) in patients with hereditary haemorrhagic telangiectasia ( hht ) . data on the direct cardiovascular haemodynamic changes induced by this treatment are scarce.objectiveswe investigated the direct haemodynamic effects of transcatheter embolisation of pavms , using non - invasive finger pressure measurements.methodsduring the procedure , blood pressure , heart rate ( hr ) , stroke volume ( sv ) , cardiac output ( co ) , total peripheral resistance ( tpr ) and delta pressure / delta time ( dp / dt ) were continuously monitored using a finometer. potential changes in these haemodynamic parameters were calculated from the pressure registrations using modelflow methodology . absolute and relative changes were calculated and compared using the paired sample t-test.resultsthe present study includes 29 hht patients ( mean age 39 15 years , 11 men ) who underwent transcatheter embolotherapy of pavms . the total number of embolisations was 72 ( mean per patient 2.5 ) . directly after pavm closure , sv and co decreased significantly by 11.9 % ( p = 0.01 ) and 9.5 % ( p = 0.01 ) respectively , without a significant change in hr ( 1.8 % ) . mean arterial blood pressure increased by 4.1 % ( p = 0.02 ) , while the tpr and dp / dt did not increase significantly ( 5.8 % and 0.2 % , respectively).conclusionssignificant haemodynamic changes occur directly after transcatheter embolisation of pavms , amongst which a decrease in stroke volume and cardiac output are most important .

Introduction Methods Patient population Transcatheter embolotherapy of PAVMs Haemodynamic changes after PAVM embolisation, using non-invasive finger pressure measurements Data registration and analysis Statistical analysis Results Patient population Haemodynamic changes using non-invasive finger pressure measurements Discussion Study limitations Conclusion Funding Conflict of interest

hereditary haemorrhagic telangiectasia ( hht ) is an autosomal dominant inherited disease characterised by vascular malformations , ranging from small telangiectases in skin and mucosal membranes to large visceral arteriovenous malformations ( avms ) predominantly localised in the lungs , brain and liver [ 13 ] . pulmonary arteriovenous malformations ( pavms ) are abnormally dilated vessels between pulmonary arteries and veins that cause a permanent extra - cardiac right - to - left shunt , which carries the risk of cerebral paradoxical embolisation of both thrombotic and septic origin . therefore , the present study investigated the direct haemodynamic effects of pavm embolisation , using non - invasive finger pressure measurements . between 2008 and 2010 , we included 29 patients who underwent transcatheter embolisation of pavms in the st . d amplatzer vascular plug during the transcatheter embolisation of pavms , arterial pressure was measured on a finger of the left hand using a finometer device ( fms , finapres medical systems , amsterdam , the netherlands ) . five minute averages of systolic ( sbp ) , diastolic ( dbp ) and mean blood pressure ( mbp ) , heart rate ( hr ) , stroke volume ( sv ) , cardiac output ( co ) , total peripheral resistance ( tpr ) and delta pressure / delta time ( dp / dt ) were calculated after an electronic calibration procedure . sv and co ( co is the product of sv and hr ) were calculated from the finger pressure wave using the modelflow methodology . between 2008 and 2010 , we included 29 patients who underwent transcatheter embolisation of pavms in the st . during the transcatheter embolisation of pavms , arterial pressure was measured on a finger of the left hand using a finometer device ( fms , finapres medical systems , amsterdam , the netherlands ) . five minute averages of systolic ( sbp ) , diastolic ( dbp ) and mean blood pressure ( mbp ) , heart rate ( hr ) , stroke volume ( sv ) , cardiac output ( co ) , total peripheral resistance ( tpr ) and delta pressure / delta time ( dp / dt ) were calculated after an electronic calibration procedure . sv and co ( co is the product of sv and hr ) were calculated from the finger pressure wave using the modelflow methodology . a total of 29 hht patients ( 62 % female , mean age 39.2 15.3 years ) were included , in which 72 pavms were embolised ( mean per patient 2.5 ) . the baseline characteristics are presented in tables 1 and 2.table 1baseline characteristics of patientstotal29gendermale11 ( 37.9)female18 ( 62.1)age ( years)39.2 15.3bmi ( kg / m)24.0 5.0bsa ( m)1.9 0.2hhtdefinite28 ( 96.6)type 119 ( 65.5)type 22 ( 6.9)type unknown8 ( 27.6)sao2 ( % ) before procedure95.5 3.5after procedure98.4 2.3shunt fraction ( % ) before procedure13.6 8.3after procedure4.9 6.6all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformationtable 2baseline characteristics of embolisation proceduretreated pavms1 pavm9 ( 31.0)2 pavms9 ( 31.0)3 pavms2 ( 6.9)4 pavms8 ( 27.6)>4 pavms1 ( 3.4)closure deviceamplatzer plug54 ( 75.0)coil18 ( 25.0)plug diameter < 4 ( mm)1 ( 1.9)4 ( mm)13 ( 24.1)6 ( mm)13 ( 24.1)8 ( mm)9 ( 16.7)10 ( mm)10 ( 18.5)>10 ( mm)6 ( 11.1)not known2 ( 3.7)all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know baseline characteristics of patients all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformation baseline characteristics of embolisation procedure all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know directly after pavm embolisation the sv and co decreased significantly : 6.4 13.0 ml ( range 45.9 to 17.9 ml ; 11.9 % , p = 0.01 ) and 0.4 0.8 l / min ( range 3.0 to 1.58 l / min ; 9.5 % , p = 0.01 ) . as expected , the ci decreased as well ( range 1.5 to 0.9 l / min / m ; 9.5 % , p = 0.01 ) . dbp and mbp increased significantly by 5.2 10.3 mmhg ( range 9.0 to 32.9 mmhg ; 5.9 % , p = 0.01 ) for dbp and 5.7 12.1 mmhg ( range 12.7 to 32.6 mmhg ; 4.1 % , p = 0.02 ) for mbp , respectively . there was no significant change in sbp ( 4.0 16.3 mmhg ( range 25.5 to 38.5 mmhg ; 1.7 % , p = 0.20 ) . the dp / dt did not change significantly : 2.1 290.0 mmhg / s ( range 750.0 to 848.2 mmhg / s ; 0.2 % , p = 0.97 ) . hr and tpr increased , but this appeared to be non - significant : 1.6 7.4 beats / min ( range 17.4 to 21.2 beats / min ; 1.8 % , p = 0.24 ) and 0.1 0.5 woods units ( range 1.62 to 1.39 woods units ; 5.8 % , p = 0.16 ) . these data are summarised in table 3.table 3haemodynamic measurements before and after embolisation with absolute and relative changesbefore sdafter sddelta absolutedelta percentpsbp ( mmhg)144.9 25.3148.9 28.44.01.70.20dpb ( mmhg)85.5 11.590.7 16.35.25.90.01mbp ( mmhg)108.0 16.0113.7 19.95.74.10.02hr ( beats / min)78.8 14.680.4 13.91.61.80.24sv ( ml)70.9 20.964.5 19.36.411.90.01co ( l / min)5.5 1.65.1 1.40.49.50.01ci ( l / min / m)3.0 0.82.8 0.80.29.50.01tpr ( woods units)1.4 0.51.5 0.50.15.80.16dp / dt ( mmhg / s)1,233.7 463.81,235.8 481.72.10.20.97 sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second haemodynamic measurements before and after embolisation with absolute and relative changes sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second a total of 29 hht patients ( 62 % female , mean age 39.2 15.3 years ) were included , in which 72 pavms were embolised ( mean per patient 2.5 ) . the baseline characteristics are presented in tables 1 and 2.table 1baseline characteristics of patientstotal29gendermale11 ( 37.9)female18 ( 62.1)age ( years)39.2 15.3bmi ( kg / m)24.0 5.0bsa ( m)1.9 0.2hhtdefinite28 ( 96.6)type 119 ( 65.5)type 22 ( 6.9)type unknown8 ( 27.6)sao2 ( % ) before procedure95.5 3.5after procedure98.4 2.3shunt fraction ( % ) before procedure13.6 8.3after procedure4.9 6.6all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformationtable 2baseline characteristics of embolisation proceduretreated pavms1 pavm9 ( 31.0)2 pavms9 ( 31.0)3 pavms2 ( 6.9)4 pavms8 ( 27.6)>4 pavms1 ( 3.4)closure deviceamplatzer plug54 ( 75.0)coil18 ( 25.0)plug diameter < 4 ( mm)1 ( 1.9)4 ( mm)13 ( 24.1)6 ( mm)13 ( 24.1)8 ( mm)9 ( 16.7)10 ( mm)10 ( 18.5)>10 ( mm)6 ( 11.1)not known2 ( 3.7)all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know baseline characteristics of patients all characteristics are written in number of patients with percentage or mean with standard deviation bmi body mass index , bsa body surface area , kg kilogram , kg / m kilogram per square meter , hht hereditary haemorrhagic telangiectasia , sao 2 saturation level of oxygen in haemoglobin , p avm pulmonary arteriovenous malformation baseline characteristics of embolisation procedure all characteristics are written in number with percentage pavm pulmonary arteriovenous malformation , mm millimetre diameter coils are not know directly after pavm embolisation the sv and co decreased significantly : 6.4 13.0 ml ( range 45.9 to 17.9 ml ; 11.9 % , p = 0.01 ) and 0.4 0.8 l / min ( range 3.0 to 1.58 l / min ; 9.5 % , p = 0.01 ) . as expected , the ci decreased as well ( range 1.5 to 0.9 l / min / m ; 9.5 % , p = 0.01 ) . dbp and mbp increased significantly by 5.2 10.3 mmhg ( range 9.0 to 32.9 mmhg ; 5.9 % , p = 0.01 ) for dbp and 5.7 12.1 mmhg ( range 12.7 to 32.6 mmhg ; 4.1 % , p = 0.02 ) for mbp , respectively . there was no significant change in sbp ( 4.0 16.3 mmhg ( range 25.5 to 38.5 mmhg ; 1.7 % , p = 0.20 ) . the dp / dt did not change significantly : 2.1 290.0 mmhg / s ( range 750.0 to 848.2 mmhg / s ; 0.2 % , p = 0.97 ) . hr and tpr increased , but this appeared to be non - significant : 1.6 7.4 beats / min ( range 17.4 to 21.2 beats / min ; 1.8 % , p = 0.24 ) and 0.1 0.5 woods units ( range 1.62 to 1.39 woods units ; 5.8 % , p = 0.16 ) . these data are summarised in table 3.table 3haemodynamic measurements before and after embolisation with absolute and relative changesbefore sdafter sddelta absolutedelta percentpsbp ( mmhg)144.9 25.3148.9 28.44.01.70.20dpb ( mmhg)85.5 11.590.7 16.35.25.90.01mbp ( mmhg)108.0 16.0113.7 19.95.74.10.02hr ( beats / min)78.8 14.680.4 13.91.61.80.24sv ( ml)70.9 20.964.5 19.36.411.90.01co ( l / min)5.5 1.65.1 1.40.49.50.01ci ( l / min / m)3.0 0.82.8 0.80.29.50.01tpr ( woods units)1.4 0.51.5 0.50.15.80.16dp / dt ( mmhg / s)1,233.7 463.81,235.8 481.72.10.20.97 sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second haemodynamic measurements before and after embolisation with absolute and relative changes sbp systolic blood pressure , dbp diastolic blood pressure , mbp mean blood pressure , hr heart rate , sv stroke volume , co cardiac output , ci cardiac index , tpr total peripheral resistance , dp / dt delta pressure / delta time , sd standard deviation , min minutes , ml millilitres , l / min litres per minute , mmhg millimetres of mercury , s second to our knowledge , this is the first study describing the occurrence of significant haemodynamic changes directly after transcatheter embolisation of pavms . using the finometer and modelflow methodology , our study accurately recorded beat - to - beat non - invasive finger pressure measurements and thereby the immediate haemodynamic changes after pavm embolisation , amongst which a decrease in sv and co were most important . this seems to be in line with the results in our current study in 29 hht patients who all underwent transcatheter embolisation of pavms . unfortunately , we could not find a significant association between plug size ( size of the pavm ) and the changes in haemodynamic parameters . due to the decrease in pulmonary right - to - left shunt from 14 to 5 % after pavm embolisation , a consequent decrease in preload and sv can be expected ( mean change 12 % in the current study ) . as there was no change in hr , the mbp increased by 4 % after pavm embolisation , which is probably due to the non - significant increase in tpr ( 6 % ) , as the mbp is a product of tpr and co. as blood pressure is inversely related to indoor temperature , the fall in ambient temperature during the procedure may have caused further vasoconstriction and thereby an increase in blood pressure . in a previous study about the association between brachial pulse dp / dt and other haemodynamic parameters in a chronic haemodialysis population , a pearson coefficient of r = 0.6 ( r = 0.36 , p < 0.001 ) was reported for the correlation between the delta blood pressure and the delta dp / dt . this is in line with the results found in our study ( r = 0.73 , r = 0.53 , p < 0.0001 ) and demonstrates that the dp / dt is responsible for more than 50 % of variance in the blood pressure . the present study shows that significant haemodynamic changes occur directly after embolisation of pulmonary arteriovenous malformations , amongst which a decrease in stroke volume and cardiac output are most important .

sealants have been developed to protect pits and fissures from caries by acting as a physical barrier to protect vulnerable areas . the introduction of fluoride - releasing sealants has widen the perspectives in the prevention of pit and fissure caries21 . fluoride - releasing materials can provide benefits in areas near to the sealant margins where the acid challenge may result in enamel demineralization10 . in addition , the anticariogenic effect of fluoride - releasing fissure sealants depends on the amount of released fluoride , but obviously even more on the longevity of this release5 . in addition to traditional fluoride - releasing resin sealants13 , other fluoride - releasing materials have been used as fissure sealants , such as conventional glass ionomer cements ( gics)10 and resin - modified gics ( rmgics)13 . these materials should provide constant fluoride release for a prolonged period of time and act as a reservoir of fluoride ion in order to promote fluorapatite formation in enamel9 . another factor in the clinical performance of sealant materials is related to biodegradation in the oral environment . it can be caused by acid challenges , including those produced by the cariogenic biofilm6 , acidic diet25 and salivary enzymes6 . each type of solution will mainly degrade components of the resin - based material . according to yap , et al.25 ( 2000 ) moreover , the inorganic filler may be damaged by water and weak intraoral acids , such as citric and lactic acids25 . in saliva they undergo a surface reaction that leads to the precipitation of calcium and phosphate ions at the outermost layer17 . in acid conditions , matrix - forming ions are found to be released into the solution , as part of a process of buffering the medium3 . in addition , the nature of the resin matrix , size , type , and distribution of filler particles regulate the biodegradation of ionomeric and resin - based materials25 . the effects of chemical degradation are surface softening and roughening , which can decrease the long - term durability of the sealer material25 . as a consequence , degraded sealants could encourage dental biofilm accumulation , which may result in superficial staining and onset of caries lesions , reducing the physical properties of the material2 . although fluoride release has been the subject of numerous in vitro studies , the roughness and hardness pattern of fluoride - releasing materials in different storage media should be accessed . the purpose of this study was to determine the effect of acidic solutions on the degradation of ionomeric and sealant materials by means of hardness , roughness and fluoride release measurements . the first hypothesis tested was that gics show greater degradation than resin sealant materials when stored in different solutions . the second hypothesis was that storage in acidic solution leads to higher fluoride release from the tested materials . three materials were used in this in vitro experiment : a resin fissure sealant ( fluroshield ) , a rmgic ( vitremer ) and a conventional gic ( ketac molar ) . the brand names , types , compositions , manufacturers and batch numbers of the tested materias are presented in figure 1 . eighteen disc - shaped specimens of each material were prepared and had their initial roughness and hardness determined for baseline information . next , 6 discs of each material were randomly selected by lottery method and assigned to one of three different storage solutions : 0.3% citric acid solution ( ca ) ; demineralizing solution ( de ) ; and remineralizing solution ( re ; artificial saliva ) . new roughness and hardness readings of the materials were done at the end of the experimental period . fifty - four disc - shaped specimens ( 6 mm in diameter and 2 mm in thickness ) were prepared according to the manufacturers ' specifications , at room temperature ( 231.0c and 505% relative humidity , iso # 7489 specification ) . the materials were inserted in plastic molds in a single increment and pressed between polyester strip and glass plates . a piece of paraffin - coated dental floss was incorporated into the cements during setting to suspend the samples in the test solution . the resin - based materials fluroshield and vitremer were light cured on both sides ( surface and bottom ) for 40 s using a light - curing unit ( elipar tri - light ; espe , seefeld , germany ) . light output was periodically checked ( 580 30mw / cm ) with a curing radiometer ( model 100 , demetron research corp . , the conventional gic , ketac molar , was allowed to set under pressure for 10 min . after setting , all surfaces of the discs , except the surface under the polyester matrix , were protected with an acid- resistant nail polish ( colorama , so paulo , sp , brazil ) . all discs were stored for 24 h at 37c and 100% humidity , and polished with 600- and 1200-grit al2o3 paper ( arotec s.a . ind . and com . , so paulo , sp , brazil ) and then cloth polished with 1.0-mm diamond paste ( buheler metadi ii ; buheler , lake buff , il , usa ) before starting the experimental phase . each specimen was gently dabbed dry with absorbent paper and the surface roughness was analyzed with a surface roughness - measuring instrument ( surfcorder se1700 ; kosaka corp , tokyo , japan ) equipped with a diamond needle of 2-m radius . in order to record roughness measurements , the needle was moved at a constant speed of 0.5 mm / s under a 0.7 mn load . the surface roughness was characterized by the average roughness ( ra ) , which is the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length . ra values for each specimen were taken across the diameter over a standard length of 0.25 mm . three traces were recorded for each specimen at three different locations - parallel , perpendicular , and oblique to scan all specimen area . the roughness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta roughness ( ar ) was determined using the following equation : knoop hardness measurements were obtained on the exposed surface using a microhardness tester ( hmv-2000 , shimadzu , japan ) with a knoop diamond under a 50 g load for 10 s. the measurement of the indentation was performed immediately after the period of 10 s. three indentations spaced 1 mm from each other were made in the central area of each specimen . the hardness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta hardness ( h ) was determined using the following equation : after baseline hardness and roughness tests , all specimens were individually immersed in 3.0 ml of each solution . the storage media used were : 0.3% citric acid solution ( ph 3.2 ) ; de solution ( 2.0 mm calcium , 2.0 mm phosphate and acetate buffer 75 mm , ph 4.3 ) ; and re solution ( artificial saliva composed of 1.5 mm calcium , 0.9 mm phosphate , kcl 150 mm and tris [ tris- ( hydroxymethyl ) aminomethane ] buffer 20 mm , ph 7.0 ) . the tubes were kept under constant agitation at 120 rpm , 1.7 hz ( cientec model ct 165 , piracicaba , sp , brazil ) at controlled temperature of 25 1.0c . the storage media were changed every 24 h. duplicate aliquots of the solutions were mixed with tisab iii at a ratio of 1:0.1 and analyzed using an ion- selective electrode ( orion 96 - 09 ; orion research inc . , boston , ma , usa ) and a digital ion - analyzer ( orion ea- 940 ; orion research inc . ) , which was previously calibrated with various standard solutions ( 0.065 , 0.125 , 0.250 , 0.500 , and 1.000 mg of f / ml ) . the results obtained from each sample at 1 , 2 , 3 , 5 , 7 , 9 , 12 , 15 days were analyzed and the result was divided by the specimen area ( exposed area = 28.26 mm ) . the amount of released fluoride in the different time periods was evaluated . original data for roughness , hardness and fluoride release were transformed ( log 10 , log 10 , and 0.3 exponential , respectively ) before applying anova , repeated - measures anova and tukey 's and contrast tests , since variance was not homogeneous . a multi - factor anova was applied to the roughness and hardness data to analyze the interactions between the factors ( materials and degradation solution ) . in order to assess significant differences within these factors , the repeated - measures anova was applied to verify the interaction of the materials , degradation solution , and fluoride release day of evaluation . in addition , the contrast test was performed to verify the differences among the 1st day of fluoride release and the release at days 2 , 3 , 5 , 7 , 9 , 12 and 15 . , cary , nc , usa ) was used and the significance level was set at 5% . three materials were used in this in vitro experiment : a resin fissure sealant ( fluroshield ) , a rmgic ( vitremer ) and a conventional gic ( ketac molar ) . the brand names , types , compositions , manufacturers and batch numbers of the tested materias are presented in figure 1 . eighteen disc - shaped specimens of each material were prepared and had their initial roughness and hardness determined for baseline information . next , 6 discs of each material were randomly selected by lottery method and assigned to one of three different storage solutions : 0.3% citric acid solution ( ca ) ; demineralizing solution ( de ) ; and remineralizing solution ( re ; artificial saliva ) . new roughness and hardness readings of the materials were done at the end of the experimental period . fifty - four disc - shaped specimens ( 6 mm in diameter and 2 mm in thickness ) were prepared according to the manufacturers ' specifications , at room temperature ( 231.0c and 505% relative humidity , iso # 7489 specification ) . the materials were inserted in plastic molds in a single increment and pressed between polyester strip and glass plates . a piece of paraffin - coated dental floss was incorporated into the cements during setting to suspend the samples in the test solution . the resin - based materials fluroshield and vitremer were light cured on both sides ( surface and bottom ) for 40 s using a light - curing unit ( elipar tri - light ; espe , seefeld , germany ) . light output was periodically checked ( 580 30mw / cm ) with a curing radiometer ( model 100 , demetron research corp . , the conventional gic , ketac molar , was allowed to set under pressure for 10 min . after setting , all surfaces of the discs , except the surface under the polyester matrix , were protected with an acid- resistant nail polish ( colorama , so paulo , sp , brazil ) . all discs were stored for 24 h at 37c and 100% humidity , and polished with 600- and 1200-grit al2o3 paper ( arotec s.a . , so paulo , sp , brazil ) and then cloth polished with 1.0-mm diamond paste ( buheler metadi ii ; buheler , lake buff , il , usa ) before starting the experimental phase . each specimen was gently dabbed dry with absorbent paper and the surface roughness was analyzed with a surface roughness - measuring instrument ( surfcorder se1700 ; kosaka corp , tokyo , japan ) equipped with a diamond needle of 2-m radius . in order to record roughness measurements , the needle was moved at a constant speed of 0.5 mm / s under a 0.7 mn load . the surface roughness was characterized by the average roughness ( ra ) , which is the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length . ra values for each specimen were taken across the diameter over a standard length of 0.25 mm . three traces were recorded for each specimen at three different locations - parallel , perpendicular , and oblique to scan all specimen area . the roughness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta roughness ( ar ) was determined using the following equation : knoop hardness measurements were obtained on the exposed surface using a microhardness tester ( hmv-2000 , shimadzu , japan ) with a knoop diamond under a 50 g load for 10 s. the measurement of the indentation was performed immediately after the period of 10 s. three indentations spaced 1 mm from each other were made in the central area of each specimen . the hardness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta hardness ( h ) was determined using the following equation : after baseline hardness and roughness tests , all specimens were individually immersed in 3.0 ml of each solution . the storage media used were : 0.3% citric acid solution ( ph 3.2 ) ; de solution ( 2.0 mm calcium , 2.0 mm phosphate and acetate buffer 75 mm , ph 4.3 ) ; and re solution ( artificial saliva composed of 1.5 mm calcium , 0.9 mm phosphate , kcl 150 mm and tris [ tris- ( hydroxymethyl ) aminomethane ] buffer 20 mm , ph 7.0 ) . the tubes were kept under constant agitation at 120 rpm , 1.7 hz ( cientec model ct 165 , piracicaba , sp , brazil ) at controlled temperature of 25 1.0c . the storage media were changed every 24 h. duplicate aliquots of the solutions were mixed with tisab iii at a ratio of 1:0.1 and analyzed using an ion- selective electrode ( orion 96 - 09 ; orion research inc . , boston , ma , usa ) and a digital ion - analyzer ( orion ea- 940 ; orion research inc . ) , which was previously calibrated with various standard solutions ( 0.065 , 0.125 , 0.250 , 0.500 , and 1.000 mg of f / ml ) . the results obtained from each sample at 1 , 2 , 3 , 5 , 7 , 9 , 12 , 15 days were analyzed and the result was divided by the specimen area ( exposed area = 28.26 mm ) . the amount of released fluoride in the different time periods was evaluated . each specimen was gently dabbed dry with absorbent paper and the surface roughness was analyzed with a surface roughness - measuring instrument ( surfcorder se1700 ; kosaka corp , tokyo , japan ) equipped with a diamond needle of 2-m radius . in order to record roughness measurements , the needle was moved at a constant speed of 0.5 mm / s under a 0.7 mn load . the surface roughness was characterized by the average roughness ( ra ) , which is the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length . ra values for each specimen were taken across the diameter over a standard length of 0.25 mm . three traces were recorded for each specimen at three different locations - parallel , perpendicular , and oblique to scan all specimen area . the roughness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta roughness ( ar ) was determined using the following equation : knoop hardness measurements were obtained on the exposed surface using a microhardness tester ( hmv-2000 , shimadzu , japan ) with a knoop diamond under a 50 g load for 10 s. the measurement of the indentation was performed immediately after the period of 10 s. three indentations spaced 1 mm from each other were made in the central area of each specimen . the hardness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta hardness ( h ) was determined using the following equation : after baseline hardness and roughness tests , all specimens were individually immersed in 3.0 ml of each solution . the storage media used were : 0.3% citric acid solution ( ph 3.2 ) ; de solution ( 2.0 mm calcium , 2.0 mm phosphate and acetate buffer 75 mm , ph 4.3 ) ; and re solution ( artificial saliva composed of 1.5 mm calcium , 0.9 mm phosphate , kcl 150 mm and tris [ tris- ( hydroxymethyl ) aminomethane ] buffer 20 mm , ph 7.0 ) . the tubes were kept under constant agitation at 120 rpm , 1.7 hz ( cientec model ct 165 , piracicaba , sp , brazil ) at controlled temperature of 25 1.0c . the storage media were changed every 24 h. duplicate aliquots of the solutions were mixed with tisab iii at a ratio of 1:0.1 and analyzed using an ion- selective electrode ( orion 96 - 09 ; orion research inc . , boston , ma , usa ) and a digital ion - analyzer ( orion ea- 940 ; orion research inc . ) , which was previously calibrated with various standard solutions ( 0.065 , 0.125 , 0.250 , 0.500 , and 1.000 mg of f / ml ) . the results obtained from each sample at 1 , 2 , 3 , 5 , 7 , 9 , 12 , 15 days were analyzed and the result was divided by the specimen area ( exposed area = 28.26 mm ) . the amount of released fluoride in the different time periods was evaluated . original data for roughness , hardness and fluoride release were transformed ( log 10 , log 10 , and 0.3 exponential , respectively ) before applying anova , repeated - measures anova and tukey 's and contrast tests , since variance was not homogeneous . a multi - factor anova was applied to the roughness and hardness data to analyze the interactions between the factors ( materials and degradation solution ) . in order to assess significant differences within these factors , the repeated - measures anova was applied to verify the interaction of the materials , degradation solution , and fluoride release day of evaluation . in addition , the contrast test was performed to verify the differences among the 1st day of fluoride release and the release at days 2 , 3 , 5 , 7 , 9 , 12 and 15 . , cary , nc , usa ) was used and the significance level was set at 5% . the percentage of the increases and standard deviation of surface roughness ( r ) for comparisons among the materials and degradation solutions are shown in figure 2 . in addition , the original data of roughness mean ( ra -m ) and standard deviation ( sd ) before ( baseline measurement - gray columns ) and after storage are demonstrated in table 1 . the immersion in ca caused a greater increase in roughness for vitremer and ketac molar than for fluroshield . table 2 shows the mean values and standard deviation of hardness before and after the storage . before the storage ( baseline measurement - gray columns ) , ketac molar was significantly harder than fluroshield . vitremer showed intermediated hardness values , not differing from the other materials , regardless storage media . a significant decrease in hardness was observed for all materials in all solutions , there was no difference among the groups , considering the h . after the storage in ca and de , there was no difference among the materials . the storage in re changed the ranking of the materials as follows : ketac molar = vitremer > fluroshield . similar capital letters in a column signify no significant statistical difference among solutions , according to anova and tukey tests ( p>0.05 ) . similar small letters in a line signify no significant statistical difference among materials , according to anova and tukey tests ( p>0.05 ) , considering the same column colors . the patterns of fluoride ion release for the materials in different solutions are shown in figure 3 . all materials demonstrated higher fluoride release on the first day . over the next days , the fluoride release decreased until it reached a plateau , in which the fluoride release was lower and constant until the end of the experiment ( 15 days ) . there was no difference in fluoride release for all materials in re solution during the 15 days of the experiment ( p>0.05 ) . in the de solution , on the 1st day after 3 days , fluoride release of all materials was similar . in the ca solution , during the first 2 days , vitremer released the highest amounts of fluoride , whereas after 3 - 15 days the release was highest for ketac molar . fluroshield released the lowest amount of fluoride , and the pattern of fluoride released was the same in all solutions ( p>0.05 ) . ketac molar and vitremer released the highest amounts of fluoride in ca solution ( p<0.05 ) . ketac molar showed a quite constant release , whereas vitremer showed a significantly higher fluoride release on the first 2 days . for the other solutions ( de and re ) , a similar fluoride release pattern was observed . the effects of solutions that simulate the chemical oral environment on the surface roughness , hardness and fluoride ion release were investigated in this study . with regard to the hypotheses tested in this study , the first hypothesis that ionomeric sealant is more degraded by storage in different solutions than resin - based sealants was accepted . the increase in roughness was more accentuated in the gics ; however the reduction in hardness was similar for all materials . the higher degradation of ionomeric materials by the citric acid solution can be explained by the characteristics of this acid : low ph , higher concentration , titrability , and buffer capacity , as well as by the characteristics of the ionomeric materials . the main effect of citric acid on ionomeric materials seems to rely on the dissolution / disintegration of the glass particles14 . moreover , citric acid is a carboxylic acid capable of chelating ions present in the cement and forming complexes of reasonable solubility in water14 . the influence of citric acid on the surface texture of ketac molar has been demonstrated by previous data15 . this material was deteriorated when stored in low ph solutions , which makes it more susceptible to clinical failure15 . the absence of increase in roughness for fluroshield , after exposure to the storage solutions , is explained by the relationship between resin matrix and filler particles . this material presents silanized filler particles in a smaller size and content compared to gics . based on the effect of the storage solution on the degradation of the materials , it could be assumed that the acidic condition has a major effect on roughness due to the ionic dissolution of the filler particles . the decrease in hardness of fluroshield seems to be related to the water sorption and hydrolysis , since all solutions , regardless of their ph , had a similar effect on material hardness . the presence of water has a key role in the deterioration of resin - based materials . it may be suggested that the main effect of the solutions on resin - based materials is the swelling of the matrix caused by hydrolysis . according to rtengren , et al.18 ( 2001 ) , matrix swelling causes the formation of pores inside the material from which organic substances can be released , resulting in mass loss . the diffusion of water into the material may also lead to filler degradation and debonding . the water sorption process is dependent on the hydrophilicity of the polymer matrix and on the presence and location of hydrolysable groups on the matrix chains8 . the matrix of fluroshield is composed basically of bisgma and tegdma , which are are hydrophilic monomers . the reduction in hardness may also affect the leaching of residual monomers into the solutions , especially tegdma7 . in the rmgic , a similar pattern of water sorption / degradation is expected . the elution of organic compounds such as bisgma , udma , tegdma , hema and additives could be related to the observed reduction in hardness after storage in all solutions23 . with regard to hardness , the differences among materials at baseline are mainly related to differences in their composition and microstructure24 . according to xie , et al.24 ( 2000 ) , there is a relationship between knoop hardness number ( khn ) and gic 's microstructure . ketac molar presents a very dense surface texture , with tightly packed glass particles in the matrix . the presence of different sizes and shapes of the glass particles dispersed on the matrix is another reason for the higher hardness24 . the marked reduction in hardness observed in ketac molar could be the result of a pronounced erosion of the polyacid matrix , the leaching of inorganic components , and an increased hydrolytic degradation at the matrix - filler interface1 . in addition , an increase has been observed in the leaching of inorganic components , such as fluoride , and a reduction of surface hardness in acidic conditions , corroborating the results of behrend and geurtsen1 ( 2001 ) . the crosslinks of rmgics are formed between two chains of the polyacid , often via a hema molecule . in this region , carboxylate groups may be too far apart to be cross - linked via calcium ions . thus , the reaction of the carbon double bond may physically separate the chains sufficiently to minimize the ionic interactions , providing a greater hardening and stiffening of the matrix19 . considering the intermediate microstructure of this resin - based ionomeric material , intermediate values of hardness , this material is a resin - based sealant with relatively low filler content ( about 50% ) . furthermore , the resin matrix , composed basically of bisgma and tegdma , appears to be more resilient than the ionomeric matrix . the second hypothesis tested in this study that acidic solutions lead to a higher fluoride release , was partially accepted , because this assumption worked only for the ionomeric materials . the fluoride release rates and periods of sealer materials are clinically important in establishing the extent to which dental caries can be prevented . the common finding for all the evaluated materials was the similar pattern of fluoride release in the three storage media . the kinetic profile of fluoride release was characterized by a rapid initial increase ( first 24 h ) and , within 15 days of storage , a plateau was reached ; however the materials continued to release low fluoride rates until the end of the experiment . the burst effect was observed for all materials , but it was more pronounced for vitremer , which is consistent with the results of the previous studies1,4 . the first is a relatively fast release from the surface and the second mechanism is a long - term diffusion from the bulk of the material through cement pores and cracks12 . vitremer and ketac molar presented great amounts of fluoride in the acid media ( de and ca solutions ) , in accordance with other studies11,16 . this phenomenon is caused by an accentuated erosion of the polysalt matrix of the glass ionomer and because of the dissolution of this material , which increases with low ph5 . the pattern of fluoride ion release found in vitremer suggested that its fluoride release mechanism was similar to that of ketac molar , although the process of elution of fluoride seemed to take longer , probably because of the resin matrix , which reduces the elution rate of the fluoride ions . fluroshiled material was characterized by a low fluoride release rate , regardless of the storage solutions . these results may be explained by the characteristics of the resin sealant matrix , which is less hydrophilic than that of conventional and rmgics , making fluoride release more difficult20 and slower after polymerization22 . the low fluoride release of fluroshiled raises the question of whether this resin - based sealant has a significant preventive effect in high caries - risk patients . the findings of this study suggest that the mechanical and chemical properties of the sealant materials could be influenced by acidic solutions . although the levels of fluoride release from conventional and rmgics in these solutions were higher , these materials were more susceptible to acidic degradation ( increase in roughness ) than the resin sealant , suggesting that the choice for the sealant material must be based on the characteristics of the material , such as mechanical properties , fluoride release , and long - term durability , as well as the caries risk / activity of the patient . nevertheless , periodic evaluation must be carried out because degradation is expected for all materials placed in the oral environment . based on the results of this study , it may be concluded that : 1 . under acidic conditions , the ionomeric materials were more susceptible to degradation ( increase in roughness ) than the resin fissure sealant ; 2 . however , a higher degradation ( increase in roughness and decrease in hardness ) was also observed in this condition ; 3 . the resin fissure sealant was not affected by the acidic environment and showed similar roughness and fluoride release after storage in all tested media .

objectives : the aim of this study was to determine the effect of environmental conditions on the degradation of ionomeric and resin sealant materials.material and methods : fluroshield , vitremer , and ketac molar disc - shaped specimens ( n=18/material ) were prepared , polished , subjected to initial hardness and roughness readings . six discs of each material were randomly assigned to one of three different storage solutions : 0.3% citric acid ( ca ) , demineralization solution ( de ) , and remineralization solution ( re ) . the specimens were individually immersed in 3 ml of the test solutions , which were daily changed . after 15 days of storage , new surface roughness and hardness readings were done . fluoride release in the solutions was measured within 15 days . data were analyzed by anova and tukey 's and contrast tests ( =0.05).results : the storage in ca increased the roughness of vitremer and ketac molar . a significant reduction in hardness was observed for all materials after storage in all solutions . for all materials , the greatest amounts of fluoride release occurred during the 1st day . fluroshield presented the same patterns of fluoride release in all solutions . ketac molar and vitremer released the highest amounts of fluoride in the ca solution.conclusions:ionomeric materials are more susceptible to degradation than resin - based materials under acidic conditions . acidic conditions lead to a higher fluoride release from ionomeric materials .

INTRODUCTION MATERIAL AND METHODS Experimental Design Specimen Preparation Experimental Phase Surface Roughness Test Knoop Hardness test Fluoride Analysis of the Storage Media Statistical Analysis RESULTS DISCUSSION CONCLUSIONS

in addition , the anticariogenic effect of fluoride - releasing fissure sealants depends on the amount of released fluoride , but obviously even more on the longevity of this release5 . these materials should provide constant fluoride release for a prolonged period of time and act as a reservoir of fluoride ion in order to promote fluorapatite formation in enamel9 . another factor in the clinical performance of sealant materials is related to biodegradation in the oral environment . each type of solution will mainly degrade components of the resin - based material . in addition , the nature of the resin matrix , size , type , and distribution of filler particles regulate the biodegradation of ionomeric and resin - based materials25 . the effects of chemical degradation are surface softening and roughening , which can decrease the long - term durability of the sealer material25 . although fluoride release has been the subject of numerous in vitro studies , the roughness and hardness pattern of fluoride - releasing materials in different storage media should be accessed . the purpose of this study was to determine the effect of acidic solutions on the degradation of ionomeric and sealant materials by means of hardness , roughness and fluoride release measurements . the first hypothesis tested was that gics show greater degradation than resin sealant materials when stored in different solutions . the second hypothesis was that storage in acidic solution leads to higher fluoride release from the tested materials . three materials were used in this in vitro experiment : a resin fissure sealant ( fluroshield ) , a rmgic ( vitremer ) and a conventional gic ( ketac molar ) . eighteen disc - shaped specimens of each material were prepared and had their initial roughness and hardness determined for baseline information . next , 6 discs of each material were randomly selected by lottery method and assigned to one of three different storage solutions : 0.3% citric acid solution ( ca ) ; demineralizing solution ( de ) ; and remineralizing solution ( re ; artificial saliva ) . new roughness and hardness readings of the materials were done at the end of the experimental period . fifty - four disc - shaped specimens ( 6 mm in diameter and 2 mm in thickness ) were prepared according to the manufacturers ' specifications , at room temperature ( 231.0c and 505% relative humidity , iso # 7489 specification ) . a piece of paraffin - coated dental floss was incorporated into the cements during setting to suspend the samples in the test solution . the resin - based materials fluroshield and vitremer were light cured on both sides ( surface and bottom ) for 40 s using a light - curing unit ( elipar tri - light ; espe , seefeld , germany ) . the surface roughness was characterized by the average roughness ( ra ) , which is the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length . three traces were recorded for each specimen at three different locations - parallel , perpendicular , and oblique to scan all specimen area . the roughness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta roughness ( ar ) was determined using the following equation : knoop hardness measurements were obtained on the exposed surface using a microhardness tester ( hmv-2000 , shimadzu , japan ) with a knoop diamond under a 50 g load for 10 s. the measurement of the indentation was performed immediately after the period of 10 s. three indentations spaced 1 mm from each other were made in the central area of each specimen . the hardness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta hardness ( h ) was determined using the following equation : after baseline hardness and roughness tests , all specimens were individually immersed in 3.0 ml of each solution . the storage media used were : 0.3% citric acid solution ( ph 3.2 ) ; de solution ( 2.0 mm calcium , 2.0 mm phosphate and acetate buffer 75 mm , ph 4.3 ) ; and re solution ( artificial saliva composed of 1.5 mm calcium , 0.9 mm phosphate , kcl 150 mm and tris [ tris- ( hydroxymethyl ) aminomethane ] buffer 20 mm , ph 7.0 ) . the storage media were changed every 24 h. duplicate aliquots of the solutions were mixed with tisab iii at a ratio of 1:0.1 and analyzed using an ion- selective electrode ( orion 96 - 09 ; orion research inc . the results obtained from each sample at 1 , 2 , 3 , 5 , 7 , 9 , 12 , 15 days were analyzed and the result was divided by the specimen area ( exposed area = 28.26 mm ) . original data for roughness , hardness and fluoride release were transformed ( log 10 , log 10 , and 0.3 exponential , respectively ) before applying anova , repeated - measures anova and tukey 's and contrast tests , since variance was not homogeneous . a multi - factor anova was applied to the roughness and hardness data to analyze the interactions between the factors ( materials and degradation solution ) . in order to assess significant differences within these factors , the repeated - measures anova was applied to verify the interaction of the materials , degradation solution , and fluoride release day of evaluation . in addition , the contrast test was performed to verify the differences among the 1st day of fluoride release and the release at days 2 , 3 , 5 , 7 , 9 , 12 and 15 . eighteen disc - shaped specimens of each material were prepared and had their initial roughness and hardness determined for baseline information . next , 6 discs of each material were randomly selected by lottery method and assigned to one of three different storage solutions : 0.3% citric acid solution ( ca ) ; demineralizing solution ( de ) ; and remineralizing solution ( re ; artificial saliva ) . new roughness and hardness readings of the materials were done at the end of the experimental period . fifty - four disc - shaped specimens ( 6 mm in diameter and 2 mm in thickness ) were prepared according to the manufacturers ' specifications , at room temperature ( 231.0c and 505% relative humidity , iso # 7489 specification ) . a piece of paraffin - coated dental floss was incorporated into the cements during setting to suspend the samples in the test solution . the resin - based materials fluroshield and vitremer were light cured on both sides ( surface and bottom ) for 40 s using a light - curing unit ( elipar tri - light ; espe , seefeld , germany ) . the surface roughness was characterized by the average roughness ( ra ) , which is the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length . three traces were recorded for each specimen at three different locations - parallel , perpendicular , and oblique to scan all specimen area . the roughness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta roughness ( ar ) was determined using the following equation : knoop hardness measurements were obtained on the exposed surface using a microhardness tester ( hmv-2000 , shimadzu , japan ) with a knoop diamond under a 50 g load for 10 s. the measurement of the indentation was performed immediately after the period of 10 s. three indentations spaced 1 mm from each other were made in the central area of each specimen . the hardness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta hardness ( h ) was determined using the following equation : after baseline hardness and roughness tests , all specimens were individually immersed in 3.0 ml of each solution . the storage media used were : 0.3% citric acid solution ( ph 3.2 ) ; de solution ( 2.0 mm calcium , 2.0 mm phosphate and acetate buffer 75 mm , ph 4.3 ) ; and re solution ( artificial saliva composed of 1.5 mm calcium , 0.9 mm phosphate , kcl 150 mm and tris [ tris- ( hydroxymethyl ) aminomethane ] buffer 20 mm , ph 7.0 ) . the storage media were changed every 24 h. duplicate aliquots of the solutions were mixed with tisab iii at a ratio of 1:0.1 and analyzed using an ion- selective electrode ( orion 96 - 09 ; orion research inc . , which was previously calibrated with various standard solutions ( 0.065 , 0.125 , 0.250 , 0.500 , and 1.000 mg of f / ml ) . the surface roughness was characterized by the average roughness ( ra ) , which is the arithmetical average value of all absolute distances of the roughness profile from the centerline within the measuring length . three traces were recorded for each specimen at three different locations - parallel , perpendicular , and oblique to scan all specimen area . the roughness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta roughness ( ar ) was determined using the following equation : knoop hardness measurements were obtained on the exposed surface using a microhardness tester ( hmv-2000 , shimadzu , japan ) with a knoop diamond under a 50 g load for 10 s. the measurement of the indentation was performed immediately after the period of 10 s. three indentations spaced 1 mm from each other were made in the central area of each specimen . the hardness test was performed at baseline ( b ) and after 15 days ( f ) , and the delta hardness ( h ) was determined using the following equation : after baseline hardness and roughness tests , all specimens were individually immersed in 3.0 ml of each solution . the storage media used were : 0.3% citric acid solution ( ph 3.2 ) ; de solution ( 2.0 mm calcium , 2.0 mm phosphate and acetate buffer 75 mm , ph 4.3 ) ; and re solution ( artificial saliva composed of 1.5 mm calcium , 0.9 mm phosphate , kcl 150 mm and tris [ tris- ( hydroxymethyl ) aminomethane ] buffer 20 mm , ph 7.0 ) . the storage media were changed every 24 h. duplicate aliquots of the solutions were mixed with tisab iii at a ratio of 1:0.1 and analyzed using an ion- selective electrode ( orion 96 - 09 ; orion research inc . , which was previously calibrated with various standard solutions ( 0.065 , 0.125 , 0.250 , 0.500 , and 1.000 mg of f / ml ) . the amount of released fluoride in the different time periods was evaluated . original data for roughness , hardness and fluoride release were transformed ( log 10 , log 10 , and 0.3 exponential , respectively ) before applying anova , repeated - measures anova and tukey 's and contrast tests , since variance was not homogeneous . a multi - factor anova was applied to the roughness and hardness data to analyze the interactions between the factors ( materials and degradation solution ) . in order to assess significant differences within these factors , the repeated - measures anova was applied to verify the interaction of the materials , degradation solution , and fluoride release day of evaluation . in addition , the contrast test was performed to verify the differences among the 1st day of fluoride release and the release at days 2 , 3 , 5 , 7 , 9 , 12 and 15 . in addition , the original data of roughness mean ( ra -m ) and standard deviation ( sd ) before ( baseline measurement - gray columns ) and after storage are demonstrated in table 1 . the immersion in ca caused a greater increase in roughness for vitremer and ketac molar than for fluroshield . before the storage ( baseline measurement - gray columns ) , ketac molar was significantly harder than fluroshield . a significant decrease in hardness was observed for all materials in all solutions , there was no difference among the groups , considering the h . after the storage in ca and de , there was no difference among the materials . the storage in re changed the ranking of the materials as follows : ketac molar = vitremer > fluroshield . similar capital letters in a column signify no significant statistical difference among solutions , according to anova and tukey tests ( p>0.05 ) . similar small letters in a line signify no significant statistical difference among materials , according to anova and tukey tests ( p>0.05 ) , considering the same column colors . the patterns of fluoride ion release for the materials in different solutions are shown in figure 3 . all materials demonstrated higher fluoride release on the first day . over the next days , the fluoride release decreased until it reached a plateau , in which the fluoride release was lower and constant until the end of the experiment ( 15 days ) . there was no difference in fluoride release for all materials in re solution during the 15 days of the experiment ( p>0.05 ) . in the de solution , on the 1st day after 3 days , fluoride release of all materials was similar . in the ca solution , during the first 2 days , vitremer released the highest amounts of fluoride , whereas after 3 - 15 days the release was highest for ketac molar . fluroshield released the lowest amount of fluoride , and the pattern of fluoride released was the same in all solutions ( p>0.05 ) . ketac molar and vitremer released the highest amounts of fluoride in ca solution ( p<0.05 ) . ketac molar showed a quite constant release , whereas vitremer showed a significantly higher fluoride release on the first 2 days . for the other solutions ( de and re ) , a similar fluoride release pattern was observed . the effects of solutions that simulate the chemical oral environment on the surface roughness , hardness and fluoride ion release were investigated in this study . with regard to the hypotheses tested in this study , the first hypothesis that ionomeric sealant is more degraded by storage in different solutions than resin - based sealants was accepted . the increase in roughness was more accentuated in the gics ; however the reduction in hardness was similar for all materials . the higher degradation of ionomeric materials by the citric acid solution can be explained by the characteristics of this acid : low ph , higher concentration , titrability , and buffer capacity , as well as by the characteristics of the ionomeric materials . the main effect of citric acid on ionomeric materials seems to rely on the dissolution / disintegration of the glass particles14 . moreover , citric acid is a carboxylic acid capable of chelating ions present in the cement and forming complexes of reasonable solubility in water14 . the influence of citric acid on the surface texture of ketac molar has been demonstrated by previous data15 . this material was deteriorated when stored in low ph solutions , which makes it more susceptible to clinical failure15 . the absence of increase in roughness for fluroshield , after exposure to the storage solutions , is explained by the relationship between resin matrix and filler particles . based on the effect of the storage solution on the degradation of the materials , it could be assumed that the acidic condition has a major effect on roughness due to the ionic dissolution of the filler particles . the decrease in hardness of fluroshield seems to be related to the water sorption and hydrolysis , since all solutions , regardless of their ph , had a similar effect on material hardness . the presence of water has a key role in the deterioration of resin - based materials . it may be suggested that the main effect of the solutions on resin - based materials is the swelling of the matrix caused by hydrolysis . the reduction in hardness may also affect the leaching of residual monomers into the solutions , especially tegdma7 . the elution of organic compounds such as bisgma , udma , tegdma , hema and additives could be related to the observed reduction in hardness after storage in all solutions23 . the presence of different sizes and shapes of the glass particles dispersed on the matrix is another reason for the higher hardness24 . the marked reduction in hardness observed in ketac molar could be the result of a pronounced erosion of the polyacid matrix , the leaching of inorganic components , and an increased hydrolytic degradation at the matrix - filler interface1 . in addition , an increase has been observed in the leaching of inorganic components , such as fluoride , and a reduction of surface hardness in acidic conditions , corroborating the results of behrend and geurtsen1 ( 2001 ) . considering the intermediate microstructure of this resin - based ionomeric material , intermediate values of hardness , this material is a resin - based sealant with relatively low filler content ( about 50% ) . the second hypothesis tested in this study that acidic solutions lead to a higher fluoride release , was partially accepted , because this assumption worked only for the ionomeric materials . the fluoride release rates and periods of sealer materials are clinically important in establishing the extent to which dental caries can be prevented . the common finding for all the evaluated materials was the similar pattern of fluoride release in the three storage media . the kinetic profile of fluoride release was characterized by a rapid initial increase ( first 24 h ) and , within 15 days of storage , a plateau was reached ; however the materials continued to release low fluoride rates until the end of the experiment . the burst effect was observed for all materials , but it was more pronounced for vitremer , which is consistent with the results of the previous studies1,4 . vitremer and ketac molar presented great amounts of fluoride in the acid media ( de and ca solutions ) , in accordance with other studies11,16 . this phenomenon is caused by an accentuated erosion of the polysalt matrix of the glass ionomer and because of the dissolution of this material , which increases with low ph5 . the pattern of fluoride ion release found in vitremer suggested that its fluoride release mechanism was similar to that of ketac molar , although the process of elution of fluoride seemed to take longer , probably because of the resin matrix , which reduces the elution rate of the fluoride ions . fluroshiled material was characterized by a low fluoride release rate , regardless of the storage solutions . these results may be explained by the characteristics of the resin sealant matrix , which is less hydrophilic than that of conventional and rmgics , making fluoride release more difficult20 and slower after polymerization22 . the low fluoride release of fluroshiled raises the question of whether this resin - based sealant has a significant preventive effect in high caries - risk patients . the findings of this study suggest that the mechanical and chemical properties of the sealant materials could be influenced by acidic solutions . although the levels of fluoride release from conventional and rmgics in these solutions were higher , these materials were more susceptible to acidic degradation ( increase in roughness ) than the resin sealant , suggesting that the choice for the sealant material must be based on the characteristics of the material , such as mechanical properties , fluoride release , and long - term durability , as well as the caries risk / activity of the patient . nevertheless , periodic evaluation must be carried out because degradation is expected for all materials placed in the oral environment . based on the results of this study , it may be concluded that : 1 . under acidic conditions , the ionomeric materials were more susceptible to degradation ( increase in roughness ) than the resin fissure sealant ; 2 . however , a higher degradation ( increase in roughness and decrease in hardness ) was also observed in this condition ; 3 . the resin fissure sealant was not affected by the acidic environment and showed similar roughness and fluoride release after storage in all tested media .

obesity may be considered as the result of a positive energy balance in conditions of energy excess . correlated with economic , social , and lifestyle changes , it represents a common condition of different populations living in environments characterised by abundant calorie - rich food and low physical activity . hence , obesity is rapidly arriving at epidemic proportions in many parts of world and is becoming one of the major public health problems . more than 1 billion individuals are overweight and more than 300 million worldwide subjects can be classified as obese ( with body mass index ( bmi ) of 30 kg / m or higher ) . more than two thirds of american population is overweight , a common condition of other western populations . in particular , the highest frequency of obesity is observed in the united states , europe and the middle east and the lowest in sub - saharan africa and east asia . in these parts of world , this condition is very alarming , because it occurs in children and adolescents [ 4 , 5 ] . hence , the current obesity may be only considered the tip of the iceberg , which will see young subjects develop the typical age - related diseases , because obesity predisposes to a variety of age - related inflammatory diseases , including insulin resistance ( ir ) , type 2 diabetes , atherosclerosis and its complications , fatty liver diseases , osteoarthritis , rheumatoid arthritis , and cancer . the obesity state is , indeed , characterized by what has been called low - grade systemic inflammation , induced by different inflammatory mediators , as demonstrated for the first time by hotamisligil et al . in 1993 . the growing evidence on the obesity and the associated pathologies has led to understand the role of adipose tissue as an active potential participant in controlling the physiological and pathological processes . to date , the adipose tissue is considered as an endocrine organ able to mediate biological effects on metabolism and inflammation , contributing to the maintenance of energy homeostasis and , probably , pathogenesis of obesity - related metabolic and inflammatory complications . this review describes the role of adipose tissue and evidences the biological effects and clinical significance of many adipokines in obesity - related inflammatory diseases . it represents the major source of fatty acids ( ffa ) in the postprandial fasting state for energy use and heat production . two types of adipose tissue are present in mammals : white adipose tissue ( wat ) and brown adipose tissue ( bat ) . they have not only different functions , but also a different cellular composition and localization . wat constitutes the major component of body 's adipose tissue , provides most of the total body fat and is the source of ffa , used as energy substrates for the generation through oxidative phosphorylation of adenosine triphosphate ( atp ) high - energy bonds . its major depots are intraabdominal around the omentum , intestines and peri - renal areas , and subcutaneous in the buttocks , thighs and abdomen . hence , it is possible to identify several local wat subgroups , including visceral , muscle , epicardial , perivascular and kidney . furthermore , wat seems to have two key functions , as recently demonstrated by worzniak et al . and juge - aubry et al . [ 8 , 10 ] . it is involved in the control of the metabolism through energy homeostasis , adipocyte differentiation , and insulin sensitivity . besides , it affects inflammation , through a control mechanism mediated by antiinflammatory molecules and the activation of anti - inflammatory metabolic and immune pathways [ 8 , 10 ] . its excessive accumulation in these body 's sites might arise and determine the development of obesity and the obesity - related diseases . much common is the wat excess in the upper parts of body , the so - called android obesity or central obesity , which represents a strong risk factor for some inflammatory pathologies . the wat excess in other lower body 's sites gives rise to the so - called gynoid obesity with no metabolic complications [ 9 , 11 ] . to understand the different wat distribution and its different link with metabolic and inflammatory complications , several theories have been advanced . among these , the first is based on the anatomy of central obesity and its capacity to drain ffa and inflammatory mediators into the portal circulation , where they can act preferentially on the liver to affect metabolism . the second considers cell biology and different properties of wat cells linked with a major or minor risk to develop metabolic and inflammatory diseases . significant differences in expression of several genes between the different body 's deposits of wat in both rodents and human have been detected [ 1315 ] . interestingly , a different mediator profile has also been observed between visceral and peripheral wat . several types of cells constitute wat : mature adipocytes and a variety of other cells ( i.e. , preadipocytes , fibroblasts , endothelial cells , and macrophages ) , usually grouped and described as the stroma vascular fraction [ 9 , 11 , 16 ] . the adipocytes , preadipocytes , and macrophages have metabolic and inflammatory functions , which render wat able to release several mediators with different biological effects in the wat itself or other tissues , acting in paracrine or endocrine way [ 8 , 10 , 1620 ] . in particular , the macrophages are responsible for the circulating levels of specific inflammatory molecules , determining the low - grade chronic obesity - related inflammation [ 8 , 11 , 16 , 17 , 21 ] . unlike wat , bat provides energy expenditure from nonoxidative phosphorylation in form of heat largely for cold adaptation . the uncoupling of phosphorylation in bat is due to the activity of uncoupling protein-1 , expressed on the internal mitochondrial membrane , which by creating a proton leak exhausts the electrochemical gradient needed for oxidative phosphorylation . as consequence , bat affects energy use by producing heat from uncoupled oxidative phosphorylation . unlike wat , bat also presents a smaller number of fat cells , which have richer vascular supplies with more abundant mitochondrial chromogens , responsible for the brown colour . bat with a richer vascular supply responds more rapidly to sympathetic nervous system ( sns ) stimulation , which then elicits heat production , rather than atp production , from nonshivering cold adaptive thermogenesis . hence , bat shows a different function respect to wat , and , in most mammals , it is responsible of the heat for fever , the arousal state from hibernation and cold - induced - thermogenesis . in humans , bat is difficult to find postnatally . however , the positron emission tomography has clearly shown in adult humans metabolically active bat depots in cervical , supraclavicular , axillary and paraventral body 's regions . these depots can be induced in response to cold and sns activation [ 2224 ] . this highlights bat as a potential relevant target for both pharmacological and gene expression manipulation to combat human obesity [ 23 , 24 ] . to understand the potential mechanisms involved in energy - rich condition under overnutrition , it is necessary to known the processes of energy homeostasis . adequate body fat and energy homeostasis are ensured through a dynamic process of fat remodelling , without excessive weight gain or loss . conditions of increased appetite and food intake determine a positive energy balance with weight gain . in contrast , satiety limits food consumption determining a negative energy balance and weight loss . energy expenditure is regulated by central and autonomic nervous systems , which achieve a balanced energy homeostasis depending on physiological needs . hence , conditions of more energy expenditure determine wat lipolysis and the consequent augment of ffa , or , conversely , less energy expenditure states allow an increased fat storage . bat responds to cold - induced sns activity with increased production of heat from uncoupled oxidative phosphorylation [ 27 , 28 ] . wat stimulated by upregulated sns activity in response to cold increases the thermogenesis from oxidative phosphorylation of ffa within liver , muscle and fat cells , which is increased in obesity . conditions of increased calorie intake associated with decreased energy use induce obesity . in obese conditions , bat mass and function are strongly decreased . obese individuals who are heat intolerant are less able to dissipate heat from both wat and bat . a regulated interaction between metabolic and immunity system exists . both over and undernutrition conditions influence metabolism and immune functions [ 30 , 31 ] . malnutrition conditions can suppress immune system and increase susceptibility to infections [ 30 , 31 ] . obesity , an energy - rich condition associated with overnutrition , impairs systemic metabolic homeostasis and elicits stress . an inflammatory process is simultaneously activated by increased wat mass in metabolically active sites , such as wat itself , liver and immune cells [ 11 , 1618 , 20 ] . this response determines a strong increase in circulating levels of proinflammatory cytokines , hormone - like molecules and other inflammatory markers , collectively defined adipokines [ 8 , 18 , 19 ] . to counteract the obesity - related stress , hypothalamic - pituitary - adrenal axis and central and peripheral components of autonomic nervous system this leads to an increased level of glucocorticoid , a steroid hormone able also to induce the development and differentiation of preadipocytes , favouring consequently the further increase of wat mass . on the other hand , the secretion of pro - inflammatory cytokines by wat may act as additional chronic stimulus for activation of hypothalamic - pituitary - adrenal axis . hence , a vicious cycle between metabolic and immune responses in obesity state is promoted , inducing a chronic active inflammatory condition able to determine the onset of obesity - related pathologies [ 29 , 31 ] . the causes and mechanisms involved in obesity - induced inflammatory state are not fully understood , even if the link between inflammation and obesity has been indicated by epidemiological studies from 1950s onwards . the discovery of the production of proinflammatory cytokines in the wat , such as tumour necrosis factor ( tnf)- , and tnf- capacity to regulate insulin action has systemically also represented a driving force in this field . today , current opinion proposes that , under normal wat conditions , adipocytes store lipids and regulate metabolic homeostasis , and resident tissue macrophages , with polarization essentially of m2 type , release anti - inflammatory cytokines . m2 macrophages produce arginase ( enzyme involved in the inhibition of nitric oxide synthase , inos ) and il-10 , il-1ra anti - inflammatory cytokines [ 21 , 34 , 35 ] . in contrast , m1 type macrophages have a specific surface marker ( cd11c+ ) and release inos and classical proinflammatory cytokines [ 21 , 34 , 35 ] . normal wat is , so , characterised by an anti - inflammatory tissue milieu able to protect from the development of obesity - related inflammation and ir , most likely also due to activity of members of peroxisome proliferator - activated receptor-(ppar)s ( particularly ppar- and - ) and liver x receptor-(lxr ) families , molecules involved in nutrient transport and metabolism and able to antagonize inflammatory activity [ 36 , 37 ] . to contribute to this physiological wat condition is a new adipokine , the lipocalin-2 ( lcn2 ) . lcn2 upregulates ppar , increases the release of adiponectin and also antagonizes tnf- effects on inflammatory and metabolic gene expression in adipocytes and macrophages . conversely , knocking down lcn2 expression , using lentiviral shrna gene silencing , results in decreased expression of ppar and its target genes , adiponectin , and leptin . hence , lcn2 , seems to act as an antagonist to the effect of inflammatory molecules on inflammation and secretion of adipokines . in obesity conditions , wat becomes inflamed , state determined by a crosstalk principally between adipocytes and macrophages [ 8 , 16 , 21 ] . the obesity - related inflammatory state occurs in several sequential stages , characterised by a cellular wat composition remodelling . an increase in number ( hyperplasia ) and size ( hypertrophy ) of adipocytes , a macrophage infiltration , and fibrosis characterise wat in obesity human conditions [ 3945 ] . adipocyte hypertrophy is induced by two factors : increased fat storage in fully differentiated adipocytes and increased expression of proinflammatory mediators [ 4345 ] . on the other hand , hypertrophic adipocytes shift the immune balance towards the production of proinflammatory molecules [ 40 , 4649 ] . the shift in the cytokines profile creates a tissue milieu responsible of the strong modification of the wat macrophages pool from activated m2 type to classically - activated m1 type [ 21 , 34 , 35 , 40 , 4649 ] . in addition , the m1 macrophage wat pool considerably increases because of the differentiation of monocytes recruited in inflamed wat . in obese wat , macrophages also aggregate in crown - like structures constituted by necrotic - like adipocytes and adipocyte cellular fragments [ 4651 ] . an increased infiltration of macrophages , indeed , occurs in the inflamed wat , preferentially into visceral wat , which contributes of the wat inflammation state and its exacerbation [ 10 , 16 , 17 , 20 , 21 , 34 , 35 , 46 , 47 ] . it seems also to be directly correlated with both hyperplasia and hypertrophy of adipocytes and inflammatory mediator production [ 10 , 16 , 17 , 20 , 21 ] . however , the mechanisms responsible for attracting monocyte / macrophage cells and their entry into the fat mass remain unclear . the group of sengens et al . has recently evidenced a key role of endothelial cells in the control of the inflammatory wat process . it has also been described the potential involvement of wat - endothelial cells as further factors involved in the regulation of macrophage phenotype in the inflamed fat tissue . inflammatory wat cytokine profile seems to be responsible of the activation of endothelial cells and their expression of a series of adhesion molecules involved in the recruitment of monocyte / macrophages . furthermore , it has been demonstrated an association between angiogenesis and adipogenesis . concerning the shift of cytokines ( m2/m1 cytokine profile ) , the exact mechanisms involved have not yet been clarified [ 21 , 34 , 35 , 40 , 4649 ] . it has been proposed that adipocytes from different body depots show differences in their inflammatory phenotype , with visceral fat characterised by more inflammatory phenotype respect to subcutaneous fat , as discussed above [ 9 , 12 ] . besides , the increased obesity - associated preadipocyte differentiation process seems also to contribute to this inflammatory cytokine profile [ 9 , 12 ] . however , human wat macrophage subsets show no strict m1 or m2 polarization , as recently demonstrated by bourlier et al . and zeyda et al . furthermore , another characteristic phenomenon , the local hypoxia induced by hypoperfusion due to rapid fat mass expanding seems to contribute to wat inflammatory state [ 5355 ] . adipose hypoxia , indeed , induces the release of proinflammatory mediators [ 5355 ] . however , on the whole , these observations give no a clear dissection of the cause and effect relationship between obesity and inflammation . on the other hand , it has , recently , been demonstrated that increased adipose tissue mass is not essentially related with wat inflammation state , as observed in two studies performed in adiponectin transgenic mice and lipocalin-2 knockout mice [ 5658 ] . this suggests that , although obesity is directly linked to inflammation , the specific role of adipokines and related pathways might be clarified . wat releases hundreds of biologically active molecules , the adipokines , including more than 50 cytokines , chemokines , hormone - like factors and other mediators [ 8 , 18 , 19 ] not exclusively produced by wat cells , these mediators are released by other different body 's tissues and organs with functions unrelated to those within wat [ 18 , 19 ] . adipokines affect appetite and satiety , glucose and lipid metabolism , blood pressure regulation , inflammation and immune functions [ 8 , 18 , 19 ] . precisely , they work as a network to regulate inflammation , insulin action , and glucose metabolism locally and systemically . this adipokine / cytokine networking system is altered in obesity , contributing to inflammation state and impaired adipocyte metabolism . however , how adipokines and cytokines coordinately regulate obesity - related inflammation and metabolism is not clearly understood [ 8 , 18 , 19 ] . different mechanisms are involved in the adipokine secretion . the production of inflammatory adipokines ( such as proinflammatory cytokines , chemokines , molecules associated with thrombosis , and hypertension , etc . ) seems to be complex and involves several inflammatory pathways , activated by both extracellular mediators and intracellular stressors . among extracellular factors , , they are chronically elevated ( by determining lipotoxicity ) , because of blunted incapacity of insulin to inhibit the lipolysis and the excessive consumption of dietary lipids . innate immunity receptors , such as toll - like receptor ( tlr)-4 and -2 , are expressed in wat ( particularly by adipocytes , preadipocytes , macrophages , and endothelial cells ) and are involved in this obesity - related inflammatory process . ffa and other molecules produced by hypoxic conditions during obesity activate these receptors , particularly tlr4 [ 60 , 61 ] . it is continually produced within the gut by death of gram - negative bacteria and is absorbed into intestinal capillaries to be transported by lipoproteins [ 63 , 64 ] . on the other hand , it has been observed that a high - fat diet given to mice increases the proportion of gut lps [ 63 , 64 ] . these data indicate the gut microbiotia may have an important role in the induction of chronic obesity - related inflammation . hence , ffa , particularly via tlr4 induce the pro - inflammatory adipokine production in adipocytes [ 65 , 66 ] . ffa also activate macrophages , referentially of the cd11c+ subset , through the tlr4 pathway , exacerbating their pro - inflammatory activity [ 6668 ] . furthermore , a paracrine loop between hypertrophied adipocytes and macrophages has been evidenced , able to induce a vicious circle of inflammatory exacerbated wat state . this paracrine loop involves ffa and tnf-. as a result , macrophages secrete the pro - inflammatory tnf-. tnf- in turn , acting particularly on tnf- receptor 1 subtype , induces inflammatory changes in hypertrophied adipocytes as well as increased release of ffa . in addition , this paracrine cross - talk could be further improved in obese subjects through the adipocyte hyperresponsiveness to tnf- and subsequent hyperactivation of inflammatory pathway . ffa may also be active on the adipocyte in an autocrine way to evoke an inflammatory state and chemokine / adipokine overproduction at least in part via tlr4 . this autocrine mechanism has been proposed to be an initial event of the inflammatory wat cascade , but this issue is still controversial . in obese wat the cells and the intracellular organelles are also exposed to increased stress , mainly as a result of metabolic overload . in particular , mitochondria and the endoplasmic reticulum appear to be the most sensitive organelles to metabolic stressors . in addition , the development of hypoxic conditions in the expanded wat during obesity results in an increased production of reactive oxygen species and the corresponding development of oxidative stress . signals mediated by both extracellular and intracellular factors culminate predominantly in the activation , principally via tlr4 receptor , of nf-b transcriptional factor , responsible of the production of inflammatory mediators , as well as the direct inhibition of insulin signaling [ 60 , 61 , 71 ] . hence , nf-b pathway represents the crucial and major factor responsible of obesity - induced inflammation . to amplify inflammation - mediated inhibition of insulin action are also other pathways , such as those mediated through the suppressor of cytokine signaling-(socs)-proteins and inos [ 72 , 73 ] in contrast , anti - inflammatory adipokine molecules seem to be released through the activation of different transcription factors induced via ppar- and lxr receptors , as mentioned above [ 3638 ] . in conditions of overnutrition , fatty acid - binding proteins , fabps , likely sequester ligands of ppar- and lxrs and induce no activation of these transcription factors . in this paper , the attention has particularly been focussed on adipokines involved in the obesity - related inflammatory diseases . the biological effects of metabolic and inflammatory adipokines are reported in tables 1 and 2 . several pathologies are associated with obesity , such as type 2 diabetes and cardiovascular diseases ( cvd ) . more recently , the obesity - related risk has also been extended to cancer , including prostate , breast , liver , kidney , colon , ovarian and endometrial cancers [ 7581 ] . the obesity - related diseases are characterised by inflammatory pathophysiology induced by several risk factors ( environmental stressors , genetic factors , etc . ) and an onset generally correlated with ageing process . epidemiological studies have revealed that a common yet preventable risk factor for these diseases is the increase of the visceral fat , a characterised hallmark of ageing in humans ( figure 1 ) [ 8 , 11 , 12 , 82 ] . using either waist circumference and/or waist - to - hip ratio as a proxy of visceral obesity , the role of visceral fat as stronger risk factor for these diseases , than bmi or other fat depots , has been confirmed . obesity - related risk is not only limited to these diseases , but also to cognitive decline , alzheimer 's disease ( ad ) and disability , as recently demonstrated [ 6 , 8385 ] . recent evidence also reports an association of obesity with increased risk of disease specific and all - cause mortality , and with a reduced life expectancy . for example , the group of fontaine reported that caucasian men and women with a bmi > 40 and age range 2029 years , could expect a reduction in remaining years of life expected by approximately 6 and 12 years , respectively . an increment of mortality and a reduction of life expectancy correlated with obesity , especially in old subjects , has been , indeed , proposed . on the other hand , obesity , and precisely visceral obesity , it has been demonstrated that obese women have telomeres of 240 bp shorter lean women of a similar age . in view of the hypothesis that telomere length in vivo represents cellular turnover and exposure to oxidative and inflammatory damage , this difference in telomere length between being lean and being obese might correspond to 8.8 years of ageing . the increased evidence on visceral obesity , as a stronger predictor of these diseases , has led to assess the mortality risk correlated with abdominal obesity [ 8890 ] . in 2008 , a large european study reported that both general ( bmi ) and abdominal adiposity ( waist circumference ; waist - to - hip ratio ) are strong predictors of mortality risk . however , the importance of visceral obesity was most remarkable among persons with a low bmi . the question of visceral fat , as factor capable of reducing life expectancy , has been recently clarified in an animal model study . the extrapolation of its data in humans suggests the key role of visceral fat and the possibility through its depletion to favour the survival and , hence , longevity , as demonstrated in calorie restriction rats . in human beings , its beneficial effects might be greater , since humans visceral fat depots have direct portal access and , so , a greater potential to harm the liver [ 82 , 92 ] . this has recently led huffman and barzilai to hypothesise the presumed link of visceral fat with both age - related diseases and lifespan . accumulation of visceral fat represents a greater risk for the development of ir and other processes of metabolic syndrome than other fat depots due to its anatomical location , high lipolytic rate and secretion of inflammatory adipokines . this determines some specific perturbations to tissue including hepatic ir , impaired glucose uptake by skeletal muscle and increased basal lipolytic rate and wat free fatty acid release . the long - term consequences are an increased risk for obesity - related inflammatory diseases and mortality and a reduced lifespan . the association of obesity with obesity - related inflammatory diseases might be explained through evolutionary speculations . it is appropriate , hence , to consider the fundamental biological necessities for the survival of an organism : ( 1 ) the ability to resist to starvation and ( 2 ) to evoke an efficient immune response to pathogens . to this aim , several metabolic and immune pathways and nutrient- and pathogen - sensing systems have been selected and evolutionarily conserved throughout species . the metabolic systems have been selected to assure energy efficiency through the storage of excess calories particularly under intermittent food uptake conditions . in contrast , under continuous overnutritional conditions , these systems induce a fat excess , as no advantageous state associated with the onset of several chronic disorders , such as obesity and its complications . at the same time , the necessity to defend against the infections has determined the selection of strong immune components , particularly induced by the epidemic and pandemic infections [ 93 , 94 ] . the combination of these efficient systems has likely created a fundamental biological instrument able to store energy and to evoke immune - inflammatory responses . its existence is showed by the several pathways with metabolic and immune functions evolved from common and ancestral units . a characteristic ancestral unit is the body fat of drosophila , having metabolic functions and an intimate control of immune responses . it has given rise after about 600 million years to homologous mammalian tissues , such as haematopoietic and immune systems , liver and adipose tissue [ 9598 ] . these mammalian tissues have conserved their development heritage and show metabolic and immune cellular components with an architectural organization able to have immediate access into blood vessels . hence , they have continuous and dynamic interactions with other important metabolic and immune sites , such as pancreas . this contributes to understand the mechanisms involved in metabolic diseases , such as type 2 diabetes . they have common regulatory molecular pathways and pathogen - sensing systems able to regulate both metabolic and immune functions . characteristic example is the tlr4-nf- pathway , evolved by drosophila homologous toll and able to mediate efficient immune responses also metabolically or nutritionally induced and lipid - related pathways able to respond to the energy necessities of particular conditions , such as during the induction of immune or inflammatory response [ 66 , 93 , 95 ] . its dysfunction is dangerous and responsible of the development of some diseases . in particular , overnutritional conditions , fruit of current nutritional habits and lifestyles of most modern western populations , are responsible of the development of the obesity - related inflammatory diseases . in the light of these observations and suggestions , we described the role of adipokines in the pathophysiology of obesity - related inflammatory diseases . the term metabolic syndrome ( ms ) assembles some abnormalities , including visceral obesity , dyslipidemia , hyperglycaemia and hypertension . the criteria to define ms have been established by international diabetes federation ( idf ) . in the idf consensus , ms is defined by the presence of visceral obesity plus two of the described components [ 101 , 102 ] . the presence of any two of the four next components is also required : elevated circulating levels of triglycerides , reduced levels of hdl - cholesterol , high blood pressure and impaired fasting glycaemia [ 101 , 102 ] . the idf eventually recommends other criteria to diagnose ms , such as increased levels of circulating inflammatory and/or thrombotic markers ( crp , saa , tnf- , il-6 , and pai ) or reduced levels of anti - inflammatory molecules , such as adiponectin . this syndrome is currently considered one of the major public health challenges , as demonstrated by two large studies performed , respectively , in 2.600 american individuals ( age range 2564 years ) and 3000 european subjects ( age range 55 years ) . in the two populations , the 25%40% and 30% ( of an italian cohort ) , respectively , were affected by ms . several reports have demonstrated in ms patients increased il-6 levels related to bmi [ 100 , 101 ] . in particular , the involvement of il-6 in ir and its complications has been evidenced , even if its role remains controversial [ 104106 ] . . however , il-6 seems to induce ir , impairing hepatic signaling through the increased expression of socs-3 and affecting the phosphorylation of insulin receptor substrate 1 ( irs-1 ) and the transcription factor pkb / akt [ 104106 ] . socs-3 has the capacity to bind and to inhibit the insulin receptor and to induce the proteosomal degradation of irs proteins . using 3t3-l1 adipocytes , it has been demonstrated the il-6 capacity to induce partial resistance in insulin - dependent glucose uptake through down - regulation of the phosphorylation of irs-1 and the expression of irs-1 and glucose transporter 4 ( glut4 ) [ 10 , 6668 , 107 ] . furthermore , in 3t3-l1 adipocytes , il-6 reduces the activity of lipoprotein lipase - lpl [ 52 , 107 ] . the relationship between high levels of tnf- and ms is related to the tnf- capacity to induce a c - jun nh2-terminal kinase to mediate a serine phosphorylation of irs-1 . this determines the inhibition of normal tyrosine phosphorylation of irs-1 and downstream insulin signaling . furthermore , in obesity , an overexpression of angiotensinogen and an increased activity of vasoconstrictor renin - angiotensin system have been demonstrated . this phenomenon seems also to contribute to the alteration of insulin sensitivity and to increase the incidence of type 2 diabetes and ms . recent evidence also demonstrates the association of elevated levels of systemic inflammatory molecules , such as saa and crp , with type 2 diabetes and ms . ( i.e. , proopiomelanocortin and corticotrophin - releasing hormone ) and , reciprocally the inhibition of orexigenic peptides ( i.e. , neuropeptide y and agouti - related protein ) , thereby limiting food intake . in obesity conditions , hypothalamic resistance to leptin has been found and ascribed to reduced transport of leptin across the blood - brain barrier and to increased levels of socs-3 and er stress , which inhibit leptin signaling [ 112 , 113 ] . this evokes profound changes in energy balance and hormone production via the hypothalamus , analogous to those induced in response to fasting . hence , a response of adaptation to low levels of leptin is induced , determining overfeeding and inhibition of energy expenditure , thyroid and reproductive hormones , and immunity . hypothetically , this response may be evolved as a protection against the threat of starvation . in obese subjects , these changes determine reduced energy expenditure and to regain weight , associated with lipid accumulation . ectopic lipid storage ( in liver , epicardial , and muscle fat ) is also induced in obese conditions because of leptin resistance , which may in turn further impair insulin sensitivity . like leptin effects its role may be evidenced in rodents , as suggested by an interesting theory [ 18 , 19 , 52 ] . however , successive studies in both rodents and humans have reported contradictory data [ 18 , 19 ] . an interesting role in the ms pathophysiology and its complications seems mediated through a more recent discovery adipokine , visfatin , which its plasma levels are correlated to lipid metabolism and inflammatory response [ 18 , 115 ] . it mediates a nicotinamide adenine dinucleotide ( nad ) biosynthetic activity in pancreatic cells . hence , visfatin acts as nicotinamide phosphoribosyltransferase ( nampt ) , the rate - limiting enzyme that converts nicotinamide ( a form of vitamin b3 ) to nicotinamide mononucleotide ( nmn ) , a nad precursor . it has been reported a decline with advanced age of nampt - mediated systemic nad biosynthesis , able to determine a reduced sirtuin-1 activity . this mechanism might contribute to decreased function of pancreatic cells in aged subjects . furthermore , another adipokine recently identified with a key role in insulin resistance is lcn-2 , as demonstrated in lcn-2 knockout mice . in contrast , a protective role of adiponectin against ms ( and the other obesity - related pathologies ) has recently been demonstrated . this molecule reduces m1 macrophage functions , by inhibiting phagocyte activity and release of il-6 and tnf- , and increases the il-10 and il-1ra production in adipocytes and macrophages [ 10 , 18 , 19 ] . increased plasma and wat levels of apelin have been detected [ 18 , 19 ] . tnf- seems to be the responsible of these increased levels both in plasma and wat [ 18 , 19 ] . this molecule seems to reestablish glucose tolerance and increased glucose utilization , as demonstrated in a mice study . studies on animal models particularly in the apelin - knockout mice have evidenced that loss of apelin determines heart diseases in response to pressure overload . growing evidence highlights the link of systemic - obesity inflammatory state with both cvd onset and cvd risk [ 110 , 121 ] . several studies have demonstrated a link between wat excess and cvd mortality in young ( particularly in adolescents ) and old subjects [ 110 , 121 ] . furthermore , a relationship between wat excess and coronary artery calcium ( a marker of coronary atherosclerosis ) measurement has been reported . imaging approaches have also been confirmed this association . how obesity determines the cvd development , it is until now not clearly understood . complex and numerous obesity - mediated mechanisms are identified , as well as the cvd risk obesity - related factors ( including hypertension , ir and dyslipidemia ) . systemic and wat adipokines seem also to affect vessel wall , by determining adverse effects [ 110 , 121 , 122 ] . precisely , proinflammatory cytokines , hormone - like molecules and other wat adipokines act in the liver , causing changes in the production and the release of lipoproteins , coagulation factors and inflammatory molecules [ 110 , 121 , 122 ] . in particular , they induce an increase of very - low - density lipoprotein , apolipoprotein b ( apob ) , and triglyceride secretion . these liver - released molecules act on endothelial , arterial smooth muscle and macrophages cells , by inducing atherogenic effects on the vessel wall through the regulation of their gene expression and functions [ 110 , 121 , 122 ] . in addition , visceral fat seems to be particularly involved in the activation of these pathways [ 122 , 123 ] . interestingly , among adipokines , leptin mediates different effects on cells of vessel wall . it evokes on endothelial cells oxidative stress , increased production of adhesion molecules and chemokines and proliferation [ 110 , 121 , 122 ] . for example , an increased blood release of mcp-1 in obese condition has been observed . it seems to increase the number of cd11c+ monocytes , favouring the binding of monocytes / macrophages to the vessel wall . acting also on smooth muscle cells , leptin induces their migration , proliferation , and hypertrophy [ 110 , 121 , 122 ] . it also induces a further activation and cytokine production of macrophages , neutrophils , and t cells and it seems also involved in the calcification of cells of vessel wall and the thrombosis through the increase of platelet aggregation [ 110 , 121 , 122 ] . these effects are also indirectly mediate of leptin through leptin resistance ( mentioned above ) . it induces on endothelial cells an increased expression of adhesion molecules , proinflammatory cytokines , and pentraxin .on smooth muscle cells , it evokes their migration . an increased expression of cd36 on macrophages seems to be also mediated by resistin , facilitating lipid accumulation and formation of foam cells [ 126 , 127 ] . on macrophages it also mediates an increased production of proinflammatory cytokines , through via tlr4 and nf-b pathway . among the adipokines of recent discovery , visfatin , and apelin visfatin has a key role in plaque destabilization , associated with its increased expression in macrophages of human unstable carotid and coronary atherosclerosis . have demonstrated that visfatin can , however , prolong the life of human smooth muscle cells . unlike visfatin , apelin is associated with a positive hemodynamic profile and has positive inotropic effects in normal and failing rat hearts and in isolated cardiomyocytes [ 132134 ] . in patients affected by single atrial fibrillation and chronic heart failure , reduced plasma apelin levels have been found [ 135 , 136 ] . in vessel wall and cardiovascular tissue of rats , apelin production seems to be upregulated by hypoxia and ischemic cardiomyopathy , likely as a compensatory mechanism [ 137 , 138 ] . its levels are positively correlated with hdl levels , and negatively with triglyceride levels , ir , and systemic circulating inflammatory markers [ 139 , 140 ] . furthermore , a negative correlation between adiponectin and coronary artery calcium has been observed . it also promotes several anti - atherogenic and anti - inflammatory effects on vessel cells : it downregulates the expression of adhesion molecules on endothelial cells ; it decreases endothelial oxidative stress and increases enos activity ; in smooth muscle cells it inhibits proliferation by suppressing the release of growth factors ; and in macrophages it reduces lipid accumulation and the expression of scavenger receptors . furthermore , crp , usually increased in cdv , has atherogenic effects on vessel wall [ 146 , 147 ] . this atherogenic effect is also increased by other wat molecules , such as agt , angiotensin - converting enzyme and pai-1 . agt ii has vasoconstrictive actions and also promotes systemic inflammation [ 121 , 122 ] ; agt contributes to the activation of renin - angiotensin system ( ras ) and both these molecules induce a hypertensive response [ 117 , 118 ] . ad is a heterogeneous and progressive neurodegenerative disease which in western societies mainly accounts for clinical dementia . the ad prevalence is below 1% in individuals aged 60 years , but shows an almost exponential increase with age , so that , in the western world , in people aged 85 years or older the prevalence is between 24% and 33% . it prevalence is expected to quadruple by the year 2047 in the united stated . there is currently no cure for ad and its pathogenesis remains the subject of many theories involving genetic as well as environmental factors . recent mounting evidence has been supposed the involvement of modifiable risk factors in ad neurodegeneration , such as lifestyle factors . among these several potential mechanisms seem to link obesity with ad : hyperglycemia , advanced glycosylation products , adipokine action , and the influence of obesity on vascular risk and cerebrovascular disease . ir and hyperinsulinemia seem to represent the key causes for the development of some age - related diseases , such as ad . precisely , insulin , crossing the blood brain barrier from periphery to central nervous system , seems to compete with a amyloid peptide for insulin degrading enzyme ( ide ) in the brain , including also the hippocampus . in contrast , insulin produced in the brain seems to have an advantageous effect on amyloid clearance . opposing effects seem to be mediated by conditions of peripheral hyperinsulinemia . they seem to determine the inhibition of brain insulin production , which in turn results in impaired amyloid clearance and a higher ad risk . these data suggest that reducing peripheral hyperinsulinemia and increasing brain insulin levels , beneficial effects might be attained on ad neurodegeneration . therapy strategies able to reduce blood insulin levels in humans have been demonstrated to affect cognition and levels of amyloid in the cerebrospinal fluid , supporting the potential direct role of insulin in ad [ 153 , 154 ] . hyperglycemia seems to be also responsible of the increased levels of advanced glycosylation end products ( ages ) . an increased glycation of amyloid has been demonstrated to improve its aggregation in vitro . furthermore , age receptors seem also to be specific cell surface receptors for amyloid , thus potentially facilitating neuronal damage . concerning the role of adipokines in ad , it is not clear whether their involvement in the ad pathophysiology are direct or associated with ir and hyperinsulinemia . several effects of leptin on the brain development and potentially on brain health in cognition and ageing have also been observed . this evidences the capacity of leptin to affect the function of the hypothalamus and learning and memory processes controlled by the hippocampus . leptin receptors have been observed in the hippocampus , hypothalamus , amygdala , cerebellum , and brain stem , supporting its capacity to mediate regulatory mechanisms . a direct interaction between leptin and adiponectin and however , other roles of leptin and related adipose - derived factors in the ad brain are not clear . fasting plasma leptin has been inversely correlated with grey matter volume in areas of the brain in which obese have reduced grey matter in comparison with lean individuals . current opinion proposes cd as additional brain damage to amyloid neurotoxicity [ 161 , 162 ] ras system regulates the blood pressure . both human brain and wat express ras , with wat ras involved in adipocyte growth , differentiation and metabolism [ 163 , 164 ] . its interaction with specific receptors induces the activation of ras , determining an increased of blood pressure . in the brain , angiotensin ii continues its conversion to angiotensin iv , which enhances learning and memory in animal models [ 165 , 166 ] . , some prospective studies have examined total ldl and hdl cholesterol levels as possible risk ad factors . the association of cholesterol with dementia may vary depending upon when cholesterol is measured in the life - span and/or relative to the course of disease . high cholesterol may be a risk factor if measured in midlife many years before clinical onset , but then as the disease pathology progresses , cholesterol levels may fall such that it appears that high cholesterol is protective . two finnish studies have , indeed , observed that high total cholesterol levels in mid - life are associated with an increased risk of ad more than 20 years later [ 167 , 168 ] . two studies each with more than 25 years of follow - up , did not find an association between mid - life total and hdl cholesterol and incident ad [ 169 , 170 ] . three other studies in elderly populations also did not find associations between ldl and/or hdl cholesterol and incident ad after follow - ups of 2 years and 7 years [ 171 , 172 ] . in fact , one of these studies reported an inverse association between total cholesterol and ad , such that those in the lowest quartile had the greatest risk . similarly , higher cholesterol levels have been reported to be associated with a reduced risk for ad [ 173 , 174 ] . prostate cancer ( pc ) is the most common cancer in western elderly male populations . the development of pc is based on the interaction between genetic factors and the host exposure to environmental factors , such as infectious agents , dietary carcinogens and hormonal imbalances . in this complex situation as reported above , the risk associated with obesity has also been extended to several malignancies . data on the association between obesity and pc incidence are inconsistent , and in some studies obesity is associated with an increase in risk of low - grade tumours . the reasons of these contrasting results may be due , in part , to variation of the methods of anthropometric measurement , such as bmi and the waist - to - hip ratio . however , a recent study has revealed visceral fat accumulation as specific risk factor for pc [ 6 , 76 ] . more consistently , it has recently been suggested that obesity reduces the risk of nonaggressive pc disease and increases the risk of aggressive pc disease . hence , it is possible that rather than increasing the absolute risk of pc development , obesity may be associated with the progression of latent or microscopic pc to clinically significant and metastatic pc . furthermore , the differential effects of obesity on pc subtypes suggest aetiological heterogeneity of these tumours and complex interaction between androgen metabolism and several putative risk factors , including ir , diabetes , inflammation , and genetic susceptibility , on pc risk . the molecular mechanisms liking obesity and pc pathophysiology are numerous and occur at several levels . however , the relationship between sex steroid hormones and obesity is complex and biological processes involved are unclear . current opinion suggests a decline in men of serum testosterone levels in obese conditions . in addition , increased peripheral aromatization of androgens to oestrogens , correlated with fat overload , is also involved in the decline of androgens . on the other hand , recent data have shown that higher serum levels of total testosterone are associated with a reduced risk of high - grade pc , but with an increased risk of low - grade pc . this emphasizes the complex relationship between obesity and serum sex steroid , and their differential effect on pc , but further supports the differential effect of obesity on pc subtypes . another mechanism is correlated to capacity of obesity to modify the production of other hormones , such as insulin - like growth factors ( igfs ) , having mitogenic properties . in particular , leptin , il-6 and tnf- seem able to enhance tumour growth . the obtained data have reported a positive association between high leptin levels and the risk of large volume prostatic tumours . stattin et al . have evidenced an association between moderately high leptin levels and later pc development . furthermore , the association between leptin and pc seems particularly confined to male subjects having a with waist - to - hip ratio > 0.87 . this datum evidences the interaction of leptin with other molecules correlated with abdominal obesity , such as sex hormones bioavailability and igf-1 levels . another study has demonstrated in a relatively small number of pc patients an association between serum leptin levels and prostate specific antigen and gleason score . in vitro studies have evidenced a role of leptin in pc carcinogenesis and its capacity to promote the proliferation of androgen - independent pc cell lines . in vitro , it has been also observed the capacity of leptin to induce vascular endothelial cell proliferation , and in vivo angiogenesis , key processes involved in cancer progression , invasion , and metastasis . the proliferative response of pc cells to leptin has been shown to involve intracellular signaling molecules such as phosphatidyl - inositol 3-kinase ( pi3-k ) and c - jun nh2-terminal kinase ( jnk ) . alterations in these signaling pathways are not only critical in processes of prostate carcinogenesis and malignant transformation , but also important in obesity , diabetes , and ir . difference effects seem to be mediated by the other adipokines , such as adiponectin proposed as an anticancer factor in some tumours , pc included . in support of this , significant lower levels of adiponectin have been observed in pc patients respect to subjects with benign prostatic hyperplasia or healthy controls . this study also evidences a negative association between plasma adiponectin and gleason score and pc stage . adiponectin receptors have been found in both benign and malignant human prostate tissue . in the lncap and pc3 pc cell lines , it has been evidenced that androgens , oestrogen , tnf- , leptin and adiponectin seem all to act and regulate adiponectin receptors . these results might to suggest a complex role of adiponectin in the pc carcinogenesis , mediated through its interaction with sex hormones and cytokines . human visceral obesity represents one of the major risk factors for obesity - related diseases . possible strategies for the prevention and the development of new therapeutic treatments are , hence , crucial medical challenge . in researching potential strategies , crucial questions remains open . furthermore , it is not also clear whether inflammation can simply be considered a state activated by altered nutrient clearance . to date , the literature evidence leads to consider the regulator molecular pathways , evolutionary well - conserved and able to control the evocation of immune responses and metabolic processes and , as possible ways for therapeutic approaches . however , their selection is difficult , as well as their manipulation with possible pharmacological agents to interfere with immune and metabolic systems , without to determine severe consequences on key mechanisms of organism . a possible candidate might be the tlr4-nf-b pathway , having the role of hub in the induction of both metabolic and inflammatory processes , as described . hence , its antagonists might be used to block the release of metabolic and inflammatory adipokines . on the other hand , tlr4-nf-b pathway has a key role in the pathophysiology of age - related inflammatory diseases , such as cvd , ad , and pc , as we have recently demonstrated [ 62 , 176 , 186 , 187 ] . other possible pathways might be the lipid - related pathways , such as ppar- and lxr pathways . it is already established with success that the use of thiazolidinediones or statins ( ligands of this pathway and insulin sensitising compounds ) is able to regulate lipid metabolism and to induce anti - inflammatory effects . another possible way for the development of possible pharmacological approaches for inflammatory obesity - related diseases might involve the adipokines , even if several their effects and functions remain unclear . in this case , the strategy might hypothetically have as aim the control of the bioavailability of some adipokines , such as leptin and adiponectin , in obese conditions . exogenous administration of adiponectin might counteract the consequences of obesity state , such as leptin - induced inflammation , or activate its antiatherogenic , vasoprotective and anticancer actions . another alternative avenue might be the inhibition of leptin receptors through monoclonal antibodies or mutant leptin . other possible targets might be pro - inflammatory cytokines and chemokines or their receptors , through the use of their agonists or monoclonal antibodies . in summary , these observations emphasize the necessity to discover the metabolic and immune pathways , including also the molecules involved in metabolic and pathogen sensing systems , involved in the delicate balance of interplay between metabolic and immune systems . this might be useful to clarify and to understand the mechanisms induced and to open possible ways for therapeutic approaches able to enhance the capacity of endogenous molecules to prevent stress and inflammatory responses induced by metabolic signals .

obesity is an energy - rich condition associated with overnutrition , which impairs systemic metabolic homeostasis and elicits stress . it also activates an inflammatory process in metabolically active sites , such as white adipose tissue , liver , and immune cells . as consequence , increased circulating levels of proinflammatory cytokines , hormone - like molecules , and other inflammatory markers are induced . this determines a chronic active inflammatory condition , associated with the development of the obesity - related inflammatory diseases . this paper describes the role of adipose tissue and the biological effects of many adipokines in these diseases .

1. Introduction 2. Obesity and Inflammation: Causes, Mechanisms and Consequences 3. Molecules Produced by WAT: Adipokines 4. Role of Visceral Obesity in Ageing and Obesity-Related Inflammatory Diseases 5. Adipokines and Obesity-Related Inflammatory Diseases 6. Conclusions: Possible Strategies for New Therapeutic Treatments for Obesity-Related Inflammatory Diseases

correlated with economic , social , and lifestyle changes , it represents a common condition of different populations living in environments characterised by abundant calorie - rich food and low physical activity . hence , obesity is rapidly arriving at epidemic proportions in many parts of world and is becoming one of the major public health problems . in particular , the highest frequency of obesity is observed in the united states , europe and the middle east and the lowest in sub - saharan africa and east asia . in these parts of world , this condition is very alarming , because it occurs in children and adolescents [ 4 , 5 ] . hence , the current obesity may be only considered the tip of the iceberg , which will see young subjects develop the typical age - related diseases , because obesity predisposes to a variety of age - related inflammatory diseases , including insulin resistance ( ir ) , type 2 diabetes , atherosclerosis and its complications , fatty liver diseases , osteoarthritis , rheumatoid arthritis , and cancer . the obesity state is , indeed , characterized by what has been called low - grade systemic inflammation , induced by different inflammatory mediators , as demonstrated for the first time by hotamisligil et al . the growing evidence on the obesity and the associated pathologies has led to understand the role of adipose tissue as an active potential participant in controlling the physiological and pathological processes . to date , the adipose tissue is considered as an endocrine organ able to mediate biological effects on metabolism and inflammation , contributing to the maintenance of energy homeostasis and , probably , pathogenesis of obesity - related metabolic and inflammatory complications . this review describes the role of adipose tissue and evidences the biological effects and clinical significance of many adipokines in obesity - related inflammatory diseases . two types of adipose tissue are present in mammals : white adipose tissue ( wat ) and brown adipose tissue ( bat ) . wat constitutes the major component of body 's adipose tissue , provides most of the total body fat and is the source of ffa , used as energy substrates for the generation through oxidative phosphorylation of adenosine triphosphate ( atp ) high - energy bonds . it is involved in the control of the metabolism through energy homeostasis , adipocyte differentiation , and insulin sensitivity . besides , it affects inflammation , through a control mechanism mediated by antiinflammatory molecules and the activation of anti - inflammatory metabolic and immune pathways [ 8 , 10 ] . its excessive accumulation in these body 's sites might arise and determine the development of obesity and the obesity - related diseases . the adipocytes , preadipocytes , and macrophages have metabolic and inflammatory functions , which render wat able to release several mediators with different biological effects in the wat itself or other tissues , acting in paracrine or endocrine way [ 8 , 10 , 1620 ] . in particular , the macrophages are responsible for the circulating levels of specific inflammatory molecules , determining the low - grade chronic obesity - related inflammation [ 8 , 11 , 16 , 17 , 21 ] . as consequence , bat affects energy use by producing heat from uncoupled oxidative phosphorylation . hence , bat shows a different function respect to wat , and , in most mammals , it is responsible of the heat for fever , the arousal state from hibernation and cold - induced - thermogenesis . to understand the potential mechanisms involved in energy - rich condition under overnutrition , it is necessary to known the processes of energy homeostasis . wat stimulated by upregulated sns activity in response to cold increases the thermogenesis from oxidative phosphorylation of ffa within liver , muscle and fat cells , which is increased in obesity . obesity , an energy - rich condition associated with overnutrition , impairs systemic metabolic homeostasis and elicits stress . an inflammatory process is simultaneously activated by increased wat mass in metabolically active sites , such as wat itself , liver and immune cells [ 11 , 1618 , 20 ] . this response determines a strong increase in circulating levels of proinflammatory cytokines , hormone - like molecules and other inflammatory markers , collectively defined adipokines [ 8 , 18 , 19 ] . to counteract the obesity - related stress , hypothalamic - pituitary - adrenal axis and central and peripheral components of autonomic nervous system this leads to an increased level of glucocorticoid , a steroid hormone able also to induce the development and differentiation of preadipocytes , favouring consequently the further increase of wat mass . hence , a vicious cycle between metabolic and immune responses in obesity state is promoted , inducing a chronic active inflammatory condition able to determine the onset of obesity - related pathologies [ 29 , 31 ] . the discovery of the production of proinflammatory cytokines in the wat , such as tumour necrosis factor ( tnf)- , and tnf- capacity to regulate insulin action has systemically also represented a driving force in this field . today , current opinion proposes that , under normal wat conditions , adipocytes store lipids and regulate metabolic homeostasis , and resident tissue macrophages , with polarization essentially of m2 type , release anti - inflammatory cytokines . normal wat is , so , characterised by an anti - inflammatory tissue milieu able to protect from the development of obesity - related inflammation and ir , most likely also due to activity of members of peroxisome proliferator - activated receptor-(ppar)s ( particularly ppar- and - ) and liver x receptor-(lxr ) families , molecules involved in nutrient transport and metabolism and able to antagonize inflammatory activity [ 36 , 37 ] . the obesity - related inflammatory state occurs in several sequential stages , characterised by a cellular wat composition remodelling . in obese wat , macrophages also aggregate in crown - like structures constituted by necrotic - like adipocytes and adipocyte cellular fragments [ 4651 ] . an increased infiltration of macrophages , indeed , occurs in the inflamed wat , preferentially into visceral wat , which contributes of the wat inflammation state and its exacerbation [ 10 , 16 , 17 , 20 , 21 , 34 , 35 , 46 , 47 ] . has recently evidenced a key role of endothelial cells in the control of the inflammatory wat process . it has also been described the potential involvement of wat - endothelial cells as further factors involved in the regulation of macrophage phenotype in the inflamed fat tissue . however , on the whole , these observations give no a clear dissection of the cause and effect relationship between obesity and inflammation . this suggests that , although obesity is directly linked to inflammation , the specific role of adipokines and related pathways might be clarified . wat releases hundreds of biologically active molecules , the adipokines , including more than 50 cytokines , chemokines , hormone - like factors and other mediators [ 8 , 18 , 19 ] not exclusively produced by wat cells , these mediators are released by other different body 's tissues and organs with functions unrelated to those within wat [ 18 , 19 ] . however , how adipokines and cytokines coordinately regulate obesity - related inflammation and metabolism is not clearly understood [ 8 , 18 , 19 ] . the production of inflammatory adipokines ( such as proinflammatory cytokines , chemokines , molecules associated with thrombosis , and hypertension , etc . ) innate immunity receptors , such as toll - like receptor ( tlr)-4 and -2 , are expressed in wat ( particularly by adipocytes , preadipocytes , macrophages , and endothelial cells ) and are involved in this obesity - related inflammatory process . these data indicate the gut microbiotia may have an important role in the induction of chronic obesity - related inflammation . in obese wat the cells and the intracellular organelles are also exposed to increased stress , mainly as a result of metabolic overload . in addition , the development of hypoxic conditions in the expanded wat during obesity results in an increased production of reactive oxygen species and the corresponding development of oxidative stress . to amplify inflammation - mediated inhibition of insulin action are also other pathways , such as those mediated through the suppressor of cytokine signaling-(socs)-proteins and inos [ 72 , 73 ] in contrast , anti - inflammatory adipokine molecules seem to be released through the activation of different transcription factors induced via ppar- and lxr receptors , as mentioned above [ 3638 ] . in conditions of overnutrition , fatty acid - binding proteins , fabps , likely sequester ligands of ppar- and lxrs and induce no activation of these transcription factors . in this paper , the attention has particularly been focussed on adipokines involved in the obesity - related inflammatory diseases . the biological effects of metabolic and inflammatory adipokines are reported in tables 1 and 2 . several pathologies are associated with obesity , such as type 2 diabetes and cardiovascular diseases ( cvd ) . more recently , the obesity - related risk has also been extended to cancer , including prostate , breast , liver , kidney , colon , ovarian and endometrial cancers [ 7581 ] . the obesity - related diseases are characterised by inflammatory pathophysiology induced by several risk factors ( environmental stressors , genetic factors , etc . ) epidemiological studies have revealed that a common yet preventable risk factor for these diseases is the increase of the visceral fat , a characterised hallmark of ageing in humans ( figure 1 ) [ 8 , 11 , 12 , 82 ] . using either waist circumference and/or waist - to - hip ratio as a proxy of visceral obesity , the role of visceral fat as stronger risk factor for these diseases , than bmi or other fat depots , has been confirmed . obesity - related risk is not only limited to these diseases , but also to cognitive decline , alzheimer 's disease ( ad ) and disability , as recently demonstrated [ 6 , 8385 ] . the extrapolation of its data in humans suggests the key role of visceral fat and the possibility through its depletion to favour the survival and , hence , longevity , as demonstrated in calorie restriction rats . accumulation of visceral fat represents a greater risk for the development of ir and other processes of metabolic syndrome than other fat depots due to its anatomical location , high lipolytic rate and secretion of inflammatory adipokines . the long - term consequences are an increased risk for obesity - related inflammatory diseases and mortality and a reduced lifespan . the association of obesity with obesity - related inflammatory diseases might be explained through evolutionary speculations . in contrast , under continuous overnutritional conditions , these systems induce a fat excess , as no advantageous state associated with the onset of several chronic disorders , such as obesity and its complications . it has given rise after about 600 million years to homologous mammalian tissues , such as haematopoietic and immune systems , liver and adipose tissue [ 9598 ] . hence , they have continuous and dynamic interactions with other important metabolic and immune sites , such as pancreas . this contributes to understand the mechanisms involved in metabolic diseases , such as type 2 diabetes . characteristic example is the tlr4-nf- pathway , evolved by drosophila homologous toll and able to mediate efficient immune responses also metabolically or nutritionally induced and lipid - related pathways able to respond to the energy necessities of particular conditions , such as during the induction of immune or inflammatory response [ 66 , 93 , 95 ] . its dysfunction is dangerous and responsible of the development of some diseases . in particular , overnutritional conditions , fruit of current nutritional habits and lifestyles of most modern western populations , are responsible of the development of the obesity - related inflammatory diseases . in the light of these observations and suggestions , we described the role of adipokines in the pathophysiology of obesity - related inflammatory diseases . the presence of any two of the four next components is also required : elevated circulating levels of triglycerides , reduced levels of hdl - cholesterol , high blood pressure and impaired fasting glycaemia [ 101 , 102 ] . the idf eventually recommends other criteria to diagnose ms , such as increased levels of circulating inflammatory and/or thrombotic markers ( crp , saa , tnf- , il-6 , and pai ) or reduced levels of anti - inflammatory molecules , such as adiponectin . using 3t3-l1 adipocytes , it has been demonstrated the il-6 capacity to induce partial resistance in insulin - dependent glucose uptake through down - regulation of the phosphorylation of irs-1 and the expression of irs-1 and glucose transporter 4 ( glut4 ) [ 10 , 6668 , 107 ] . recent evidence also demonstrates the association of elevated levels of systemic inflammatory molecules , such as saa and crp , with type 2 diabetes and ms . in obesity conditions , hypothalamic resistance to leptin has been found and ascribed to reduced transport of leptin across the blood - brain barrier and to increased levels of socs-3 and er stress , which inhibit leptin signaling [ 112 , 113 ] . hence , a response of adaptation to low levels of leptin is induced , determining overfeeding and inhibition of energy expenditure , thyroid and reproductive hormones , and immunity . in obese subjects , these changes determine reduced energy expenditure and to regain weight , associated with lipid accumulation . ectopic lipid storage ( in liver , epicardial , and muscle fat ) is also induced in obese conditions because of leptin resistance , which may in turn further impair insulin sensitivity . in contrast , a protective role of adiponectin against ms ( and the other obesity - related pathologies ) has recently been demonstrated . complex and numerous obesity - mediated mechanisms are identified , as well as the cvd risk obesity - related factors ( including hypertension , ir and dyslipidemia ) . precisely , proinflammatory cytokines , hormone - like molecules and other wat adipokines act in the liver , causing changes in the production and the release of lipoproteins , coagulation factors and inflammatory molecules [ 110 , 121 , 122 ] . it evokes on endothelial cells oxidative stress , increased production of adhesion molecules and chemokines and proliferation [ 110 , 121 , 122 ] . it also induces a further activation and cytokine production of macrophages , neutrophils , and t cells and it seems also involved in the calcification of cells of vessel wall and the thrombosis through the increase of platelet aggregation [ 110 , 121 , 122 ] . it induces on endothelial cells an increased expression of adhesion molecules , proinflammatory cytokines , and pentraxin .on smooth muscle cells , it evokes their migration . on macrophages it also mediates an increased production of proinflammatory cytokines , through via tlr4 and nf-b pathway . among the adipokines of recent discovery , visfatin , and apelin visfatin has a key role in plaque destabilization , associated with its increased expression in macrophages of human unstable carotid and coronary atherosclerosis . its levels are positively correlated with hdl levels , and negatively with triglyceride levels , ir , and systemic circulating inflammatory markers [ 139 , 140 ] . it also promotes several anti - atherogenic and anti - inflammatory effects on vessel cells : it downregulates the expression of adhesion molecules on endothelial cells ; it decreases endothelial oxidative stress and increases enos activity ; in smooth muscle cells it inhibits proliferation by suppressing the release of growth factors ; and in macrophages it reduces lipid accumulation and the expression of scavenger receptors . this atherogenic effect is also increased by other wat molecules , such as agt , angiotensin - converting enzyme and pai-1 . recent mounting evidence has been supposed the involvement of modifiable risk factors in ad neurodegeneration , such as lifestyle factors . among these several potential mechanisms seem to link obesity with ad : hyperglycemia , advanced glycosylation products , adipokine action , and the influence of obesity on vascular risk and cerebrovascular disease . ir and hyperinsulinemia seem to represent the key causes for the development of some age - related diseases , such as ad . therapy strategies able to reduce blood insulin levels in humans have been demonstrated to affect cognition and levels of amyloid in the cerebrospinal fluid , supporting the potential direct role of insulin in ad [ 153 , 154 ] . hyperglycemia seems to be also responsible of the increased levels of advanced glycosylation end products ( ages ) . concerning the role of adipokines in ad , it is not clear whether their involvement in the ad pathophysiology are direct or associated with ir and hyperinsulinemia . the development of pc is based on the interaction between genetic factors and the host exposure to environmental factors , such as infectious agents , dietary carcinogens and hormonal imbalances . data on the association between obesity and pc incidence are inconsistent , and in some studies obesity is associated with an increase in risk of low - grade tumours . the reasons of these contrasting results may be due , in part , to variation of the methods of anthropometric measurement , such as bmi and the waist - to - hip ratio . hence , it is possible that rather than increasing the absolute risk of pc development , obesity may be associated with the progression of latent or microscopic pc to clinically significant and metastatic pc . furthermore , the differential effects of obesity on pc subtypes suggest aetiological heterogeneity of these tumours and complex interaction between androgen metabolism and several putative risk factors , including ir , diabetes , inflammation , and genetic susceptibility , on pc risk . however , the relationship between sex steroid hormones and obesity is complex and biological processes involved are unclear . on the other hand , recent data have shown that higher serum levels of total testosterone are associated with a reduced risk of high - grade pc , but with an increased risk of low - grade pc . another mechanism is correlated to capacity of obesity to modify the production of other hormones , such as insulin - like growth factors ( igfs ) , having mitogenic properties . this datum evidences the interaction of leptin with other molecules correlated with abdominal obesity , such as sex hormones bioavailability and igf-1 levels . alterations in these signaling pathways are not only critical in processes of prostate carcinogenesis and malignant transformation , but also important in obesity , diabetes , and ir . difference effects seem to be mediated by the other adipokines , such as adiponectin proposed as an anticancer factor in some tumours , pc included . human visceral obesity represents one of the major risk factors for obesity - related diseases . possible strategies for the prevention and the development of new therapeutic treatments are , hence , crucial medical challenge . a possible candidate might be the tlr4-nf-b pathway , having the role of hub in the induction of both metabolic and inflammatory processes , as described . on the other hand , tlr4-nf-b pathway has a key role in the pathophysiology of age - related inflammatory diseases , such as cvd , ad , and pc , as we have recently demonstrated [ 62 , 176 , 186 , 187 ] . other possible pathways might be the lipid - related pathways , such as ppar- and lxr pathways . another possible way for the development of possible pharmacological approaches for inflammatory obesity - related diseases might involve the adipokines , even if several their effects and functions remain unclear . in this case , the strategy might hypothetically have as aim the control of the bioavailability of some adipokines , such as leptin and adiponectin , in obese conditions . exogenous administration of adiponectin might counteract the consequences of obesity state , such as leptin - induced inflammation , or activate its antiatherogenic , vasoprotective and anticancer actions .

it is clear that superstitious behaviours are commonplace in sport , with many amateur and professional sportspeople engaging in a range of apparently bizarre behaviours that can range from wearing lucky items of clothing to engaging in specific rituals before competition ( bleak & frederick , 1998 ) . it has also been suggested that sportspeople may be more prone to developing these beliefs ( vyse , 1997 ) . these practices may appear to be innocuous for the most part , but may be deleterious to an athlete on occasion , i did n't tie my laces right and i did n't bounce the ball five times and i did n't bring my shower sandals to the court with me . ( serena williams explaining her exit in the french open ( syed , 2009 ) ) . a superstition can be defined as acting as though there is , or believing there is , a connection between a cause and an effect where there is no rational direct association . given the potential influence of superstitions on sporting performance , it is surprising that there is not more research about them ; yet , there are not many sporting studies that indicate how these irrational beliefs and behaviours develop and how they might affect an individual 's behaviour . the primary explanation for how people might acquire superstitious behaviours can be found in the work of skinner ( skinner , 1948 ) . in lecture demonstrations , he would place a pigeon into a skinner box and by the end of the lecture would have created a superstitious pigeon that produced apparently random behaviours ( e.g. thrusting its head into the corner of the cage , turning in circles , etc . ) . skinner proposed that the pigeon would learn these behaviours via accidental operant conditioning , the bird happens to be executing some response as the hopper appears ; as a result it tends to repeat this response that is , the pigeon appeared to associate its irrelevant behaviour with the food 's appearance and then proceeded to initiate the irrelevant behaviour in an attempt to elicit more food . it might be tempting to assume that pigeons are not analogous to humans in this regard except several comparable studies have revealed similar findings using human participants ( e.g. ono , 1987 ; rudski , 2001 ) . in ono 's study ( 1987 ) , participants were asked to achieve as high a score as possible in a task involving moving three levers . participants developed a range of beliefs about how the levers caused the score to increase ; yet , just as with skinner 's pigeons , the behaviour produced by the participants had no influence on the reward ; the score simply increased on an arbitrary schedule . it should be noted that skinner himself saw the parallels of the superstitious pigeon in the development of sporting superstitions . he suggested that 10-pin bowlers who roll the ball down the lane can often be observed to continue twisting their hands after the ball is released in an apparent attempt to influence the spin of the ball ( skinner , 1948 ) . whilst some element of this could be argued to represent a normal follow - through action , the superstitious action referred to by skinner can be taken to refer to an extended action , beyond a period where a natural follow through could feasibly operate and be of benefit . skinner would see these types of behaviours as superstitious based on reinforcement after previous attempts to help guide the ball to its target . further work with random reinforcement regimes indicated that superstitious behaviour could also be related to an irrelevant stimulus ( morse & skinner , 1957 ) . this has been taken to be akin to the development of a belief in lucky items ( see vyse , 1997 ) . to date , most studies on superstition in sport have been correlational in nature relying on questionnaire measures ( e.g. bleak & frederick , 1998 ; brevers , dan , noel , & nils , 2011 ; burger & lynn , 2005 ; todd & brown , 2003 ) . for example , in the study by bleak and fredericks ( 1998 ) , it was found that chance locus of control was negatively correlated with increases with superstitious behaviours in athletes . that is , those who believed less in chance were more likely to engage in superstitious behaviour . this is a seemingly contradictory outcome , but can be interpreted as showing that those who do not believe in chance engage in superstitious behaviours as a means to control random events in the world . when athletes are uncertain of success , a greater emphasis is placed on the importance of carrying out superstitious rituals as this reduces psychological tension ( brevers et al . , 2011 ; schippers & van lang , 2006 ) . studies such as these can identify possible associations with personality traits , but more experimentally controlled studies are needed to identify the potential causes of superstition , such as that proposed by skinner . there are , however , very few studies which have attempted to study sporting superstitions in a laboratory setting , one of the reasons being that it is difficult to create representative designs in this area . notable exceptions to this are studies by van raalte , brewer , nemeroff , and linder ( 1991 ) , wright and erdal ( 2008 ) and damisch , stoberock , and mussweiler ( 2010 ) , who use a lucky ball procedure in a laboratory setting . in the study by damisch et al . ( 2010 ) , participants performed better on a golf putting task when using a ball that was said to be lucky . they also considered superstitious beliefs in other situations ; participants performed a motor - dexterity task better when the experimenter told the participant that they would keep their fingers crossed for them and had better memory when in the presence of a lucky charm . they suggested that in the presence of a lucky charm , a good luck superstition is activated , giving participants greater self - efficacy in mastering a task , leading to greater persistence and ultimately higher performance . van raalte et al.s ( 1991 ) lucky ball procedure involved asking participants to putt on an artificial putting green with one of four differently coloured balls ( red , blue , pink and green ) . in an attempt to remove participant colour preferences , these colours were selected on the basis that they were the most popular colour golf balls overall as determined in a pre - test of 135 participants . after a putt , the experimenter noted whether the putt was successful or not , then for the next trial , the ball was returned to the participant who therefore made their selection for their subsequent putt from the initial pool of four coloured golf balls . this method supposes that superstitious behaviour has occurred if the participant selects the same coloured ball after a successful performance . in line with skinner 's prediction , the participant would associate the irrelevant colour with the holed putt , thereby creating a superstition for that particular ball , and indeed this is the outcome that van raalte et al . were wise to try to remove the possibility that participants might have preferences for colours , but by still having four colour choices available , it does still leave open the possibility that a participant might make their selection wholly , or in part , on the basis of a colour preference and not their putting performance . to illustrate this issue , a participant who used the blue ball consistently throughout the study because they preferred it , and holed 10 putts , but missed 10 , would register as showing 10 examples of superstitious behaviour , where , clearly , there was no difference between their success colour choice and their fail - to - putt colour choice . van raalte 's study may therefore , in part , demonstrate superstitious preference based on colour choice rather than on the basis of putting success . the present study investigates potential mechanisms for superstition development in sport within the context of skinner 's ( 1948 ) theory of operant conditioning . based on the paucity of well - controlled sport superstition studies , the objective of the current study was to determine the extent to which it was possible to induce superstitious behaviour and beliefs in a golf putting task in a laboratory . in the present study , an experimental manipulation was employed in which inherent biases to the sports equipment were removed by simply using identical equipment , in this case , three identical golf putting clubs . given the clubs were identical , there needed to be some mechanism by which they could be told apart ; we therefore referred to them based on their location leant against a wall in the laboratory : the club on the left , the club in the centre and the club on the right . the experimental manipulation involved providing a regime of false feedback to the participant such that they thought that they had performed differently with the three clubs ( good , average and poor performance ) . putter on the basis of this reinforcement , demonstrated by choosing it in a competition phase of the experiment over other putters , and by perceiving it to have better qualities . a one - factor repeated - measures design was employed where feedback regarding performance was manipulated for a given putter being positive , honest or negative in nature . the dependent measures were the perceptions of the putters used in the task and putter selection by the participant for a competition phase . participants were 17 males and 11 females aged between 19 and 41 ( m = 21.8 , sd = 4.35 ) . all were right handed , as assessed using items from the edinburgh handedness inventory ( oldfield , 1971 ) . more importantly for the current task , all used a putter in a right - handed manner . after the experimental deception was revealed , all participants indicated consent for their data to be used for analysis . the primary research institution provided ethical approval . putting took place in a laboratory with a level floor on short pile carpet tiles , providing a surface comparable to a fast putting green . all putters were , apart from to - be - expected minimal manufacturing differences , identical in appearance . the balls used throughout the study were white wilson staff ti - dna - spin balls . the task itself required participants to putt towards a 7 7 grid comprising of 0.15 0.15 m squares marked with tape ( see figure 1 ) . the centre of the central square was 5 m from the participants striking point . in front of the participant ( 1 m away ) was an opaque divider screen ( 1.95 m in height , 1.80 m in width ) preventing visual feedback of the task outcome , but allowing passage of the golf ball underneath . attached to the divider screen was a chart of the target grid allowing the participant to easily understand the verbal feedback regarding ball position , provided during the experiment ( see procedure ) . a series of items were asked about the physical qualities and perceptions experienced whilst using the positive , negative and honest putters . all items were responded to on a 5-point likert scale for each putter , for example , for item 1 , not at all comfortable , slightly comfortable , moderately comfortable , very comfortable the 10 items were : overall , how comfortable did each of the putters feel?overall , how clean was the contact on the ball with each of the putters?in general , how easy was each of the putters to use?overall , how smooth was the roll of the golf ball from each of the putters?overall , how comfortable was the weight of each of the putters?in general , how confident did each of the putters make you feel?overall , how accurately did each of the putters control the distance of the ball?overall , how accurately did each of the putters control the direction of the ball?overall , how accurately did each of the putters control the speed of the ball?in general , how well do you feel you performed with each of the putters ? overall , how comfortable did each of the putters feel ? overall , how clean was the contact on the ball with each of the putters ? overall , how smooth was the roll of the golf ball from each of the putters ? overall , how accurately did each of the putters control the distance of the ball ? overall , how accurately did each of the putters control the direction of the ball ? overall , how accurately did each of the putters control the speed of the ball ? in general , how well do you feel you performed with each of the putters ? prior to commencing the study , participants read briefing instructions which explained that they would be asked to perform putts using three identical putters . to explain the experimental procedure , participants then used the acquisition putter , without the divider screen , to learn the basics of the task in 30 practice trials , thus ensuring that participants were familiar with the strength and line required for the task . a 7 7 grid marked on the floor with tape was used to rate putting performance ( each square being 0.15 m 0.15 m ) , the centre of which ( d4 ) was considered the target outcome . in a practice trial , the participant would place the ball at a starting point and putt towards the grid ; when the ball came to a stop , the experimenter would call out the grid reference . for example , a1 meant they had overhit the ball to the left and g7 would mean they had underhit to the right . any balls that were out of the grid were called as out of bounds and the participant did that trial again ; however , feedback was still provided on the magnitude and direction of error . in this way , the participant could see how the outcome of their putt was translated into the verbal feedback scheme . after this practice session , the divider screen was introduced and the three identical putters were leant against a wall ( 0.30 m apart ) next to the participant . the putters were referred to as the left , centre and right - hand putters to ensure that there was no natural preference for any of them ( compared to if they had been labelled with a number or colour coded ) . at this stage , participants were reminded verbally that the putters were identical . unbeknownst to the participant , the experimenter also used a random order to determine whether the feedback for this putter would be positive , honest or negative . the participant was told that they would be required to produce about 20 putts within the grid before the next putter was used . the procedure for honest feedback was as had occurred in the practice condition , the grid reference called out after each putt . after sufficient trials had been collected , participants were asked to replace the putter they had been using in its previous location and select another specified by the experimenter . the procedure for the negative feedback condition was to exaggerate feedback away from the centre , for example , balls that landed in d2 were feedback to the participant as d1 , b4 became a4 , b2 became a1 . balls in d4 were called as the square they were nearest to , balls in the outer ring of the grid were called out of bounds . the procedure for positive feedback was to exaggerate towards the centre , d2 was fed back as d3 , b4 became c4 and any square outside d4 was called as d4 . balls that were out of bounds were still called as such , however . after the third putter had been used , it was replaced in its position by the wall and the divider screen was removed . participants were informed that as thanks for participating in the study , a competition would take place where the person who could get the best putting score out of 10 putts would be given a small cash prize . they were asked to select any putter ( the selection was noted by the experimenter ) and their putting score was assessed over 10 putts for the purpose of the competition . after this competition phase , participants were asked to fill in the likert scales assessing qualities of the putters ( see materials ) and were then debriefed and thanked for their time . participants afterwards reported that they had not suspected the feedback had not been honest and had not realised the true nature of the study . to analyse the data , a chi statistic was generated for putter preference , and a one - way repeated - measures analysis of variance ( anova ) , followed by linear contrasts , was used for the questionnaire data . a one - factor repeated - measures design was employed where feedback regarding performance was manipulated for a given putter being positive , honest or negative in nature . the dependent measures were the perceptions of the putters used in the task and putter selection by the participant for a competition phase . participants were 17 males and 11 females aged between 19 and 41 ( m = 21.8 , sd = 4.35 ) . all were right handed , as assessed using items from the edinburgh handedness inventory ( oldfield , 1971 ) . more importantly for the current task , all used a putter in a right - handed manner . after the experimental deception was revealed , all participants indicated consent for their data to be used for analysis . the primary research institution provided ethical approval . putting took place in a laboratory with a level floor on short pile carpet tiles , providing a surface comparable to a all putters were , apart from to - be - expected minimal manufacturing differences , identical in appearance . an additional putter ( cleveland vas ) the balls used throughout the study were white wilson staff ti - dna - spin balls . the task itself required participants to putt towards a 7 7 grid comprising of 0.15 0.15 m squares marked with tape ( see figure 1 ) . the centre of the central square was 5 m from the participants striking point . in front of the participant ( 1 m away ) was an opaque divider screen ( 1.95 m in height , 1.80 m in width ) preventing visual feedback of the task outcome , but allowing passage of the golf ball underneath . attached to the divider screen was a chart of the target grid allowing the participant to easily understand the verbal feedback regarding ball position , provided during the experiment ( see procedure ) . a series of items were asked about the physical qualities and perceptions experienced whilst using the positive , negative and honest putters . all items were responded to on a 5-point likert scale for each putter , for example , for item 1 , not at all comfortable , slightly comfortable , moderately comfortable the 10 items were : overall , how comfortable did each of the putters feel?overall , how clean was the contact on the ball with each of the putters?in general , how easy was each of the putters to use?overall , how smooth was the roll of the golf ball from each of the putters?overall , how comfortable was the weight of each of the putters?in general , how confident did each of the putters make you feel?overall , how accurately did each of the putters control the distance of the ball?overall , how accurately did each of the putters control the direction of the ball?overall , how accurately did each of the putters control the speed of the ball?in general , how well do you feel you performed with each of the putters ? overall , how clean was the contact on the ball with each of the putters ? overall , how smooth was the roll of the golf ball from each of the putters ? overall , how accurately did each of the putters control the distance of the ball ? overall , how accurately did each of the putters control the direction of the ball ? overall , how accurately did each of the putters control the speed of the ball ? in general , how well do you feel you performed with each of the putters ? prior to commencing the study , participants read briefing instructions which explained that they would be asked to perform putts using three identical putters . to explain the experimental procedure , participants then used the acquisition putter , without the divider screen , to learn the basics of the task in 30 practice trials , thus ensuring that participants were familiar with the strength and line required for the task . a 7 7 grid marked on the floor with tape was used to rate putting performance ( each square being 0.15 m 0.15 m ) , the centre of which ( d4 ) was considered the target outcome . in a practice trial , the participant would place the ball at a starting point and putt towards the grid ; when the ball came to a stop , the experimenter would call out the grid reference . for example , a1 meant they had overhit the ball to the left and g7 would mean they had underhit to the right . any balls that were out of the grid were called as out of bounds and the participant did that trial again ; however , feedback was still provided on the magnitude and direction of error . in this way , the participant could see how the outcome of their putt was translated into the verbal feedback scheme . after this practice session , the divider screen was introduced and the three identical putters were leant against a wall ( 0.30 m apart ) next to the participant . the putters were referred to as the left , centre and right - hand putters to ensure that there was no natural preference for any of them ( compared to if they had been labelled with a number or colour coded ) . at this stage , participants were reminded verbally that the putters were identical . unbeknownst to the participant , the experimenter also used a random order to determine whether the feedback for this putter would be positive , honest or negative . the participant was told that they would be required to produce about 20 putts within the grid before the next putter was used . the procedure for honest feedback was as had occurred in the practice condition , the grid reference called out after each putt . for reference , after sufficient trials had been collected , participants were asked to replace the putter they had been using in its previous location and select another specified by the experimenter . the procedure for the negative feedback condition was to exaggerate feedback away from the centre , for example , balls that landed in d2 were feedback to the participant as d1 , b4 became a4 , b2 became a1 . balls in d4 were called as the square they were nearest to , balls in the outer ring of the grid were called out of bounds . the procedure for positive feedback was to exaggerate towards the centre , d2 was fed back as d3 , b4 became c4 and any square outside d4 was called as d4 . after the third putter had been used , it was replaced in its position by the wall and the divider screen was removed . participants were informed that as thanks for participating in the study , a competition would take place where the person who could get the best putting score out of 10 putts would be given a small cash prize . they were asked to select any putter ( the selection was noted by the experimenter ) and their putting score was assessed over 10 putts for the purpose of the competition . after this competition phase , participants were asked to fill in the likert scales assessing qualities of the putters ( see materials ) and were then debriefed and thanked for their time . participants afterwards reported that they had not suspected the feedback had not been honest and had not realised the true nature of the study . to analyse the data , a chi statistic was generated for putter preference , and a one - way repeated - measures analysis of variance ( anova ) , followed by linear contrasts , was used for the questionnaire data . overall , the selection of putter for the competition phase was more likely to be the positive putter ( n = 22 ) , although the honest and negative feedback putter were also selected ( n = 5 and n = 1 , respectively ) . this selection was not random as determined by a contingency table , ( 2 , n = 28 ) = 26.65 , p < 0.01 . one - way repeated - measures anova indicated that the perceptions of the putter also varied depending on the feedback provided , with all main effects significant at p < .001 , the effect sizes were all large ( see table 1 ) . in addition , all linear contrasts were significant for all questions at p < .001 . the positive putter was perceived most favourably , followed by the honest , then the negative feedback putter . assumptions of normality were met as indicated by skewness and kurtosis values within the range deemed acceptable ( tabachnick & fidell , 2007).where the sphericity assumption was violated , greenhouse - geisser statistics are reported . for the sake of brevity , mean response on a 15 scale to questions about the club ( standard deviation in parenthesis ) , with main effect of putter type followed by linear contrast ( effect size , p , in parenthesis).questionnegativehonestpositivef ( 2 , 54 ) = f ( 1 , 27 ) = how comfortable to hold?2.43 ( 0.88)3.00 ( 0.77)3.93 ( 0.47)33.45 * * * ( 0.55)31.50 * * * ( 0.64)how clean was contact?2.64 ( 0.91)3.11 ( 0.74)3.71 ( 0.81)16.89 * * * ( 0.38)24.20 * * * ( 0.47)how easy was it to use?2.71 ( 1.01)3.36 ( 0.78)4.18 ( 0.61)29.98 * * * ( 0.53)41.60 * * * ( 0.61)how smooth was roll?2.75 ( 0.89)3.14 ( 0.71)3.86 ( 0.80)30.36 * * * ( 0.53)40.86 * * * ( 0.60)how comfortable was weight?2.39 ( 0.79)3.11 ( 0.79)4.07 ( 0.66)37.96 * * * ( 0.58)39.45 * * * ( 0.66)how confident did you feel?2.11 ( 0.96)2.96 ( 0.64)4.11 ( 0.74)61.01 * * * ( 0.69)75.60 * * * ( 0.74)how accurate ( distance)?2.14 ( 0.85)2.86 ( 0.76)3.64 ( 0.62)39.03 * * * ( 0.59)63.00 * * * ( 0.70)how accurate ( direction)?2.11 ( 0.96)2.79 ( 0.74)3.64 ( 0.78)38.11 * * * ( 0.58)57.58 * * * ( 0.68)how accurate ( speed)?1.86 ( 0.80)2.82 ( 0.77)3.64 ( 0.83)58.65 * * * ( 0.68)97.54 * * * ( 0.78)how well did you perform?1.71 ( 0.76)2.79 ( 0.88)3.96 ( 0.69)80.67 * * * ( 0.75)164.61 * * * ( 0.86)note : a higher number indicates more preference . where appropriate , adjusted degrees of freedom ( greenhouse - geisser ) were used , all effects remained significant at the < 0.001 level.***p < 0.001 . where appropriate , adjusted degrees of freedom ( greenhouse - geisser ) were used , all effects remained significant at the < 0.001 level . finally , analysis of putting performance during the manipulation phase indicated a significant condition effect ( f 2 , 54 = 38.88 , p < .001 , ) , with performance best with the positive putter ( m = 56.25 , s = 3.15 ) , followed by the honest ( m = 61.18 , s = 4.06 ) , then the negative putter ( m = 64.36 , s = 4.56 ) . the results strongly suggest that it is possible to induce a superstitious belief in a laboratory setting in a short period of time . as predicted , the participants in the present study preferred to use the positive putter for the competition and had more positive perceptions about it . for example , items relating to physical properties of the putter such as comfort and weight were rated as significantly higher in the positive putter , meaning that they were rated as more comfortable to hold and having a better weight for the task . it must be borne in mind that this was in spite of the fact that the putters themselves are all identical . items relating to the function of the putter were also higher in the positive putter ; it was considered to contact the ball better and produce more smoothness . again , considering the putter was identical to the others , it has no more capacity for better function than the others did . it might be argued , however , that any identical items will , upon closer scrutiny , reveal slight manufacturing differences . hence , it might be that one of the clubs was , in fact , slightly we do not doubt that there are slight manufacturing differences between the clubs used in this particular study , for example , the rubber handle was 2 mm longer on one club compared to the other two . it is unlikely , however , that these minimal manufacturing differences could account for such differences in perception and behaviour . more importantly , this potential alternative explanation was guarded against by randomly allocating clubs to the feedback regime . hence , the fact that the participants responded that a particular club was more comfortable to hold was due to the feedback condition and not the club itself because all three clubs were employed within each feedback condition during the study . the finding that participants performed better with the positive putter during the feedback phase of the study can similarly not be due to the club . we believe , we were successful at producing a situation where a performer perceives a run of good performance ( with the positive putter ) or bad performance ( with the negative putter ) with a club , with false feedback serving to exaggerate the perceived performance differences between the clubs . since the clubs were identical , any perceived difference about the quality of the putters as a consequence of feedback is an irrational belief , even if performance with a certain club is better than with others . a comparable scenario would be a craps player who perceives a certain die to be superior to others due to a run of good performance with it . it is perhaps easier to understand the irrational thinking in this scenario since there is obviously no difference between dice . its use was avoided in the competition phase by all but one participant and the physical and functional perceptions of it were more negative . it was considered to have poorer weight for the task , was less comfortable to hold and produced poorer accuracy . sporting superstitions , in some instances , can take the form of unlucky items . where performance is particularly poor with an item , they tend to be avoided in the future , as was the case in the present study , suggesting a possible connection between poor performance and perceptions of unluckiness . in line with this , a study by brevers et al . ( 2011 ) also indicated that increases in uncertainty were related to increased rates of superstitious behaviour . the categorisation of putters in this study into these dichotomous positions occurred in the space of about 2030 minutes in a laboratory setting . this was by necessity an artificial situation , but by extension , one could see how this situation could occur in real sporting situations when , by chance , sporting performance happens to be better with one specific item of equipment . for example , tennis players who have served an ace have been known to request the exact same ball back from the ball boy or girl for the next serve . given the quality testing for these items of equipment , it is unlikely that particular ball differs from any of the others in play , they are more than likely all the same . in addition , these dichotomous positions were developed in spite of the fact that participants were told that the putters were identical , and indeed they could also see the putters were the same make and model . these results can be seen in the context of the work on conditioning and the development of superstitious behaviour ( ono , 1987 ; rudski , 2001 ; skinner , 1948 ) . the experimental manipulation involved providing a regime of false feedback to the participant such that they thought they had performed differently with the clubs . we argue that this can be considered a surrogate circumstance for real - world performance , in that using a putter under these varying circumstances causes the irrational decision - making . we would see our procedure as akin to a situation where a sportsperson gets a run of good or bad performance , and how this can cause superstitious preferences . our study is comparable to that of skinner ( 1948 ) since it shows that participants behave as if there is a causal relation between the putter used and the quality of their performance . in skinner 's study , pigeons behaved as if there was a causal relation between their behaviour ( e.g. turning , twisting , pecking ) and the presentation of food . in both studies , no such causal relation was apparent and , therefore , according to skinner , a superstition is demonstrated . the present study extends the only other laboratory - based work that examines the development of superstitions in golf conducted by van raalte et al . , it was argued that participants developed a superstitious preference for a lucky golf ball based on previous good performance with that ball . however , a preference for a ball may have been primarily due to the colour of the ball rather than previous performance . in our study , we employed an experimental manipulation in which inherent biases to the sports equipment were removed by simply using identical equipment . despite participants putting with three identical golf clubs , a superstitious preference was developed for one of the clubs based on perceived superior performance with that putter . it would be interesting to know whether other features of operant conditioning could be mirrored in the development of superstitions . for example , the operant conditioning literature has explored various regimes of reinforcement ( variable vs. fixed and interval vs. ratio ) . although some work has taken place along these lines ( e.g. rudski , 2001 , found no major influence between different variable interval schedules and superstitious belief ) , it was not specifically sport - based , instead using competitive button pressing tasks . more sport - like situations would be needed to conclude whether varying schedules exert the same effects on sporting superstitions as they do on conditioned behaviour . such findings would lend more support to the idea that operant conditioning was a factor in the development of these beliefs . one further line of research would be to consider alternative procedures , perhaps involving real - world variability , to create an even more representative experimental situation . in summary , an experiment was conducted in which participants developed a superstitious belief towards a particular putter ( and against another ) . not only did participants assume the putter had superior qualities compared to other identical putters , but they felt more confident and preferred to use it in a competition situation . in addition , this preference was developed in a relatively short time in the laboratory , approximately 2030 minutes .

the objective was to determine the extent to which it was possible to induce superstitious behaviour and beliefs in a golf putting task in a laboratory . participants ( n = 28 ) took part in a putting task using three identical clubs in which visual feedback regarding performance was restricted . participants were provided with verbal feedback of their performance , which was honest when they used one putter , negative with a second putter ( they did better than they were told ) and positive with a third ( they did worse than they were told ) . after this initial acquisition phase , a competition was announced and participants were asked to select a putter they would like to use . the participants were then asked to rate various qualities of the putters . significantly more participants selected the positive putter for the competition ( n = 22 ) compared to the negative putter ( n = 1 ) , p < .001 . in addition , participants claimed that the positive putter had a better weight , was more comfortable and easier to use than the negative putter ( all p < .001 ) . overall , this evidence can be taken to show that a superstitious belief can be formed in a short amount of time within a laboratory setting and that it can affect both the perceptions and choices of an individual .

Introduction Method Design Participants Measures, materials and apparatus Procedure Results Discussion

in ono 's study ( 1987 ) , participants were asked to achieve as high a score as possible in a task involving moving three levers . whilst some element of this could be argued to represent a normal follow - through action , the superstitious action referred to by skinner can be taken to refer to an extended action , beyond a period where a natural follow through could feasibly operate and be of benefit . there are , however , very few studies which have attempted to study sporting superstitions in a laboratory setting , one of the reasons being that it is difficult to create representative designs in this area . notable exceptions to this are studies by van raalte , brewer , nemeroff , and linder ( 1991 ) , wright and erdal ( 2008 ) and damisch , stoberock , and mussweiler ( 2010 ) , who use a lucky ball procedure in a laboratory setting . ( 2010 ) , participants performed better on a golf putting task when using a ball that was said to be lucky . after a putt , the experimenter noted whether the putt was successful or not , then for the next trial , the ball was returned to the participant who therefore made their selection for their subsequent putt from the initial pool of four coloured golf balls . to illustrate this issue , a participant who used the blue ball consistently throughout the study because they preferred it , and holed 10 putts , but missed 10 , would register as showing 10 examples of superstitious behaviour , where , clearly , there was no difference between their success colour choice and their fail - to - putt colour choice . based on the paucity of well - controlled sport superstition studies , the objective of the current study was to determine the extent to which it was possible to induce superstitious behaviour and beliefs in a golf putting task in a laboratory . in the present study , an experimental manipulation was employed in which inherent biases to the sports equipment were removed by simply using identical equipment , in this case , three identical golf putting clubs . putter on the basis of this reinforcement , demonstrated by choosing it in a competition phase of the experiment over other putters , and by perceiving it to have better qualities . a one - factor repeated - measures design was employed where feedback regarding performance was manipulated for a given putter being positive , honest or negative in nature . the dependent measures were the perceptions of the putters used in the task and putter selection by the participant for a competition phase . more importantly for the current task , all used a putter in a right - handed manner . putting took place in a laboratory with a level floor on short pile carpet tiles , providing a surface comparable to a fast putting green . the centre of the central square was 5 m from the participants striking point . in front of the participant ( 1 m away ) was an opaque divider screen ( 1.95 m in height , 1.80 m in width ) preventing visual feedback of the task outcome , but allowing passage of the golf ball underneath . attached to the divider screen was a chart of the target grid allowing the participant to easily understand the verbal feedback regarding ball position , provided during the experiment ( see procedure ) . a series of items were asked about the physical qualities and perceptions experienced whilst using the positive , negative and honest putters . all items were responded to on a 5-point likert scale for each putter , for example , for item 1 , not at all comfortable , slightly comfortable , moderately comfortable , very comfortable the 10 items were : overall , how comfortable did each of the putters feel?overall , how clean was the contact on the ball with each of the putters?in general , how easy was each of the putters to use?overall , how smooth was the roll of the golf ball from each of the putters?overall , how comfortable was the weight of each of the putters?in general , how confident did each of the putters make you feel?overall , how accurately did each of the putters control the distance of the ball?overall , how accurately did each of the putters control the direction of the ball?overall , how accurately did each of the putters control the speed of the ball?in general , how well do you feel you performed with each of the putters ? overall , how comfortable did each of the putters feel ? overall , how clean was the contact on the ball with each of the putters ? overall , how smooth was the roll of the golf ball from each of the putters ? overall , how accurately did each of the putters control the distance of the ball ? overall , how accurately did each of the putters control the direction of the ball ? overall , how accurately did each of the putters control the speed of the ball ? prior to commencing the study , participants read briefing instructions which explained that they would be asked to perform putts using three identical putters . to explain the experimental procedure , participants then used the acquisition putter , without the divider screen , to learn the basics of the task in 30 practice trials , thus ensuring that participants were familiar with the strength and line required for the task . a 7 7 grid marked on the floor with tape was used to rate putting performance ( each square being 0.15 m 0.15 m ) , the centre of which ( d4 ) was considered the target outcome . in this way , the participant could see how the outcome of their putt was translated into the verbal feedback scheme . after this practice session , the divider screen was introduced and the three identical putters were leant against a wall ( 0.30 m apart ) next to the participant . the putters were referred to as the left , centre and right - hand putters to ensure that there was no natural preference for any of them ( compared to if they had been labelled with a number or colour coded ) . at this stage , participants were reminded verbally that the putters were identical . unbeknownst to the participant , the experimenter also used a random order to determine whether the feedback for this putter would be positive , honest or negative . after sufficient trials had been collected , participants were asked to replace the putter they had been using in its previous location and select another specified by the experimenter . the procedure for the negative feedback condition was to exaggerate feedback away from the centre , for example , balls that landed in d2 were feedback to the participant as d1 , b4 became a4 , b2 became a1 . participants were informed that as thanks for participating in the study , a competition would take place where the person who could get the best putting score out of 10 putts would be given a small cash prize . they were asked to select any putter ( the selection was noted by the experimenter ) and their putting score was assessed over 10 putts for the purpose of the competition . after this competition phase , participants were asked to fill in the likert scales assessing qualities of the putters ( see materials ) and were then debriefed and thanked for their time . to analyse the data , a chi statistic was generated for putter preference , and a one - way repeated - measures analysis of variance ( anova ) , followed by linear contrasts , was used for the questionnaire data . a one - factor repeated - measures design was employed where feedback regarding performance was manipulated for a given putter being positive , honest or negative in nature . the dependent measures were the perceptions of the putters used in the task and putter selection by the participant for a competition phase . more importantly for the current task , all used a putter in a right - handed manner . putting took place in a laboratory with a level floor on short pile carpet tiles , providing a surface comparable to a all putters were , apart from to - be - expected minimal manufacturing differences , identical in appearance . in front of the participant ( 1 m away ) was an opaque divider screen ( 1.95 m in height , 1.80 m in width ) preventing visual feedback of the task outcome , but allowing passage of the golf ball underneath . attached to the divider screen was a chart of the target grid allowing the participant to easily understand the verbal feedback regarding ball position , provided during the experiment ( see procedure ) . a series of items were asked about the physical qualities and perceptions experienced whilst using the positive , negative and honest putters . all items were responded to on a 5-point likert scale for each putter , for example , for item 1 , not at all comfortable , slightly comfortable , moderately comfortable the 10 items were : overall , how comfortable did each of the putters feel?overall , how clean was the contact on the ball with each of the putters?in general , how easy was each of the putters to use?overall , how smooth was the roll of the golf ball from each of the putters?overall , how comfortable was the weight of each of the putters?in general , how confident did each of the putters make you feel?overall , how accurately did each of the putters control the distance of the ball?overall , how accurately did each of the putters control the direction of the ball?overall , how accurately did each of the putters control the speed of the ball?in general , how well do you feel you performed with each of the putters ? overall , how clean was the contact on the ball with each of the putters ? overall , how smooth was the roll of the golf ball from each of the putters ? overall , how accurately did each of the putters control the distance of the ball ? overall , how accurately did each of the putters control the direction of the ball ? overall , how accurately did each of the putters control the speed of the ball ? prior to commencing the study , participants read briefing instructions which explained that they would be asked to perform putts using three identical putters . to explain the experimental procedure , participants then used the acquisition putter , without the divider screen , to learn the basics of the task in 30 practice trials , thus ensuring that participants were familiar with the strength and line required for the task . after this practice session , the divider screen was introduced and the three identical putters were leant against a wall ( 0.30 m apart ) next to the participant . the putters were referred to as the left , centre and right - hand putters to ensure that there was no natural preference for any of them ( compared to if they had been labelled with a number or colour coded ) . at this stage , participants were reminded verbally that the putters were identical . for reference , after sufficient trials had been collected , participants were asked to replace the putter they had been using in its previous location and select another specified by the experimenter . the procedure for the negative feedback condition was to exaggerate feedback away from the centre , for example , balls that landed in d2 were feedback to the participant as d1 , b4 became a4 , b2 became a1 . participants were informed that as thanks for participating in the study , a competition would take place where the person who could get the best putting score out of 10 putts would be given a small cash prize . they were asked to select any putter ( the selection was noted by the experimenter ) and their putting score was assessed over 10 putts for the purpose of the competition . after this competition phase , participants were asked to fill in the likert scales assessing qualities of the putters ( see materials ) and were then debriefed and thanked for their time . to analyse the data , a chi statistic was generated for putter preference , and a one - way repeated - measures analysis of variance ( anova ) , followed by linear contrasts , was used for the questionnaire data . overall , the selection of putter for the competition phase was more likely to be the positive putter ( n = 22 ) , although the honest and negative feedback putter were also selected ( n = 5 and n = 1 , respectively ) . this selection was not random as determined by a contingency table , ( 2 , n = 28 ) = 26.65 , p < 0.01 . one - way repeated - measures anova indicated that the perceptions of the putter also varied depending on the feedback provided , with all main effects significant at p < .001 , the effect sizes were all large ( see table 1 ) . in addition , all linear contrasts were significant for all questions at p < .001 . the positive putter was perceived most favourably , followed by the honest , then the negative feedback putter . for the sake of brevity , mean response on a 15 scale to questions about the club ( standard deviation in parenthesis ) , with main effect of putter type followed by linear contrast ( effect size , p , in parenthesis).questionnegativehonestpositivef ( 2 , 54 ) = f ( 1 , 27 ) = how comfortable to hold?2.43 ( 0.88)3.00 ( 0.77)3.93 ( 0.47)33.45 * * * ( 0.55)31.50 * * * ( 0.64)how clean was contact?2.64 ( 0.91)3.11 ( 0.74)3.71 ( 0.81)16.89 * * * ( 0.38)24.20 * * * ( 0.47)how easy was it to use?2.71 ( 1.01)3.36 ( 0.78)4.18 ( 0.61)29.98 * * * ( 0.53)41.60 * * * ( 0.61)how smooth was roll?2.75 ( 0.89)3.14 ( 0.71)3.86 ( 0.80)30.36 * * * ( 0.53)40.86 * * * ( 0.60)how comfortable was weight?2.39 ( 0.79)3.11 ( 0.79)4.07 ( 0.66)37.96 * * * ( 0.58)39.45 * * * ( 0.66)how confident did you feel?2.11 ( 0.96)2.96 ( 0.64)4.11 ( 0.74)61.01 * * * ( 0.69)75.60 * * * ( 0.74)how accurate ( distance)?2.14 ( 0.85)2.86 ( 0.76)3.64 ( 0.62)39.03 * * * ( 0.59)63.00 * * * ( 0.70)how accurate ( direction)?2.11 ( 0.96)2.79 ( 0.74)3.64 ( 0.78)38.11 * * * ( 0.58)57.58 * * * ( 0.68)how accurate ( speed)?1.86 ( 0.80)2.82 ( 0.77)3.64 ( 0.83)58.65 * * * ( 0.68)97.54 * * * ( 0.78)how well did you perform?1.71 ( 0.76)2.79 ( 0.88)3.96 ( 0.69)80.67 * * * ( 0.75)164.61 * * * ( 0.86)note : a higher number indicates more preference . finally , analysis of putting performance during the manipulation phase indicated a significant condition effect ( f 2 , 54 = 38.88 , p < .001 , ) , with performance best with the positive putter ( m = 56.25 , s = 3.15 ) , followed by the honest ( m = 61.18 , s = 4.06 ) , then the negative putter ( m = 64.36 , s = 4.56 ) . the results strongly suggest that it is possible to induce a superstitious belief in a laboratory setting in a short period of time . as predicted , the participants in the present study preferred to use the positive putter for the competition and had more positive perceptions about it . for example , items relating to physical properties of the putter such as comfort and weight were rated as significantly higher in the positive putter , meaning that they were rated as more comfortable to hold and having a better weight for the task . it must be borne in mind that this was in spite of the fact that the putters themselves are all identical . items relating to the function of the putter were also higher in the positive putter ; it was considered to contact the ball better and produce more smoothness . hence , it might be that one of the clubs was , in fact , slightly we do not doubt that there are slight manufacturing differences between the clubs used in this particular study , for example , the rubber handle was 2 mm longer on one club compared to the other two . more importantly , this potential alternative explanation was guarded against by randomly allocating clubs to the feedback regime . hence , the fact that the participants responded that a particular club was more comfortable to hold was due to the feedback condition and not the club itself because all three clubs were employed within each feedback condition during the study . the finding that participants performed better with the positive putter during the feedback phase of the study can similarly not be due to the club . we believe , we were successful at producing a situation where a performer perceives a run of good performance ( with the positive putter ) or bad performance ( with the negative putter ) with a club , with false feedback serving to exaggerate the perceived performance differences between the clubs . since the clubs were identical , any perceived difference about the quality of the putters as a consequence of feedback is an irrational belief , even if performance with a certain club is better than with others . it was considered to have poorer weight for the task , was less comfortable to hold and produced poorer accuracy . the categorisation of putters in this study into these dichotomous positions occurred in the space of about 2030 minutes in a laboratory setting . in addition , these dichotomous positions were developed in spite of the fact that participants were told that the putters were identical , and indeed they could also see the putters were the same make and model . these results can be seen in the context of the work on conditioning and the development of superstitious behaviour ( ono , 1987 ; rudski , 2001 ; skinner , 1948 ) . we argue that this can be considered a surrogate circumstance for real - world performance , in that using a putter under these varying circumstances causes the irrational decision - making . however , a preference for a ball may have been primarily due to the colour of the ball rather than previous performance . despite participants putting with three identical golf clubs , a superstitious preference was developed for one of the clubs based on perceived superior performance with that putter . rudski , 2001 , found no major influence between different variable interval schedules and superstitious belief ) , it was not specifically sport - based , instead using competitive button pressing tasks . in summary , an experiment was conducted in which participants developed a superstitious belief towards a particular putter ( and against another ) . not only did participants assume the putter had superior qualities compared to other identical putters , but they felt more confident and preferred to use it in a competition situation . in addition , this preference was developed in a relatively short time in the laboratory , approximately 2030 minutes .

this population - based cohort study with a nested case - control analysis was based on information from the health improvement network ( thin ) . longitudinal primary care medical record database that includes diagnostic and prescribing data recorded by general practitioners as part of their routine clinical care . the source population included all individuals aged 2079 years in thin enrolled for at least 2 years with their general practitioner , who had the first prescription ever recorded in the computer files > 1 year before entering the study and at least one health contact in the last 2 years . we excluded all individuals with a history of cancer ( other than nonmelanoma skin cancer ) or pancreatic disease before entering the study . individuals aged 70 years with a follow - up > 1 year and less than two health contacts during their follow - up period were removed because data completeness is most likely seriously deficient in this subgroup . we identified two cohorts within this source population : the type 2 diabetes cohort and the ( diabetes - free ) general population cohort . we ascertained all patients within the study base with a read code of diabetes recorded in the database . based on type - specific read codes ( i.e. , those that denote explicitly the type of diabetes ) , the age at diagnosis , and the lifetime history of antidiabetic pharmacological treatment we were able to classify the initial 92,701 patients with a recorded diagnosis of diabetes into 7,176 ( 7.74% ) patients with type 1 and 85,525 ( 92.26% ) with type 2 diabetes . the general population cohort included 200,000 individuals randomly sampled from the source population and frequency - matched by age , sex , and calendar year to the diabetes cohort . the same eligibility criteria as for the diabetes cohort were applied with the additional condition that patients had to be free of a recorded diagnosis of diabetes . we followed all members from the diabetes and the general population cohort from the date when an individual met all eligibility criteria until the earliest occurrence of one of the following end points : a recorded diagnosis of acute pancreatitis , cancer , his or her 80th birthday , death , or the end of the study period . we reviewed the computerized patient profiles with free text comments of all individuals identified as potentially having cases of pancreatitis . after this manual review of all potential cases , we classified cases as not confirmed ( and subsequently excluded them from the study ) in situations in which acute pancreatitis was initially suspected but ruled out later on , and in individuals who met an exclusion criterion ( i.e. , cancer or prevalent case of pancreatic disease ) . all remaining cases were considered as confirmed incident cases , most cases ( 88% ) being well documented ; i.e. , the episode of acute pancreatitis either included a documented emergency admission and/or hospitalization or the diagnosis had been made by a specialist . for a random sample of 50 case subjects , we also sent out a questionnaire to the patient 's general practitioners to confirm the diagnosis and to provide all available additional data related to that event . we received valid information ( questionnaires and additional clinical documents ) for 44 case subjects , corresponding to a 88% response rate . person - time at risk in each study cohort was classified across strata by age , sex , and calendar year . age- and sex - specific incidence rates of acute pancreatitis were calculated using the corresponding person - time at risk in each cohort as denominator . crude and adjusted incidence rate ratios ( irrs ) with 95% cis associated with diabetes were computed using a poisson regression model with age , sex , and calendar year included in the model . in addition , a nested case - control analysis was performed to evaluate in more detail the role of diabetes and of antidiabetic drugs on the risk of acute pancreatitis . the individuals with confirmed cases of acute pancreatitis from both cohorts were used as case subjects and their date of diagnosis was used as the index date . control subjects were randomly sampled from the two study cohorts in which pancreatitis case subjects were ascertained . a group of 5,000 control subjects was randomly selected from the list of eligible person - time and frequency - matched to the case subjects on sex , same age ( 1 year ) , and calendar year . for all case and control subjects we ascertained demographic ( age , sex , townsend deprivation index , and bmi ) and lifestyle factors ( e.g. , smoking and alcohol intake ) as well as general comorbidity ( e.g. , chronic conditions and previous gastrointestinal diseases ) at the index date . in addition , we ascertained exposure to antidiabetic drugs using separate exposure variables for insulin , metformin , sulfonylureas , thiazolidinediones , and other antidiabetic drugs ( i.e. , acarbose , repaglinide , and nateglinide ) and other common drugs or drug classes including antibiotics , antidepressants , corticosteroids , acid - suppressing drugs , nonsteroidal anti - inflammatory drugs ( nsaids ) , aspirin , paracetamol , antihypertensive agents , lipid - lowering drugs , and hormone replacement therapy . individuals were classified as current users if they were taking the drug at the index date or had taken it within the previous 30 days . those not currently exposed but who had used the drug within the year before the index date were classified as past users . we also explored the effect of drug duration ( < 1 year , 13 years , and > 3 years ) on the risk of acute pancreatitis . unconditional regression analyses were used to assess odds ratios ( ors ) together with 95% cis . the fully adjusted model included the matching variables age and sex and the following predictors of acute pancreatitis : smoking status , alcohol intake , ischemic heart disease , previous gastrointestinal disease , exposure to antidiabetic drugs , antibiotics , acid - suppressing drugs , nsaids ( including aspirin and coxibs ) , paracetamol , and antihypertensive drugs , bmi , and townsend deprivation index . all analyses were done using stata se 10.0 ( statacorp , college station , tx ) . we followed all members from the diabetes and the general population cohort from the date when an individual met all eligibility criteria until the earliest occurrence of one of the following end points : a recorded diagnosis of acute pancreatitis , cancer , his or her 80th birthday , death , or the end of the study period . we reviewed the computerized patient profiles with free text comments of all individuals identified as potentially having cases of pancreatitis . after this manual review of all potential cases , we classified cases as not confirmed ( and subsequently excluded them from the study ) in situations in which acute pancreatitis was initially suspected but ruled out later on , and in individuals who met an exclusion criterion ( i.e. , cancer or prevalent case of pancreatic disease ) . all remaining cases were considered as confirmed incident cases , most cases ( 88% ) being well documented ; i.e. , the episode of acute pancreatitis either included a documented emergency admission and/or hospitalization or the diagnosis had been made by a specialist . for a random sample of 50 case subjects , we also sent out a questionnaire to the patient 's general practitioners to confirm the diagnosis and to provide all available additional data related to that event . we received valid information ( questionnaires and additional clinical documents ) for 44 case subjects , corresponding to a 88% response rate . person - time at risk in each study cohort was classified across strata by age , sex , and calendar year . age- and sex - specific incidence rates of acute pancreatitis were calculated using the corresponding person - time at risk in each cohort as denominator . crude and adjusted incidence rate ratios ( irrs ) with 95% cis associated with diabetes were computed using a poisson regression model with age , sex , and calendar year included in the model . potential interactions were studied by including these variables in the model as interaction terms . in addition , a nested case - control analysis was performed to evaluate in more detail the role of diabetes and of antidiabetic drugs on the risk of acute pancreatitis . the individuals with confirmed cases of acute pancreatitis from both cohorts were used as case subjects and their date of diagnosis was used as the index date . control subjects were randomly sampled from the two study cohorts in which pancreatitis case subjects were ascertained . a group of 5,000 control subjects was randomly selected from the list of eligible person - time and frequency - matched to the case subjects on sex , same age ( 1 year ) , and calendar year . for all case and control subjects we ascertained demographic ( age , sex , townsend deprivation index , and bmi ) and lifestyle factors ( e.g. , smoking and alcohol intake ) as well as general comorbidity ( e.g. , chronic conditions and previous gastrointestinal diseases ) at the index date . in addition , we ascertained exposure to antidiabetic drugs using separate exposure variables for insulin , metformin , sulfonylureas , thiazolidinediones , and other antidiabetic drugs ( i.e. , acarbose , repaglinide , and nateglinide ) and other common drugs or drug classes including antibiotics , antidepressants , corticosteroids , acid - suppressing drugs , nonsteroidal anti - inflammatory drugs ( nsaids ) , aspirin , paracetamol , antihypertensive agents , lipid - lowering drugs , and hormone replacement therapy . individuals were classified as current users if they were taking the drug at the index date or had taken it within the previous 30 days . those not currently exposed but who had used the drug within the year before the index date were classified as past users . we also explored the effect of drug duration ( < 1 year , 13 years , and > 3 years ) on the risk of acute pancreatitis . unconditional regression analyses were used to assess odds ratios ( ors ) together with 95% cis . the fully adjusted model included the matching variables age and sex and the following predictors of acute pancreatitis : smoking status , alcohol intake , ischemic heart disease , previous gastrointestinal disease , exposure to antidiabetic drugs , antibiotics , acid - suppressing drugs , nsaids ( including aspirin and coxibs ) , paracetamol , and antihypertensive drugs , bmi , and townsend deprivation index . all analyses were done using stata se 10.0 ( statacorp , college station , tx ) . person - time at risk in each study cohort was classified across strata by age , sex , and calendar year . age- and sex - specific incidence rates of acute pancreatitis were calculated using the corresponding person - time at risk in each cohort as denominator . crude and adjusted incidence rate ratios ( irrs ) with 95% cis associated with diabetes were computed using a poisson regression model with age , sex , and calendar year included in the model . in addition , a nested case - control analysis was performed to evaluate in more detail the role of diabetes and of antidiabetic drugs on the risk of acute pancreatitis . the individuals with confirmed cases of acute pancreatitis from both cohorts were used as case subjects and their date of diagnosis was used as the index date . control subjects were randomly sampled from the two study cohorts in which pancreatitis case subjects were ascertained . a group of 5,000 control subjects was randomly selected from the list of eligible person - time and frequency - matched to the case subjects on sex , same age ( 1 year ) , and calendar year . for all case and control subjects we ascertained demographic ( age , sex , townsend deprivation index , and bmi ) and lifestyle factors ( e.g. , smoking and alcohol intake ) as well as general comorbidity ( e.g. , chronic conditions and previous gastrointestinal diseases ) at the index date . in addition , we ascertained exposure to antidiabetic drugs using separate exposure variables for insulin , metformin , sulfonylureas , thiazolidinediones , and other antidiabetic drugs ( i.e. , acarbose , repaglinide , and nateglinide ) and other common drugs or drug classes including antibiotics , antidepressants , corticosteroids , acid - suppressing drugs , nonsteroidal anti - inflammatory drugs ( nsaids ) , aspirin , paracetamol , antihypertensive agents , lipid - lowering drugs , and hormone replacement therapy . individuals were classified as current users if they were taking the drug at the index date or had taken it within the previous 30 days . those not currently exposed but who had used the drug within the year before the index date were classified as past users . we also explored the effect of drug duration ( < 1 year , 13 years , and > 3 years ) on the risk of acute pancreatitis . unconditional regression analyses were used to assess odds ratios ( ors ) together with 95% cis . the fully adjusted model included the matching variables age and sex and the following predictors of acute pancreatitis : smoking status , alcohol intake , ischemic heart disease , previous gastrointestinal disease , exposure to antidiabetic drugs , antibiotics , acid - suppressing drugs , nsaids ( including aspirin and coxibs ) , paracetamol , and antihypertensive drugs , bmi , and townsend deprivation index . all analyses were done using stata se 10.0 ( statacorp , college station , tx ) . a total of 85,525 patients with type 2 diabetes were included for the cohort analyses together with the 200,000 frequency - matched individuals from the general population . the average sd age at start date in the type 2 diabetes and the general population cohort was 61.2 11.4 and 59.5 12.8 years , respectively ; 43.7% of subjects in the diabetes and 43.5% in the general population cohort were female . the total amount of follow - up time was 325,990 person - years in the type 2 diabetes and 807,453 person - years in the general population cohort , corresponding to an average follow - up time of 3.8 and 4.0 years , respectively . during follow - up , we identified a total of 456 potential cases of acute pancreatitis , 261 in the general population and 195 in the diabetes cohort . after manual review of the patients ' computerized profiles , we classified 35 cases as not confirmed cases ( 7.7% ) . the remaining 421 cases were classified as confirmed cases . in the validation study , 2 of the 44 cases in individuals for whom we received valid information were considered as not confirmed by the general practitioners . these 2 not confirmed cases were subsequently excluded , leaving 419 cases of acute pancreatitis included in the final analyses ( 243 cases in the general population and 176 cases in the diabetes cohort ) . the corresponding crude incidence rate of acute pancreatitis was 30.1 per 100,000 person - years in the general population and 54.0 per 100,000 person - years in the diabetes cohort . incidence rates stratified by age and sex for the diabetic and the general population cohort are presented in table 1 . incidence rate of acute pancreatitis by age and sex in the general population without diabetes and in type 2 diabetic patients ( cohort analysis ) * unadjusted estimate of acute pancreatitis in type 2 diabetic patients compared with the general population . these results translate into a 79% increased risk of a first - ever episode of acute pancreatitis among type 2 diabetic patients compared with the general population ( crude irr 1.79 [ 95% ci 1.482.18 ] ) . because the two cohorts were frequency matched , we obtained very similar results when we estimated the age- , sex- , and calendar year increasing age was associated with a higher risk of acute pancreatitis in the overall cohort . compared with those aged < 40 years , the risk of acute pancreatitis was increased in those aged 4059 , 6069 , and 70 years by 17 , 20 , and 50% , respectively ( ptrend = 0.02 ) . furthermore , we explored the potential effect modification of the association between diabetes and acute pancreatitis by age . we found a significant trend toward a decreasing association between diabetes and acute pancreatitis with increasing age ( p = 0.019 ) . we also evaluated whether sex could modify the association between diabetes and acute pancreatitis , but the corresponding statistical test was not significant . after adjustment for various demographic and lifestyle variables , comorbidities , and drug exposure , patients with type 2 diabetes had a nonsignificantly increased risk of acute pancreatitis compared with those free of diabetes ( adjusted or 1.37 [ 95% ci 0.991.89 ] ) ( table 2 ) . when we analyzed separately the risk associated with incident diabetes ( diabetes first diagnosed during the study period ) and prevalent diabetes ( diabetes first diagnosed before the study period ) , the adjusted ors were virtually the same ( 1.38 [ 0.981.94 ] and 1.34 [ 0.902.01 ] , respectively ) . furthermore , to explore the possibility of an increased risk of acute pancreatitis around the time of the first diagnosis of diabetes , we further divided our cohort of incident diabetic patients according to the time elapsed since the first diagnosis of diabetes . during the 1st year after the diagnosis of diabetes , the risk was somewhat higher ( 1.61 [ 1.002.60 ] ) than thereafter ( 1.26 [ 0.851.88 ] ) . this difference , however , did not reach statistical significance ( p = 0.38 ) . risk of acute pancreatitis associated with diabetes , antidiabetic drugs , and other factors ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . * adjusted for all variables included in the table plus age , sex , townsend index , ischemic heart disease , and exposure to antibiotics , h2 blockers , proton pump inhibitors , nsaids ( including aspirin and coxibs ) , and other antihypertensive drugs . includes gallstones , biliary tract disease , cholecystitis , gastroenteritis , abdominal pain , and others . among users of antidiabetic drugs , current users of insulin were at a decreased risk of acute pancreatitis ( adjusted or 0.35 [ 95% ci 0.200.61 ] ) compared with nonusers . moreover , past use of sulfonylureas was associated with a significant risk increase of acute pancreatitis compared with that for nonusers ( 2.58 [ 1.344.96 ] ) . otherwise , exposure to antidiabetic drugs was not materially associated with the risk of acute pancreatitis ( table 2 ) . we also explored the impact of treatment duration among current antidiabetic drug users ( table 3 ) . a reduced risk of acute pancreatitis was observed across all different strata of insulin duration with a similar magnitude of association , corresponding to a 6070% reduction in the acute pancreatitis risk . of interest , metformin and sulfonylureas , which overall did not seem to be associated with acute pancreatitis , were associated with decreased and increased risks , respectively , but only among long - term users of these drugs . the other antidiabetic drugs studied ( thiazolidinediones and others ) were not found to be associated with acute pancreatitis in any of these analyses although their numbers were considerably smaller . risk of acute pancreatitis and duration of current use of antidiabetic drugs ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . * adjusted for all variables included in the table plus those in the fully adjusted model of table 2 . among individuals with diabetes , metformin and sulfonylureas were the two most commonly prescribed drugs , followed by insulin . among control subjects , a total of 73% of diabetic patients were receiving specific antidiabetic drug treatment at the index date . treated diabetic patients had an adjusted or of 1.19 [ 95% ci 0.911.55 ] for acute pancreatitis compared with that for the general population , whereas those not receiving antidiabetic drug treatment ( 1.49 [ 1.062.08 ] ) seemed to concentrate the overall increased risk associated with diabetes . however , this difference was not statistically significant ( p = 0.21 ) . in addition , the risk of acute pancreatitis was significantly increased among current smokers , those taking 30 units of alcohol per week , individuals with a previous history of gastrointestinal disease , and current users of paracetamol and ace inhibitors ( table 2 ) . a total of 85,525 patients with type 2 diabetes were included for the cohort analyses together with the 200,000 frequency - matched individuals from the general population . the average sd age at start date in the type 2 diabetes and the general population cohort was 61.2 11.4 and 59.5 12.8 years , respectively ; 43.7% of subjects in the diabetes and 43.5% in the general population cohort were female . the total amount of follow - up time was 325,990 person - years in the type 2 diabetes and 807,453 person - years in the general population cohort , corresponding to an average follow - up time of 3.8 and 4.0 years , respectively . during follow - up , we identified a total of 456 potential cases of acute pancreatitis , 261 in the general population and 195 in the diabetes cohort . after manual review of the patients ' computerized profiles , we classified 35 cases as not confirmed cases ( 7.7% ) . the remaining 421 cases were classified as confirmed cases . in the validation study , 2 of the 44 cases in individuals for whom we received valid information were considered as not confirmed by the general practitioners . these 2 not confirmed cases were subsequently excluded , leaving 419 cases of acute pancreatitis included in the final analyses ( 243 cases in the general population and 176 cases in the diabetes cohort ) . the corresponding crude incidence rate of acute pancreatitis was 30.1 per 100,000 person - years in the general population and 54.0 per 100,000 person - years in the diabetes cohort . incidence rates stratified by age and sex for the diabetic and the general population cohort are presented in table 1 . incidence rate of acute pancreatitis by age and sex in the general population without diabetes and in type 2 diabetic patients ( cohort analysis ) * unadjusted estimate of acute pancreatitis in type 2 diabetic patients compared with the general population . these results translate into a 79% increased risk of a first - ever episode of acute pancreatitis among type 2 diabetic patients compared with the general population ( crude irr 1.79 [ 95% ci 1.482.18 ] ) . because the two cohorts were frequency matched , we obtained very similar results when we estimated the age- , sex- , and calendar year increasing age was associated with a higher risk of acute pancreatitis in the overall cohort . compared with those aged < 40 years , the risk of acute pancreatitis was increased in those aged 4059 , 6069 , and 70 years by 17 , 20 , and 50% , respectively ( ptrend = 0.02 ) . furthermore , we explored the potential effect modification of the association between diabetes and acute pancreatitis by age . we found a significant trend toward a decreasing association between diabetes and acute pancreatitis with increasing age ( p = 0.019 ) . we also evaluated whether sex could modify the association between diabetes and acute pancreatitis , but the corresponding statistical test was not significant . after adjustment for various demographic and lifestyle variables , comorbidities , and drug exposure , patients with type 2 diabetes had a nonsignificantly increased risk of acute pancreatitis compared with those free of diabetes ( adjusted or 1.37 [ 95% ci 0.991.89 ] ) ( table 2 ) . when we analyzed separately the risk associated with incident diabetes ( diabetes first diagnosed during the study period ) and prevalent diabetes ( diabetes first diagnosed before the study period ) , the adjusted ors were virtually the same ( 1.38 [ 0.981.94 ] and 1.34 [ 0.902.01 ] , respectively ) . furthermore , to explore the possibility of an increased risk of acute pancreatitis around the time of the first diagnosis of diabetes , we further divided our cohort of incident diabetic patients according to the time elapsed since the first diagnosis of diabetes . during the 1st year after the diagnosis of diabetes , the risk was somewhat higher ( 1.61 [ 1.002.60 ] ) than thereafter ( 1.26 [ 0.851.88 ] ) . this difference , however , did not reach statistical significance ( p = 0.38 ) . risk of acute pancreatitis associated with diabetes , antidiabetic drugs , and other factors ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . * adjusted for all variables included in the table plus age , sex , townsend index , ischemic heart disease , and exposure to antibiotics , h2 blockers , proton pump inhibitors , nsaids ( including aspirin and coxibs ) , and other antihypertensive drugs . includes gallstones , biliary tract disease , cholecystitis , gastroenteritis , abdominal pain , and others . among users of antidiabetic drugs , current users of insulin were at a decreased risk of acute pancreatitis ( adjusted or 0.35 [ 95% ci 0.200.61 ] ) compared with nonusers . moreover , past use of sulfonylureas was associated with a significant risk increase of acute pancreatitis compared with that for nonusers ( 2.58 [ 1.344.96 ] ) . otherwise , exposure to antidiabetic drugs was not materially associated with the risk of acute pancreatitis ( table 2 ) . we also explored the impact of treatment duration among current antidiabetic drug users ( table 3 ) . a reduced risk of acute pancreatitis was observed across all different strata of insulin duration with a similar magnitude of association , corresponding to a 6070% reduction in the acute pancreatitis risk . of interest , metformin and sulfonylureas , which overall did not seem to be associated with acute pancreatitis , were associated with decreased and increased risks , respectively , but only among long - term users of these drugs . the other antidiabetic drugs studied ( thiazolidinediones and others ) were not found to be associated with acute pancreatitis in any of these analyses although their numbers were considerably smaller . risk of acute pancreatitis and duration of current use of antidiabetic drugs ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . * adjusted for all variables included in the table plus those in the fully adjusted model of table 2 . among individuals with diabetes , metformin and sulfonylureas were the two most commonly prescribed drugs , followed by insulin . among control subjects , a total of 73% of diabetic patients were receiving specific antidiabetic drug treatment at the index date . treated diabetic patients had an adjusted or of 1.19 [ 95% ci 0.911.55 ] for acute pancreatitis compared with that for the general population , whereas those not receiving antidiabetic drug treatment ( 1.49 [ 1.062.08 ] ) seemed to concentrate the overall increased risk associated with diabetes . however , this difference was not statistically significant ( p = 0.21 ) . in addition , the risk of acute pancreatitis was significantly increased among current smokers , those taking 30 units of alcohol per week , individuals with a previous history of gastrointestinal disease , and current users of paracetamol and ace inhibitors ( table 2 ) . the results of this study confirm the excess risk of acute pancreatitis associated with type 2 diabetes previously reported in other observational studies ( 2,4,5 ) . in fact , the cohort analysis yielded a statistically significant 77% increased risk of acute pancreatitis associated with a prior history of diabetes . however , the magnitude of this association was reduced when it was adjusted for other risk factors in a multivariate model and became borderline significant in the nested - case control analysis . this association of an increased risk of acute pancreatitis and type 2 diabetes seems more pronounced at younger ages and has already been observed in a recent retrospective cohort study based on information from a u.s , health claims database ( 2 ) . interestingly , use of insulin and long - term use of metformin were associated with a decreased risk of pancreatitis , as opposed to long - term use of sulfonylureas , which seems to increase the risk . in a previous case - control study , blomgren et al . ( 4 ) found that the sulfonylurea glyburide increased the risk of acute pancreatitis , but neither insulin nor metformin seemed to lower the risk . in fact , there are reports of cases of acute pancreatitis in patients using metformin after an episode of acute renal failure ( 9,10 ) . to the best of our knowledge , overall , when we analyzed the risk of pancreatitis among treated and not treated diabetic patients separately , we observed that the greatest risk appeared among those without antidiabetic pharmacotherapy , who represent a quarter of our diabetic population . this result may be partially due to a slight increased risk of acute pancreatitis immediately after the diagnosis of diabetes , although this assumption could not be confirmed in our study . our data from the general population cohort could replicate results from previous epidemiological studies showing that the incidence of acute pancreatitis rises with increasing age and tends to be higher in men than women ( 1,11,12 ) . furthermore , the incidence rate found in the general population is also in line with results from a recent study from england ( 13 ) . among other risk factors studied , we found that previously reported risk factors such as smoking ( 5 ) , alcohol use ( 1,14 ) , or use of ace inhibitors ( 15,16 ) were replicated in our study . exposure to paracetamol was also associated with an increased pancreatitis risk as in previous studies ( 15 ) . first , it is based on a large database with documented high data quality and completeness ( 1719 ) . second , detailed information on important lifestyle factors , comorbidities , concomitant drug therapies , and bmi was available . third , we reviewed the patient profiles of computer - detected potential cases with all additional information included in free text comments and validated the case status in a random sample by accessing the original medical records available in general practitioner offices . fourth , we could replicate results from other studies regarding the association of various comorbidities and/or exposures and the risk of acute pancreatitis . although we adjusted our analyses for various potential risk factors of acute pancreatitis , it is possible that there is still some residual confounding we did not account for . moreover , throughout this report we present results for a large number of factors including lifestyle , comorbidity , and drug therapies ( as well as different drug durations ) . one should keep this in mind , when interpreting these results , and even more so in the case of subanalyses that do not belong to the main purpose of the study . however , because of the exploratory nature of these findings , we did not deem it necessary to adjust for multiple comparisons . in summary , we have shown that type 2 diabetes may be associated with a slight increase in the risk of acute pancreatitis . we have also found that use of insulin in type 2 diabetes might be associated with a reduced risk .

objectiveprevious observational studies have found an increased risk of acute pancreatitis among type 2 diabetic patients . however , limited information is available on this association and specifically on the role of antidiabetic treatment . our aim , therefore , was to further assess the risk of acute pancreatitis in adult patients with type 2 diabetes.research design and methodswe performed a population - based case - control analysis nested in a cohort of 85,525 type 2 diabetic patients and 200,000 diabetes - free individuals from the general population using data from the health improvement network database . subjects were followed up to ascertain incident cases of acute pancreatitis.resultswe identified 419 cases of acute pancreatitis , 243 in the general population and 176 in the diabetes cohort . incidence rates were 30.1 and 54.0 per 100,000 person - years in the general population and the diabetes cohort , respectively . in the cohort analysis , the adjusted incidence rate ratio of acute pancreatitis in diabetic patients versus that in the general population was 1.77 ( 95% ci 1.462.15 ) . the magnitude of this association decreased with adjustment for multiple factors in the nested case - control analysis ( adjusted odds ratio 1.37 [ 95% ci 0.991.89 ] ) . furthermore , we found that the risk of acute pancreatitis was decreased among insulin - treated diabetic patients ( 0.35 [ 0.200.61]).conclusionstype 2 diabetes may be associated with a slight increase in the risk of acute pancreatitis . we also found that insulin use in type 2 diabetes might decrease this risk . further research is warranted to confirm these associations .

RESEARCH DESIGN AND METHODS Follow-up for acute pancreatitis Statistical analyses Cohort analysis. Nested case-control analysis. RESULTS Cohort analysis Nested case-control analysis CONCLUSIONS

this population - based cohort study with a nested case - control analysis was based on information from the health improvement network ( thin ) . we identified two cohorts within this source population : the type 2 diabetes cohort and the ( diabetes - free ) general population cohort . , those that denote explicitly the type of diabetes ) , the age at diagnosis , and the lifetime history of antidiabetic pharmacological treatment we were able to classify the initial 92,701 patients with a recorded diagnosis of diabetes into 7,176 ( 7.74% ) patients with type 1 and 85,525 ( 92.26% ) with type 2 diabetes . the general population cohort included 200,000 individuals randomly sampled from the source population and frequency - matched by age , sex , and calendar year to the diabetes cohort . we followed all members from the diabetes and the general population cohort from the date when an individual met all eligibility criteria until the earliest occurrence of one of the following end points : a recorded diagnosis of acute pancreatitis , cancer , his or her 80th birthday , death , or the end of the study period . after this manual review of all potential cases , we classified cases as not confirmed ( and subsequently excluded them from the study ) in situations in which acute pancreatitis was initially suspected but ruled out later on , and in individuals who met an exclusion criterion ( i.e. , the episode of acute pancreatitis either included a documented emergency admission and/or hospitalization or the diagnosis had been made by a specialist . for a random sample of 50 case subjects , we also sent out a questionnaire to the patient 's general practitioners to confirm the diagnosis and to provide all available additional data related to that event . age- and sex - specific incidence rates of acute pancreatitis were calculated using the corresponding person - time at risk in each cohort as denominator . crude and adjusted incidence rate ratios ( irrs ) with 95% cis associated with diabetes were computed using a poisson regression model with age , sex , and calendar year included in the model . in addition , a nested case - control analysis was performed to evaluate in more detail the role of diabetes and of antidiabetic drugs on the risk of acute pancreatitis . the individuals with confirmed cases of acute pancreatitis from both cohorts were used as case subjects and their date of diagnosis was used as the index date . we also explored the effect of drug duration ( < 1 year , 13 years , and > 3 years ) on the risk of acute pancreatitis . the fully adjusted model included the matching variables age and sex and the following predictors of acute pancreatitis : smoking status , alcohol intake , ischemic heart disease , previous gastrointestinal disease , exposure to antidiabetic drugs , antibiotics , acid - suppressing drugs , nsaids ( including aspirin and coxibs ) , paracetamol , and antihypertensive drugs , bmi , and townsend deprivation index . we followed all members from the diabetes and the general population cohort from the date when an individual met all eligibility criteria until the earliest occurrence of one of the following end points : a recorded diagnosis of acute pancreatitis , cancer , his or her 80th birthday , death , or the end of the study period . after this manual review of all potential cases , we classified cases as not confirmed ( and subsequently excluded them from the study ) in situations in which acute pancreatitis was initially suspected but ruled out later on , and in individuals who met an exclusion criterion ( i.e. , the episode of acute pancreatitis either included a documented emergency admission and/or hospitalization or the diagnosis had been made by a specialist . age- and sex - specific incidence rates of acute pancreatitis were calculated using the corresponding person - time at risk in each cohort as denominator . crude and adjusted incidence rate ratios ( irrs ) with 95% cis associated with diabetes were computed using a poisson regression model with age , sex , and calendar year included in the model . in addition , a nested case - control analysis was performed to evaluate in more detail the role of diabetes and of antidiabetic drugs on the risk of acute pancreatitis . the individuals with confirmed cases of acute pancreatitis from both cohorts were used as case subjects and their date of diagnosis was used as the index date . we also explored the effect of drug duration ( < 1 year , 13 years , and > 3 years ) on the risk of acute pancreatitis . the fully adjusted model included the matching variables age and sex and the following predictors of acute pancreatitis : smoking status , alcohol intake , ischemic heart disease , previous gastrointestinal disease , exposure to antidiabetic drugs , antibiotics , acid - suppressing drugs , nsaids ( including aspirin and coxibs ) , paracetamol , and antihypertensive drugs , bmi , and townsend deprivation index . age- and sex - specific incidence rates of acute pancreatitis were calculated using the corresponding person - time at risk in each cohort as denominator . crude and adjusted incidence rate ratios ( irrs ) with 95% cis associated with diabetes were computed using a poisson regression model with age , sex , and calendar year included in the model . in addition , a nested case - control analysis was performed to evaluate in more detail the role of diabetes and of antidiabetic drugs on the risk of acute pancreatitis . the individuals with confirmed cases of acute pancreatitis from both cohorts were used as case subjects and their date of diagnosis was used as the index date . we also explored the effect of drug duration ( < 1 year , 13 years , and > 3 years ) on the risk of acute pancreatitis . the fully adjusted model included the matching variables age and sex and the following predictors of acute pancreatitis : smoking status , alcohol intake , ischemic heart disease , previous gastrointestinal disease , exposure to antidiabetic drugs , antibiotics , acid - suppressing drugs , nsaids ( including aspirin and coxibs ) , paracetamol , and antihypertensive drugs , bmi , and townsend deprivation index . a total of 85,525 patients with type 2 diabetes were included for the cohort analyses together with the 200,000 frequency - matched individuals from the general population . the average sd age at start date in the type 2 diabetes and the general population cohort was 61.2 11.4 and 59.5 12.8 years , respectively ; 43.7% of subjects in the diabetes and 43.5% in the general population cohort were female . the total amount of follow - up time was 325,990 person - years in the type 2 diabetes and 807,453 person - years in the general population cohort , corresponding to an average follow - up time of 3.8 and 4.0 years , respectively . during follow - up , we identified a total of 456 potential cases of acute pancreatitis , 261 in the general population and 195 in the diabetes cohort . these 2 not confirmed cases were subsequently excluded , leaving 419 cases of acute pancreatitis included in the final analyses ( 243 cases in the general population and 176 cases in the diabetes cohort ) . the corresponding crude incidence rate of acute pancreatitis was 30.1 per 100,000 person - years in the general population and 54.0 per 100,000 person - years in the diabetes cohort . incidence rates stratified by age and sex for the diabetic and the general population cohort are presented in table 1 . incidence rate of acute pancreatitis by age and sex in the general population without diabetes and in type 2 diabetic patients ( cohort analysis ) * unadjusted estimate of acute pancreatitis in type 2 diabetic patients compared with the general population . these results translate into a 79% increased risk of a first - ever episode of acute pancreatitis among type 2 diabetic patients compared with the general population ( crude irr 1.79 [ 95% ci 1.482.18 ] ) . because the two cohorts were frequency matched , we obtained very similar results when we estimated the age- , sex- , and calendar year increasing age was associated with a higher risk of acute pancreatitis in the overall cohort . compared with those aged < 40 years , the risk of acute pancreatitis was increased in those aged 4059 , 6069 , and 70 years by 17 , 20 , and 50% , respectively ( ptrend = 0.02 ) . furthermore , we explored the potential effect modification of the association between diabetes and acute pancreatitis by age . we also evaluated whether sex could modify the association between diabetes and acute pancreatitis , but the corresponding statistical test was not significant . after adjustment for various demographic and lifestyle variables , comorbidities , and drug exposure , patients with type 2 diabetes had a nonsignificantly increased risk of acute pancreatitis compared with those free of diabetes ( adjusted or 1.37 [ 95% ci 0.991.89 ] ) ( table 2 ) . when we analyzed separately the risk associated with incident diabetes ( diabetes first diagnosed during the study period ) and prevalent diabetes ( diabetes first diagnosed before the study period ) , the adjusted ors were virtually the same ( 1.38 [ 0.981.94 ] and 1.34 [ 0.902.01 ] , respectively ) . furthermore , to explore the possibility of an increased risk of acute pancreatitis around the time of the first diagnosis of diabetes , we further divided our cohort of incident diabetic patients according to the time elapsed since the first diagnosis of diabetes . during the 1st year after the diagnosis of diabetes , the risk was somewhat higher ( 1.61 [ 1.002.60 ] ) than thereafter ( 1.26 [ 0.851.88 ] ) . risk of acute pancreatitis associated with diabetes , antidiabetic drugs , and other factors ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . among users of antidiabetic drugs , current users of insulin were at a decreased risk of acute pancreatitis ( adjusted or 0.35 [ 95% ci 0.200.61 ] ) compared with nonusers . moreover , past use of sulfonylureas was associated with a significant risk increase of acute pancreatitis compared with that for nonusers ( 2.58 [ 1.344.96 ] ) . otherwise , exposure to antidiabetic drugs was not materially associated with the risk of acute pancreatitis ( table 2 ) . a reduced risk of acute pancreatitis was observed across all different strata of insulin duration with a similar magnitude of association , corresponding to a 6070% reduction in the acute pancreatitis risk . of interest , metformin and sulfonylureas , which overall did not seem to be associated with acute pancreatitis , were associated with decreased and increased risks , respectively , but only among long - term users of these drugs . the other antidiabetic drugs studied ( thiazolidinediones and others ) were not found to be associated with acute pancreatitis in any of these analyses although their numbers were considerably smaller . risk of acute pancreatitis and duration of current use of antidiabetic drugs ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . treated diabetic patients had an adjusted or of 1.19 [ 95% ci 0.911.55 ] for acute pancreatitis compared with that for the general population , whereas those not receiving antidiabetic drug treatment ( 1.49 [ 1.062.08 ] ) seemed to concentrate the overall increased risk associated with diabetes . in addition , the risk of acute pancreatitis was significantly increased among current smokers , those taking 30 units of alcohol per week , individuals with a previous history of gastrointestinal disease , and current users of paracetamol and ace inhibitors ( table 2 ) . a total of 85,525 patients with type 2 diabetes were included for the cohort analyses together with the 200,000 frequency - matched individuals from the general population . the average sd age at start date in the type 2 diabetes and the general population cohort was 61.2 11.4 and 59.5 12.8 years , respectively ; 43.7% of subjects in the diabetes and 43.5% in the general population cohort were female . the total amount of follow - up time was 325,990 person - years in the type 2 diabetes and 807,453 person - years in the general population cohort , corresponding to an average follow - up time of 3.8 and 4.0 years , respectively . during follow - up , we identified a total of 456 potential cases of acute pancreatitis , 261 in the general population and 195 in the diabetes cohort . these 2 not confirmed cases were subsequently excluded , leaving 419 cases of acute pancreatitis included in the final analyses ( 243 cases in the general population and 176 cases in the diabetes cohort ) . the corresponding crude incidence rate of acute pancreatitis was 30.1 per 100,000 person - years in the general population and 54.0 per 100,000 person - years in the diabetes cohort . incidence rates stratified by age and sex for the diabetic and the general population cohort are presented in table 1 . incidence rate of acute pancreatitis by age and sex in the general population without diabetes and in type 2 diabetic patients ( cohort analysis ) * unadjusted estimate of acute pancreatitis in type 2 diabetic patients compared with the general population . these results translate into a 79% increased risk of a first - ever episode of acute pancreatitis among type 2 diabetic patients compared with the general population ( crude irr 1.79 [ 95% ci 1.482.18 ] ) . because the two cohorts were frequency matched , we obtained very similar results when we estimated the age- , sex- , and calendar year increasing age was associated with a higher risk of acute pancreatitis in the overall cohort . compared with those aged < 40 years , the risk of acute pancreatitis was increased in those aged 4059 , 6069 , and 70 years by 17 , 20 , and 50% , respectively ( ptrend = 0.02 ) . furthermore , we explored the potential effect modification of the association between diabetes and acute pancreatitis by age . we also evaluated whether sex could modify the association between diabetes and acute pancreatitis , but the corresponding statistical test was not significant . after adjustment for various demographic and lifestyle variables , comorbidities , and drug exposure , patients with type 2 diabetes had a nonsignificantly increased risk of acute pancreatitis compared with those free of diabetes ( adjusted or 1.37 [ 95% ci 0.991.89 ] ) ( table 2 ) . when we analyzed separately the risk associated with incident diabetes ( diabetes first diagnosed during the study period ) and prevalent diabetes ( diabetes first diagnosed before the study period ) , the adjusted ors were virtually the same ( 1.38 [ 0.981.94 ] and 1.34 [ 0.902.01 ] , respectively ) . furthermore , to explore the possibility of an increased risk of acute pancreatitis around the time of the first diagnosis of diabetes , we further divided our cohort of incident diabetic patients according to the time elapsed since the first diagnosis of diabetes . during the 1st year after the diagnosis of diabetes , the risk was somewhat higher ( 1.61 [ 1.002.60 ] ) than thereafter ( 1.26 [ 0.851.88 ] ) . risk of acute pancreatitis associated with diabetes , antidiabetic drugs , and other factors ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . among users of antidiabetic drugs , current users of insulin were at a decreased risk of acute pancreatitis ( adjusted or 0.35 [ 95% ci 0.200.61 ] ) compared with nonusers . moreover , past use of sulfonylureas was associated with a significant risk increase of acute pancreatitis compared with that for nonusers ( 2.58 [ 1.344.96 ] ) . otherwise , exposure to antidiabetic drugs was not materially associated with the risk of acute pancreatitis ( table 2 ) . a reduced risk of acute pancreatitis was observed across all different strata of insulin duration with a similar magnitude of association , corresponding to a 6070% reduction in the acute pancreatitis risk . of interest , metformin and sulfonylureas , which overall did not seem to be associated with acute pancreatitis , were associated with decreased and increased risks , respectively , but only among long - term users of these drugs . the other antidiabetic drugs studied ( thiazolidinediones and others ) were not found to be associated with acute pancreatitis in any of these analyses although their numbers were considerably smaller . risk of acute pancreatitis and duration of current use of antidiabetic drugs ( nested case - control analysis ) data are n ( % ) unless otherwise indicated . treated diabetic patients had an adjusted or of 1.19 [ 95% ci 0.911.55 ] for acute pancreatitis compared with that for the general population , whereas those not receiving antidiabetic drug treatment ( 1.49 [ 1.062.08 ] ) seemed to concentrate the overall increased risk associated with diabetes . in addition , the risk of acute pancreatitis was significantly increased among current smokers , those taking 30 units of alcohol per week , individuals with a previous history of gastrointestinal disease , and current users of paracetamol and ace inhibitors ( table 2 ) . the results of this study confirm the excess risk of acute pancreatitis associated with type 2 diabetes previously reported in other observational studies ( 2,4,5 ) . in fact , the cohort analysis yielded a statistically significant 77% increased risk of acute pancreatitis associated with a prior history of diabetes . however , the magnitude of this association was reduced when it was adjusted for other risk factors in a multivariate model and became borderline significant in the nested - case control analysis . this association of an increased risk of acute pancreatitis and type 2 diabetes seems more pronounced at younger ages and has already been observed in a recent retrospective cohort study based on information from a u.s , health claims database ( 2 ) . interestingly , use of insulin and long - term use of metformin were associated with a decreased risk of pancreatitis , as opposed to long - term use of sulfonylureas , which seems to increase the risk . ( 4 ) found that the sulfonylurea glyburide increased the risk of acute pancreatitis , but neither insulin nor metformin seemed to lower the risk . in fact , there are reports of cases of acute pancreatitis in patients using metformin after an episode of acute renal failure ( 9,10 ) . to the best of our knowledge , overall , when we analyzed the risk of pancreatitis among treated and not treated diabetic patients separately , we observed that the greatest risk appeared among those without antidiabetic pharmacotherapy , who represent a quarter of our diabetic population . this result may be partially due to a slight increased risk of acute pancreatitis immediately after the diagnosis of diabetes , although this assumption could not be confirmed in our study . our data from the general population cohort could replicate results from previous epidemiological studies showing that the incidence of acute pancreatitis rises with increasing age and tends to be higher in men than women ( 1,11,12 ) . furthermore , the incidence rate found in the general population is also in line with results from a recent study from england ( 13 ) . among other risk factors studied , we found that previously reported risk factors such as smoking ( 5 ) , alcohol use ( 1,14 ) , or use of ace inhibitors ( 15,16 ) were replicated in our study . exposure to paracetamol was also associated with an increased pancreatitis risk as in previous studies ( 15 ) . fourth , we could replicate results from other studies regarding the association of various comorbidities and/or exposures and the risk of acute pancreatitis . however , because of the exploratory nature of these findings , we did not deem it necessary to adjust for multiple comparisons . in summary , we have shown that type 2 diabetes may be associated with a slight increase in the risk of acute pancreatitis . we have also found that use of insulin in type 2 diabetes might be associated with a reduced risk .

in the 21st century , cereals continue to constitute the most important crops with an annual output of 2 billion tons ( according to fao in 2006 ; http://www.fao.org ) . in today 's worldwide production , barley ranks fourth among cereals and is preferentially used as feed grain , as a raw material for beer production and , to a smaller extent , as food . initially , barley was domesticated in the fertile crescent of the neolithic near east over 10 000 years ago . in the subsequent millennia , farmers continuously adapted local populations to their needs , leading to a great variety of landraces . about 100 years ago , these formed the basis for the development of modern cultivars by cross breeding . during this time , grain yield was more than doubled with an estimated genetic contribution to this increase of about 3050% . however , to meet the future challenges imposed by a changing environment , to feed a growing world population , and to provide renewable resources to satisfy the soaring demand for energy , genomics - based technologies have to be efficiently implemented to study the genetic basis of plant performance and to isolate agronomically important genes from the genetic diversity present in the gene pool of barley . a broad spectrum of resources has been developed during the last two decades to facilitate the systematic analysis of the barley genome . these include a large number of mapped molecular markers , comprehensive est collections , bac libraries , mutant collections , dna arrays , and enabling technologies such as the large scale production of doubled haploids and efficient transformation protocols . advances made in barley genomics and recent efforts made towards physical map construction and sequencing of the barley gene space ( http://barleygenome.org ) will largely contribute to a comprehensive understanding of gene functions in the context of agronomical important phenotypes ( refer to figure 1 and table 1 ) . recently , the techniques and methods employed in cereal genomics have been reviewed [ 38 ] . in this overview , we have tried to summarize progress in structural and functional genomics of barley and put emphasis on important agronomical aspects such as grain yield , seed quality traits , and implications for malting quality improvement . the seven barley chromosomes represent the basic genome of all triticeae species . still , the large genome ( 5500 mb ) , of which 80% is composed of repetitive dna is presently not amenable to whole genome sequencing . therefore , large scale sequencing programs for the development of expressed sequence tags ( ests ) from various cdna libraries have been initiated . the progress made in the last 5 years resulted in the generation of 437,713 ests covering different cdna libraries from various stages of plant development and tissues challenged with abiotic and biotic stresses ( http://www.ncbi.nlm.nih.gov/dbest/dbest_summary.html , september 14th 2007 release ) . alignment of these ests led to the identification of a representative set of 50,453 unigenes with 23,176 tentative consensi and 27,094 singletons ( http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=barley ) , representing possibly about 75% of all genes in the barley genome . an earlier estimate of the barley gene content based on 110,000 ests led to the prediction of around 30 000 unique genes . the same est data set , which was generated from different tissues covering the plant 's life cycle , was analyzed to gain insight into differential gene expression programs in diverse plant tissues by in silico expression studies . in this way , comprehensive analysis of extensive est resources generated from large genomes provides snap shots of the transcriptome aiding in gene discovery . this also allows identifying coregulated metabolic and regulatory networks [ 10 , 11 ] and helps to establish high - density molecular maps [ 1214 ] which form the basis for comparative genomic studies , trait mapping , and map - based gene isolation . thus , in large genome cereal species like barley , est sequences facilitate a comprehensive overview of gene content and represent a resource to study the evolution and organization of a genome . regarding the latter , est - derived information remains limited as it fails to provide , for instance , regulatory information , since promoters and full length sequences are not available . physical maps represent an important link to connect the genetic level to the sequence level . similar to genetic maps , physical maps are available at different levels of resolution . wheat - barley addition lines are a useful resource to rapidly assign ests to an entire chromosome or to a chromosome arm . using this resource , 1787 genes present on the barley 1 genechip could be assigned to the six different chromosomes of barley ( 365 genes to 2h , 271 to 3h , 265 to 4h , 323 to5h , 194 to 6h , and 369 to 7h ) . at a higher resolution , a physical map of all the seven barley chromosomes has been prepared by mapping dna markers derived from both genomic as well as gene - based sequences relative to the translocation breakpoints of individual chromosomes that had been isolated using microdissection techniques . the resulting map is of particular value , as it can be directly aligned to the genetic map of barley by common markers and thus allows for the estimation of the ratio between genetic and physical distances . here the presence of a wheat gametocidal chromosome in a wheat barley addition line was exploited to select 90 progeny lines that carried differently sized fragments of barley chromosome 7h . these were subsequently used to determine the physical order and distance of markers located on barley chromosome 7h . during the past several years , core public resources have been established by generating bacterial artificial chromosome ( bac ) libraries from different barley cultivars : morex (; 313,344 clones ) , cebada capa (; 177,000 clones ) and haruno nijo ( http://www.intl-pag.org/10/abstracts/pagx_p393.html ) . based on fluorescence in situ hybridization ( fish ) techniques karyotype landmarks were derived for barley , which could be used in future to place the bac clones onto the physical map . this map shows that the genetic linkage maps are well covered with markers among all chromosomes . at the same time , the physical maps reveal large areas of the barley genome that have yet to be mapped . these unmapped areas mainly consist of heterochromatin and show very low recombination rates . in accordance with these findings , there is increasing evidence that genes are not randomly distributed across the barley genome but confined to a gene space , which mainly covers the distal parts of the chromosomes . experimental evidence for the existence of a gene space has been gained from screening a barley bac library with est - derived probes , which showed a significant nonrandom distribution across the bac clones . more direct evidence has been reached on the sequence level for barley and other triticeae species . although up to now only a limited amount of sequence data is available , the average density of annotated genes is much higher than that expected for a random distribution across the genome . the disproportionate gene number found is probably due to the preferential selection of gene containing bacs for sequence analysis . within single bacs , there is considerable variation ranging , in case of barley , from 1 gene in 12 kb up to 1 gene in 220 kb ( for review see ) . thus even the gene space itself seems to be characterized by a highly variable distribution of genes against the backdrop of noncoding , mainly repetitive dna . the existence of a gene space also opens up new opportunities to focus analyses on gene - rich regions only . recently , international efforts have been gearing up to utilize the extensive barley est resources for bac anchoring and genetic mapping . an elegant approach of screening of the morex bac library using probes resulted in the identification of gene containing bacs . upon fingerprinting of a subset of 21 161 clones , 2262 contigs could be assembled covering approximately 9.4% of the barley genome . furthermore , a database has been set up to search screening results of bac libraries as well as to provide an integrative view of data from the existing barley genetic and physical maps ( http://www.genome.clemson.edu ) . the identified bac - based gene - rich regions of the genome have been selected as a genomic reference from cultivar morex to initiate sequencing of all gene - containing regions of the barley genome by an international effort coordinated through the international barley sequencing consortium ( ibsc , http://barleygenome.org ) . despite the lack of a barley genome sequence , functional genomics efforts have been initiated by taking advantage of the available est sequence information generated by multinational coordinated efforts ( see above ) . as a first step , efforts were made to derive functional assignments of the available barley unigene set by annotation transfer from homologous sequences relying on the available plant whole genome sequences and by identifying common motifs from interpro . as a result , several ontology structures such as mips and mapman functional categories ( n. sreenivasulu , unpublished data ; http://mapman.mpimp-golm.mpg.de/index.shtml ) were developed . such computational methods also yielded putative regulatory networks as well as metabolic pathway interaction networks , but still about half of the genes have to be classified as unknown . the available barley est unigene resources played a profound role in developing several platforms for transcriptome analysis including cdna - based macroarrays [ 11 , 25 ] , microarrays , and oligonucleotide - based affymetrix arrays [ 10 , 27 ] . other profiling techniques used in barley include cdna - aflp , sage ( serial analysis of gene expression ) [ 29 , 30 ] , and igentifier . the latter method combines elements of tag sequencing such as sage and fragment display . by successfully applying these techniques , barley transcriptome data have been collected from grain development [ 11 , 25 , 32 ] , grain germination [ 33 , 34 ] , at least 15 different tissues / organs covering different growth stages , and abiotic [ 26 , 3537 ] as well as biotic stress responses [ 38 , 39 ] . the new insights gained from transcriptome analysis of host - pathogen studies have lately been reviewed by wise et al . . these large scale gene expression data sets serve as baseline experiments to generate a barley transcriptome atlas . also , an online plant expression database ( plexdb ) , previously known as barleybase ( http://www.plexdb.org/plex.php?database=barley ) has been created to store , visualize , and statistically analyze barley 1 genechip data . while transcriptomics have brought about substantial progress in elucidating biochemical pathways of barley seed metabolism ( see reviews [ 5 , 42 ] ) , very recent findings shed light on the interplay of many cellular and metabolic events that are coordinated by a complex regulatory network during barley seed development [ 10 , 11 , 25 ] . studying expression data of nearly 12 000 seed - expressed genes revealed , for instance , the participation of tissue - specific signaling networks controlling aba - mediated starch accumulation ( via snf1 kinase and a set of transcription factors ) in the endosperm and participation of aba - responsive genes in establishing embryo desiccation tolerance . cpg methylation found in the promoters of prolamin box - binding factor and b - hordein genes suppresses transcript levels during the prestorage until the intermediate phase of grain development . this process coincides with the coexpression of methyltransferases , core histones and dna - unwinding atpases . thus storage protein gene expression may be regulated by cpg methylation . using a lys 3a mutant , it has been shown that demethylation of the b - hordein promoter does not occur in the mutant , hence transcripts encoding storage proteins such as b - hordeins and c - hordeins are almost absent in the developing endosperm of this mutant . transcriptome profiling of barley embryos using the 22k affymetrix barley 1 genechip revealed activation of developmentally distinct defense related gene sets including coregulated phenylpropanoid and phytoalexin related genes around 20 days after flowering ( daf ) , followed by upregulation of antioxidant and pathogen related gene sets around 37 daf . the knowledge obtained on metabolic processes of seed quality traits could eventually be used to develop superior varieties by genetic engineering or by marker - assisted selection in conventional breeding programs . transcriptome analysis has also been carried out during barley grain germination at tissue - specific levels [ 10 , 46 ] . using cdna array technology gene expression was analyzed in germinating seed samples , collected from ten different barley genotypes showing differential malting response . based on six different malting quality parameters related to hydrolytic events connected to protein , starch , and cell wall degradation 19 candidate genes were identified , whose transcript abundance showed a significant correlation with some of the malting quality parameters . analyzed seven different sage libraries derived from malted grains and identified 100 most abundant transcripts showing differential responses during eight different time points during malting . these transcripts are related to stress and defense response , hydrolytic processes and translational events . the list of candidate genes identified in the two studies [ 30 , 46 ] was further validated by a genetical genomics approach in which gene expression studies were conducted with populations segregating for malting traits [ 34 , 47 ] . a major aim of functional genomic studies is to understand the metabolic and regulatory networks within the structural and functional context of cells , tissues , and organs often changing with time . hence in this review , we update the functional genomic resources available ( table 1 ) to study gene functions in barley using reverse genetics approaches and highlight the initial success achieved through genetic engineering based on the manipulation of individual genes . to determine gene - function relationships , large scale genome - wide reverse genetics approaches have been developed in barley ( see for review ) which includes both nontransgenic technology platforms such as tilling ( targeting induced local lesions in genomes ) and insertional mutagenesis systems based on transgenic technology [ 5054 ] . thus , the scottish crop research institute generated a large m2 tilling population in the barley cultivar optic with leaf material and seeds from 20 000 plants freeze dried and archived . ems induced mutations were scored at various growth stages under different conditions and documented [ 49 , 55 ] . mutant phenotypes , candidate genes , and observed dna sequence variations can be queried in an scri mutant database ( http://germinate.scri.ac.uk/barley/mutants/index.php?option=com_wrapper&itemid=35 ) . in a more recent attempt , ipk developed a tilling population of 10 000 m2 plants in the cultivar similarly , a collection of 5000 m3 mutants of the cultivar morex is provided by the university of bologna ( http://www.intl-pag.org/13/abstracts/pag13_p081.html ) . to aid functional gene analysis , insertional mutagenesis approaches were followed in barley during the last decade ( i ) to create loss - of - function mutations by the insertion of transposable elements into a gene of interest [ 5053 ] and ( ii ) use activation tagging ( the random genomic insertion of either promoter or enhancer sequences ) to generate dominant gain - of - function mutations [ 54 , 56 ] . insertion lines have been generated by creating transgenic plants carrying ac and ds elements , and crossed them to induce ds transposition [ 5052 ] . ds elements were preferentially found in genic regions and exhibited a high - remobilization frequency [ 52 , 53 ] . such ds launch pads , represented by barley lines with each harboring a single copy ds insertion at a well - defined position in the genome , will be valuable for future targeted gene tagging . similarly , dominant overexpression phenotypes [ 54 , 56 ] will help to study gene functions in the large barley genome where loss - of - function mutations often may not cause phenotypes because of gene redundancy . in order to functionally characterize candidate genes identified in functional genomic studies , it was mandatory to establish a stable and efficient genetic transformation technique in barley . in contrast to the biolistic gene transfer technique , a more efficient agrobacterium mediated barley genetic transformation method based on immature embryos was developed in spring barley . in a recent attempt to further improve this technology , kumlehn et al . developed a transformation method for winter barley based upon the infection with agrobacterium of androgenic pollen cultures . by this approach , homozygous double haploid plants could be immediately obtained at high frequency through chromosome doubling . during the last decade , systematic efforts were made for genetic engineering of barley to improve seed quality traits including those related to malting ( reviewed in ) . malting improvement has been addressed by altering the expression of hydrolytic enzymes related to the degradation of storage products such as starch ( and - amylases , [ 61 , 62 ] ) and cell wall components . in another approach , several enzymes such as xylanase , glucanase , endo- , and exoprotease were over expressed in transgenic barley grains and preferably the enzyme mix necessary for malting process are provided by transgenic seeds . protein engineering has been used to produce thermostable 1 , 3 ; 1 , 4 - glucanases in transgenic barley grains [ 6466 ] . such grains can be used to enhance the feed quality of barley for poultry [ 67 , 68 ] . in a similar approach , a hybrid cellulase gene driven by the endosperm specific rice glub-1 promoter was expressed and produced the enzyme up to 1.5% of total grain protein . in addition , functions of key genes involved in determining seed quality traits related to storage product accumulation were tested . for instance , antisense downregulation of limit dextrinase inhibitor showed reduced amylose over amylopectin levels and eventually reduced total starch . also overexpression of wheat thioredoxin h in the endosperm of transgenic barley grain leads to increased activity of the starch debranching enzyme limit dextrinase [ 71 , 72 ] . further , a powerful approach of antisense oligodeoxynucleotide inhibition has been used to reveal sugar signaling networks . short stretches of 1225 nucleotide long single - strand sequences have been delivered to barley leaf cells to block the effect of susiba2 , a key transcriptional activator involved in plant sugar signaling . recently , this approach has been successfully implemented to deliver antisense oligodeoxynucleotides to barley seed endosperm to suppress sugar related signaling genes . hvgamyb , a transcription factor initially identified in aleurone and shown to be upregulated by gibberellin , has been shown to be expressed also in barley anthers . the overexpressing hvgamyb transgenic lines show reduced anther size with a male sterility phenotype . our laboratory has recently characterized a new protein called jekyll , which is preferentially expressed in barley grain nucellar projection tissue . its downregulation decelerates autolysis of nurse tissue . as a result , proliferation of endosperm nuclei is impaired and less starch is finally accumulated in the endosperm . with respect to applied aspects in crop plants , a comprehensive knowledge of cellular and functional complexity as related to key agronomic traits a number of tools and databases were developed at our institute ( leibniz institute of plant genetics and crop plant research / ipk ) to store , analyze , and display the data derived from multiparallel - omics profiling studies at transcript , metabolite , and protein / enzyme level with the aim to eventually gain insight into the organization of function - related networks in barley [ 78 , 79 ] . these include cr - est ( it provides access to clustering and annotation data of ipk est projects ) , meta - all , and metacrop ( they allow to access curated metabolic pathway information and kinetic reactions of crop plants ) , vanted ( for visualization and analysis of metabolic and regulatory networks ) , hit - mds ( for screening of coexpressed genes and validation of cluster centroids ) as well as barley mapman and pageman [ http://mapman.mpimp-golm.mpg.de ; to index and visualize overrepresented functional categories and detailed metabolic pathway charts from throughput transcriptome data ] . with the focus of using the developing seed as model for systems biology studies , we investigated transcriptional and metabolic networks during grain development [ 11 , 25 , 82 ] , developed 3d models of the developing barley grain , implemented magnetic resonance - based techniques to establish 4d models as a framework to store different sets of data in their spatiotemporal context , visualized the spatial distribution of specific biochemical compounds by noninvasive nmr - based imaging methods and established kinetic models of primary metabolism ( and e. grafahrend - belau and b. junker , unpublished data ) as already worked out for potato . in addition , a proteomic platform has been successfully established to study barley grain development [ 88 , 89 ] . the emerging model ( largely qualitative ) explaining how the barley grain develops and functions has to be further validated especially by the creation and analysis of different lines of transgenic plants with perturbations at putative key metabolic and/or regulatory sites ( see figure 1 ) . almost two decades ago , rflp markers were employed to develop the first comprehensive molecular marker maps in barley [ 9092 ] . using those rflp maps , a series of agronomic traits and characters including many quality traits and resistance against several diseases have been mapped ( for review see [ 93 , 94 ] ) . later , the availability of large numbers of ests facilitated the systematic development of functional markers , for example , by extracting ests containing simple sequence repeat ( ssr ) motifs using appropriate software tools . although est - based ssr markers have been shown to be less polymorphic than their genomic counterparts , this drawback is more than compensated for by the ease of their development . also , the availability of ests from multiple - genotypes / cultivars of barley provides the possibility to identify sequence polymorphisms ( mainly single - nucleotide polymorphisms and small indels ) in the corresponding est alignments . these in turn can be exploited for the development of markers [ 96 , 97 ] . kota et al . developed the computer algorithm snipping for discovery of functional markers through browsing est assemblies in barley . also an snp2caps program has been published to facilitate the computational conversion of snp markers into caps markers . information generated from the diverse mapping projects was further enhanced by the development of consensus maps [ 14 , 100102 ] . these provide integrative genetic information by featuring high marker densities . although the gel - based genotyping platforms offer the best quality marker systems , their low throughput encouraged researchers to explore high - throughput technologies that can simultaneously assay thousands of markers based on single nucleotide polymorphisms ( snp ) . most recently , genome - wide scans using snp - based genotyping platforms such as illumina goldengate beadarrays and the diversity arrays technology ( dart ) , which do not require any sequence information have been successfully established in barley . although darts are not systematically interrogating expressed sequences , the choice of appropriate enzymes facilitates their enriched representation . based on dart technology , a high - density consensus map has recently been established . a number of recent studies also reported the use of the affymetrix barley 1 genechip for identifying single - feature polymorphisms ( sfps ) , which cover not only snps but also indels and polymorphisms generated due to alternative splicing and polyadenylation [ 34 , 106 ] . an important application of the above discussed functional markers is marker - assisted selection ( mas ) . mas is based on linking the dna polymorphisms revealed by marker analysis with agronomical traits allowing for their rapid selection in routine breeding programs . mas can be performed already at juvenile growth stages and before flowering , and thus provides breeders with the opportunity to implement faster back - crossing strategies and allele enrichment in complex crosses , which eventually reduces the time and costs required for the development of improved varieties . despite its inherent advantages , the application of mas in barley up to now has mainly been restricted to monogenic traits such as disease resistances . here , one of the most widespread examples is the marker assisted selection of the rym4 gene giving resistance to the barley yellow mosaic virus complex . for this gene , several closely linked and easily scorable markers have been developed [ 107 , 108 ] . more recently , cloning of the gene facilitated the exploitation of functional polymorphisms within the coding region of the resistance gene to differentiate between alleles . using mas , several genes providing full resistance could be readily combined in complex crosses without time consuming progeny tests in the greenhouse or in the field ( e.g. , [ 110 , 111 ] ) . ( i ) compared to monogenic traits , quantitative traits are characterized by lower heritabilities impairing their accurate scoring and entailing a less accurately defined genetic position of the corresponding quantitative trait locus ( qtl ) . as a result , large chromosomal fragment needs to be selected for , resulting in the meiotic transfer of many potentially undesired genes . the feasibility of downtracking a qtl to a single gene has been initially demonstrated in tomato and requires the stepwise size reduction of a qtl fragment and its conversion into a near isogenic line by repeated backcrossing ( for review see ) . in barley , this approach has been successfully employed to isolate the bot1 gene underlying a major qtl conferring boron tolerance . ( ii ) many of qtl alleles escape detection , when transferred into a different genetic background . the reasons for the disappearance of qtls include epistatic interactions , qtl x environment effects , the allelic states of the parental lines or the small contribution of a single qtl to the overall variance . as a result , only few common qtls were detected , when the results of mapping studies that were performed in different crosses were compared . although the number of successful examples for applying mas in barley breeding is still rather limited ( see reviews by [ 114 , 115 ] , the recent implementation of high - throughput genotyping platforms ( illumina , dart , and sfp identification by using barley 1 genechip affymetrix array ) in barley will significantly increase the identification of marker trait associations , and the subsequent identification of potential candidate genes . finally , this will allow to treat qtls as monogenic traits and thus spur their marker assisted manipulation in breeding programs . in combination with a wide range of mapping populations developed for specific agronomic traits , this comprehensive resource of markers now allows the identification of polymorphisms in functionally defined sequences [ 12 , 34 , 105 , 106 ] . functional markers will also be useful for ( i ) association studies based on linkage disequilibrium , ( ii ) detection of cis and trans - acting regulators either based on genetical genomics studies using well - defined mapping populations or by investigating allelic imbalance , ( iii ) identification of alleles influencing agronomically important traits using tilling / ecotilling approaches ( ecotilling is a means to determine the extent of natural variation in selected genes ) , and ( iv ) genomics - assisted breeding ( see figure 1 ) . linkage disequilibrium is the nonrandom distribution of alleles in a sample population and forms the basis for the construction of genetic maps and the localization of genetic loci for a variety of traits . the principles leading to ld apply to both biparental mapping populations ( f2 , rils , etc . ) and natural populations . therefore , ld mapping is the method of choice for genetic analysis in organisms like humans and animals , where experimental populations are either not available or difficult to establish . because of its inherent advantages , ld mapping approaches are increasingly being applied for plant species , in particular maize . due to the outbreeding character of this species , ld extends only over a few kb and thus leads to a high - genetic resolution , up to the level of individual candidate genes that can be associated with a given trait ( see recent reviews [ 118 , 119 ] ) . the use of association genetic analyses in inbreeding species such as barley has been limited so far however , recent studies have shown that ld extends over much longer genetic distances in barley than in maize . a european germplasm collection of 146 two - rowed spring barley cultivars was used to carry out ld mapping of yield traits using 236 aflp markers . associated markers were identified that are located in similar regions where qtls for yield had been found in barley [ 93 , 121 , 122 ] . a systematic survey of 953 gene bank accessions representing a broad spectrum of the genetic diversity in barley genetic resources revealed that ld extends up to 50 cm but is highly dependent on population structure [ 120 , 123 ] . on the one hand , the high level of ld in barley is due to the inbreeding mating type of this species ; on the other hand , the selection of germplasm plays an important role . analysis of a germplasm collection of european cultivars , land races , and wild barley accession from the fertile crescent region provided hints that the level of ld decreases from cultivars to landraces to wild barley . similarly , morrell et al . reported low levels of ld in wild barley by examining ld within and between 18 genes from 25 accessions . local differences in ld have been observed at the grain hardness locus comprising four closely linked genes ( hinb , hina , gsp , pg2 ) . here , a high level of ld was observed in the intergenic region between hinb-1 and hina probably due to transposable elements present in this region , which influence the local recombination rate . by assaying 1524 genome - wide snps in elite northwest . concluded that whole - genome association scans can be exploited for trait mapping in barley . this was further exemplified by the identification of a marker that showed an association with the winter habit and which could be tracked to a cluster of cbf ( c - repeat / dre - binding factor ) gene homologs . in a recent whole genome ld - mapping approach , steffenson et al . used 318 wild barley accessions to perform association mapping studies using dart markers to identify rust resistance genes . in addition , ld analysis has been performed based on haplotypes derived from 131 accessions by covering 83 snps within 132 kb around the gene hveif4e , which confers resistance to barley yellow mosaic virus . the authors identified three haplogroups discriminating between the alleles rym4 and rym5 . taken together , the above mentioned association studies provide starting points for a more systematic analysis of agronomic traits . these may be selected from the vast ex situ gene bank collections available for this crop . alone at the ipk gene bank some 20 000 different barley accessions represent an ample cross section of the genetic diversity present in this species . however , in order to fully exploit the potential of ld - based association analysis in this species , populations have to be carefully selected to minimize the confounding effects of population structure . this is particularly evident in modern barley germplasm , which is frequently structured into spring and winter as well as 2-rowed and 6-rowed types , forming distinct subpopulations ( e.g. , ) . if these effects are not adequately accounted for during association analysis , the risk of detecting spurious associations increases . differentially expressed genes ( but also proteins and metabolites ) involved in metabolic and regulatory pathways and identified by high - throughput technologies are treated as phenotypes , and genetic variants that influence gene expression are identified in genetically related lines . this strategy has been successfully applied also in plant systems , and relevant data were reviewed elsewhere [ 128130 ] . here , we will focus on the latest development in expression qtl ( eqtl ) mapping in barley . using the barley 1 genechip affymetrix array sfp genotyping has been performed in 35 recombinant lines of a steptoe morex doubled - haploid population , enabling eqtl studies . using a high - throughput sfp genotyping platform , genome - wide linkage analysis has been performed based on 22 000 transcript data collected from 139 dh lines ( steptoe morex ) . the most significant eqtls derived from germinating barley grain are linked to cis regulation . using the same mapping population , a serine carboxypeptidase 1 eqtl has been mapped on chromosome 3h to the same region where a qtl for the malting quality trait diastatic power has been mapped . in another study , instead of a segregating population a set of 47 bc3 dh introgression lines was employed ( wild barley [ h. spontaneum ] is introgressed in the genetic background of the elite line [ h. vulgare ] ) in order to understand gene expression networks controlling seed traits . initially , this bc3 dh population was used to identify qtls for yield and yield components . in further experiments , expression data from nearly 12 000 genes interrogated by using a barley seed specific array were used to calculate eqtls ( c. pietsch et al . , unpublished ) . although such initial studies provide evidence that genetical genomics is a promising concept which assists to expose gene - trait relationships , an extensive exploration of the technology needs the full barley genome sequence and improved high - throughput genotyping information . in recent years , we experienced a dramatic development of new tools and technologies for genome research and a concomitantly dramatic increase in data leading to a much improved and advanced knowledge base . barley research gained a lot of momentum from this development but the nonavailability of a whole genome sequence is still a serious limitation . however , due to consortial efforts ( see above ) and the rapidly developing sequencing technologies that are relevant for even complex genomes like that of barley this limitation will be largely overcome , hopefully within the next five years . high - throughput transcriptome analysis techniques have already provided numerous new insights in transcriptional networks . they will , together with rapidly improving protein and metabolite profiling techniques and in combination with new genetic analysis concepts such as genetical genomics and association genetics , improve our knowledge on the relationship between the genetic and the phenotypic architecture of agronomic traits and thus create a basis for knowledge - based molecular breeding . as a next step systems biology approaches are emerging , which attempt to model complex cellular or organismic functions in response to changing internal and external factors . until now molecular markers have had limited success in barley breeding programs , but due to recent advancement of barley genomics a stronger impact on breeding strategies is expected . for instance , marker technologies together with double haploid production have almost halved the time of variety development in australian wheat and barley breeding programs . however , new whole - genome breeding strategies have to be developed to make full use of the ever increasing knowledge about crop plant genomes and their behavior .

barley ( hordeum vulgare ) , first domesticated in the near east , is a well - studied crop in terms of genetics , genomics , and breeding and qualifies as a model plant for triticeae research . recent advances made in barley genomics mainly include the following : ( i ) rapid accumulation of est sequence data , ( ii ) growing number of studies on transcriptome , proteome , and metabolome , ( iii ) new modeling techniques , ( iv ) availability of genome - wide knockout collections as well as efficient transformation techniques , and ( v ) the recently started genome sequencing effort . these developments pave the way for a comprehensive functional analysis and understanding of gene expression networks linked to agronomically important traits . here , we selectively review important technological developments in barley genomics and related fields and discuss the relevance for understanding genotype - phenotype relationships by using approaches such as genetical genomics and association studies . high - throughput genotyping platforms that have recently become available will allow the construction of high - density genetic maps that will further promote marker - assisted selection as well as physical map construction . systems biology approaches will further enhance our knowledge and largely increase our abilities to design refined breeding strategies on the basis of detailed molecular physiological knowledge .

1. INTRODUCTION 2. BARLEY ESTS, BACS, AND PHYSICAL MAPSA SPRINGBOARD FOR THE EXPLORATION OF THE GENOME 3. A BARLEY TRANSCRIPTOME ATLAS 4. FUNCTIONAL GENOMICS APPROACHES IN BARLEY 5. FUNCTIONAL MOLECULAR MARKERS AND THEIR POTENTIAL APPLICATIONS IN THE AREA OF APPLIED GENOMICS 6. OUTLOOOK

in today 's worldwide production , barley ranks fourth among cereals and is preferentially used as feed grain , as a raw material for beer production and , to a smaller extent , as food . initially , barley was domesticated in the fertile crescent of the neolithic near east over 10 000 years ago . however , to meet the future challenges imposed by a changing environment , to feed a growing world population , and to provide renewable resources to satisfy the soaring demand for energy , genomics - based technologies have to be efficiently implemented to study the genetic basis of plant performance and to isolate agronomically important genes from the genetic diversity present in the gene pool of barley . these include a large number of mapped molecular markers , comprehensive est collections , bac libraries , mutant collections , dna arrays , and enabling technologies such as the large scale production of doubled haploids and efficient transformation protocols . advances made in barley genomics and recent efforts made towards physical map construction and sequencing of the barley gene space ( http://barleygenome.org ) will largely contribute to a comprehensive understanding of gene functions in the context of agronomical important phenotypes ( refer to figure 1 and table 1 ) . in this overview , we have tried to summarize progress in structural and functional genomics of barley and put emphasis on important agronomical aspects such as grain yield , seed quality traits , and implications for malting quality improvement . still , the large genome ( 5500 mb ) , of which 80% is composed of repetitive dna is presently not amenable to whole genome sequencing . the progress made in the last 5 years resulted in the generation of 437,713 ests covering different cdna libraries from various stages of plant development and tissues challenged with abiotic and biotic stresses ( http://www.ncbi.nlm.nih.gov/dbest/dbest_summary.html , september 14th 2007 release ) . alignment of these ests led to the identification of a representative set of 50,453 unigenes with 23,176 tentative consensi and 27,094 singletons ( http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=barley ) , representing possibly about 75% of all genes in the barley genome . this also allows identifying coregulated metabolic and regulatory networks [ 10 , 11 ] and helps to establish high - density molecular maps [ 1214 ] which form the basis for comparative genomic studies , trait mapping , and map - based gene isolation . thus , in large genome cereal species like barley , est sequences facilitate a comprehensive overview of gene content and represent a resource to study the evolution and organization of a genome . using this resource , 1787 genes present on the barley 1 genechip could be assigned to the six different chromosomes of barley ( 365 genes to 2h , 271 to 3h , 265 to 4h , 323 to5h , 194 to 6h , and 369 to 7h ) . at a higher resolution , a physical map of all the seven barley chromosomes has been prepared by mapping dna markers derived from both genomic as well as gene - based sequences relative to the translocation breakpoints of individual chromosomes that had been isolated using microdissection techniques . at the same time , the physical maps reveal large areas of the barley genome that have yet to be mapped . more direct evidence has been reached on the sequence level for barley and other triticeae species . although up to now only a limited amount of sequence data is available , the average density of annotated genes is much higher than that expected for a random distribution across the genome . an elegant approach of screening of the morex bac library using probes resulted in the identification of gene containing bacs . furthermore , a database has been set up to search screening results of bac libraries as well as to provide an integrative view of data from the existing barley genetic and physical maps ( http://www.genome.clemson.edu ) . as a first step , efforts were made to derive functional assignments of the available barley unigene set by annotation transfer from homologous sequences relying on the available plant whole genome sequences and by identifying common motifs from interpro . as a result , several ontology structures such as mips and mapman functional categories ( n. sreenivasulu , unpublished data ; http://mapman.mpimp-golm.mpg.de/index.shtml ) were developed . such computational methods also yielded putative regulatory networks as well as metabolic pathway interaction networks , but still about half of the genes have to be classified as unknown . other profiling techniques used in barley include cdna - aflp , sage ( serial analysis of gene expression ) [ 29 , 30 ] , and igentifier . the latter method combines elements of tag sequencing such as sage and fragment display . by successfully applying these techniques , barley transcriptome data have been collected from grain development [ 11 , 25 , 32 ] , grain germination [ 33 , 34 ] , at least 15 different tissues / organs covering different growth stages , and abiotic [ 26 , 3537 ] as well as biotic stress responses [ 38 , 39 ] . also , an online plant expression database ( plexdb ) , previously known as barleybase ( http://www.plexdb.org/plex.php?database=barley ) has been created to store , visualize , and statistically analyze barley 1 genechip data . while transcriptomics have brought about substantial progress in elucidating biochemical pathways of barley seed metabolism ( see reviews [ 5 , 42 ] ) , very recent findings shed light on the interplay of many cellular and metabolic events that are coordinated by a complex regulatory network during barley seed development [ 10 , 11 , 25 ] . cpg methylation found in the promoters of prolamin box - binding factor and b - hordein genes suppresses transcript levels during the prestorage until the intermediate phase of grain development . using a lys 3a mutant , it has been shown that demethylation of the b - hordein promoter does not occur in the mutant , hence transcripts encoding storage proteins such as b - hordeins and c - hordeins are almost absent in the developing endosperm of this mutant . the knowledge obtained on metabolic processes of seed quality traits could eventually be used to develop superior varieties by genetic engineering or by marker - assisted selection in conventional breeding programs . using cdna array technology gene expression was analyzed in germinating seed samples , collected from ten different barley genotypes showing differential malting response . based on six different malting quality parameters related to hydrolytic events connected to protein , starch , and cell wall degradation 19 candidate genes were identified , whose transcript abundance showed a significant correlation with some of the malting quality parameters . the list of candidate genes identified in the two studies [ 30 , 46 ] was further validated by a genetical genomics approach in which gene expression studies were conducted with populations segregating for malting traits [ 34 , 47 ] . hence in this review , we update the functional genomic resources available ( table 1 ) to study gene functions in barley using reverse genetics approaches and highlight the initial success achieved through genetic engineering based on the manipulation of individual genes . to determine gene - function relationships , large scale genome - wide reverse genetics approaches have been developed in barley ( see for review ) which includes both nontransgenic technology platforms such as tilling ( targeting induced local lesions in genomes ) and insertional mutagenesis systems based on transgenic technology [ 5054 ] . mutant phenotypes , candidate genes , and observed dna sequence variations can be queried in an scri mutant database ( http://germinate.scri.ac.uk/barley/mutants/index.php?option=com_wrapper&itemid=35 ) . in a more recent attempt , ipk developed a tilling population of 10 000 m2 plants in the cultivar similarly , a collection of 5000 m3 mutants of the cultivar morex is provided by the university of bologna ( http://www.intl-pag.org/13/abstracts/pag13_p081.html ) . to aid functional gene analysis , insertional mutagenesis approaches were followed in barley during the last decade ( i ) to create loss - of - function mutations by the insertion of transposable elements into a gene of interest [ 5053 ] and ( ii ) use activation tagging ( the random genomic insertion of either promoter or enhancer sequences ) to generate dominant gain - of - function mutations [ 54 , 56 ] . such ds launch pads , represented by barley lines with each harboring a single copy ds insertion at a well - defined position in the genome , will be valuable for future targeted gene tagging . similarly , dominant overexpression phenotypes [ 54 , 56 ] will help to study gene functions in the large barley genome where loss - of - function mutations often may not cause phenotypes because of gene redundancy . in order to functionally characterize candidate genes identified in functional genomic studies , it was mandatory to establish a stable and efficient genetic transformation technique in barley . in another approach , several enzymes such as xylanase , glucanase , endo- , and exoprotease were over expressed in transgenic barley grains and preferably the enzyme mix necessary for malting process are provided by transgenic seeds . hvgamyb , a transcription factor initially identified in aleurone and shown to be upregulated by gibberellin , has been shown to be expressed also in barley anthers . as a result , proliferation of endosperm nuclei is impaired and less starch is finally accumulated in the endosperm . with respect to applied aspects in crop plants , a comprehensive knowledge of cellular and functional complexity as related to key agronomic traits a number of tools and databases were developed at our institute ( leibniz institute of plant genetics and crop plant research / ipk ) to store , analyze , and display the data derived from multiparallel - omics profiling studies at transcript , metabolite , and protein / enzyme level with the aim to eventually gain insight into the organization of function - related networks in barley [ 78 , 79 ] . these include cr - est ( it provides access to clustering and annotation data of ipk est projects ) , meta - all , and metacrop ( they allow to access curated metabolic pathway information and kinetic reactions of crop plants ) , vanted ( for visualization and analysis of metabolic and regulatory networks ) , hit - mds ( for screening of coexpressed genes and validation of cluster centroids ) as well as barley mapman and pageman [ http://mapman.mpimp-golm.mpg.de ; to index and visualize overrepresented functional categories and detailed metabolic pathway charts from throughput transcriptome data ] . with the focus of using the developing seed as model for systems biology studies , we investigated transcriptional and metabolic networks during grain development [ 11 , 25 , 82 ] , developed 3d models of the developing barley grain , implemented magnetic resonance - based techniques to establish 4d models as a framework to store different sets of data in their spatiotemporal context , visualized the spatial distribution of specific biochemical compounds by noninvasive nmr - based imaging methods and established kinetic models of primary metabolism ( and e. grafahrend - belau and b. junker , unpublished data ) as already worked out for potato . also , the availability of ests from multiple - genotypes / cultivars of barley provides the possibility to identify sequence polymorphisms ( mainly single - nucleotide polymorphisms and small indels ) in the corresponding est alignments . although the gel - based genotyping platforms offer the best quality marker systems , their low throughput encouraged researchers to explore high - throughput technologies that can simultaneously assay thousands of markers based on single nucleotide polymorphisms ( snp ) . most recently , genome - wide scans using snp - based genotyping platforms such as illumina goldengate beadarrays and the diversity arrays technology ( dart ) , which do not require any sequence information have been successfully established in barley . based on dart technology , a high - density consensus map has recently been established . a number of recent studies also reported the use of the affymetrix barley 1 genechip for identifying single - feature polymorphisms ( sfps ) , which cover not only snps but also indels and polymorphisms generated due to alternative splicing and polyadenylation [ 34 , 106 ] . an important application of the above discussed functional markers is marker - assisted selection ( mas ) . despite its inherent advantages , the application of mas in barley up to now has mainly been restricted to monogenic traits such as disease resistances . here , one of the most widespread examples is the marker assisted selection of the rym4 gene giving resistance to the barley yellow mosaic virus complex . ( i ) compared to monogenic traits , quantitative traits are characterized by lower heritabilities impairing their accurate scoring and entailing a less accurately defined genetic position of the corresponding quantitative trait locus ( qtl ) . as a result , large chromosomal fragment needs to be selected for , resulting in the meiotic transfer of many potentially undesired genes . ( ii ) many of qtl alleles escape detection , when transferred into a different genetic background . although the number of successful examples for applying mas in barley breeding is still rather limited ( see reviews by [ 114 , 115 ] , the recent implementation of high - throughput genotyping platforms ( illumina , dart , and sfp identification by using barley 1 genechip affymetrix array ) in barley will significantly increase the identification of marker trait associations , and the subsequent identification of potential candidate genes . finally , this will allow to treat qtls as monogenic traits and thus spur their marker assisted manipulation in breeding programs . functional markers will also be useful for ( i ) association studies based on linkage disequilibrium , ( ii ) detection of cis and trans - acting regulators either based on genetical genomics studies using well - defined mapping populations or by investigating allelic imbalance , ( iii ) identification of alleles influencing agronomically important traits using tilling / ecotilling approaches ( ecotilling is a means to determine the extent of natural variation in selected genes ) , and ( iv ) genomics - assisted breeding ( see figure 1 ) . linkage disequilibrium is the nonrandom distribution of alleles in a sample population and forms the basis for the construction of genetic maps and the localization of genetic loci for a variety of traits . the use of association genetic analyses in inbreeding species such as barley has been limited so far however , recent studies have shown that ld extends over much longer genetic distances in barley than in maize . associated markers were identified that are located in similar regions where qtls for yield had been found in barley [ 93 , 121 , 122 ] . a systematic survey of 953 gene bank accessions representing a broad spectrum of the genetic diversity in barley genetic resources revealed that ld extends up to 50 cm but is highly dependent on population structure [ 120 , 123 ] . on the one hand , the high level of ld in barley is due to the inbreeding mating type of this species ; on the other hand , the selection of germplasm plays an important role . here , a high level of ld was observed in the intergenic region between hinb-1 and hina probably due to transposable elements present in this region , which influence the local recombination rate . by assaying 1524 genome - wide snps in elite northwest . taken together , the above mentioned association studies provide starting points for a more systematic analysis of agronomic traits . this is particularly evident in modern barley germplasm , which is frequently structured into spring and winter as well as 2-rowed and 6-rowed types , forming distinct subpopulations ( e.g. differentially expressed genes ( but also proteins and metabolites ) involved in metabolic and regulatory pathways and identified by high - throughput technologies are treated as phenotypes , and genetic variants that influence gene expression are identified in genetically related lines . this strategy has been successfully applied also in plant systems , and relevant data were reviewed elsewhere [ 128130 ] . here , we will focus on the latest development in expression qtl ( eqtl ) mapping in barley . using a high - throughput sfp genotyping platform , genome - wide linkage analysis has been performed based on 22 000 transcript data collected from 139 dh lines ( steptoe morex ) . in another study , instead of a segregating population a set of 47 bc3 dh introgression lines was employed ( wild barley [ h. spontaneum ] is introgressed in the genetic background of the elite line [ h. vulgare ] ) in order to understand gene expression networks controlling seed traits . although such initial studies provide evidence that genetical genomics is a promising concept which assists to expose gene - trait relationships , an extensive exploration of the technology needs the full barley genome sequence and improved high - throughput genotyping information . high - throughput transcriptome analysis techniques have already provided numerous new insights in transcriptional networks . they will , together with rapidly improving protein and metabolite profiling techniques and in combination with new genetic analysis concepts such as genetical genomics and association genetics , improve our knowledge on the relationship between the genetic and the phenotypic architecture of agronomic traits and thus create a basis for knowledge - based molecular breeding . as a next step systems biology approaches are emerging , which attempt to model complex cellular or organismic functions in response to changing internal and external factors . until now molecular markers have had limited success in barley breeding programs , but due to recent advancement of barley genomics a stronger impact on breeding strategies is expected . however , new whole - genome breeding strategies have to be developed to make full use of the ever increasing knowledge about crop plant genomes and their behavior .

free radical - mediated lipid peroxidation and antioxidants are attracting considerable research interest in many areas . lipid oxidation is one of the major deteriorative processes in many types of foods , leading to the changes in food quality and nutritional value . in particular , investigators report that free radicals , generated by oxidation , play a critical role in a variety of health disorders , including the processes of ageing , cancer , diabetes mellitus , inflammation , coronary heart , and neurological disorders , such as alzheimer 's disease . therefore , it is important to inhibit the oxidation and formation of free radicals occurring in the living body and foodstuffs . some synthetic antioxidative agents , such as butylated hydroxyanisole ( bha ) , butylated hydroxytoluene ( bht ) , and propyl gallate , are commonly used as free radical scavengers in food and biological systems . although , these synthetic antioxidants show stronger antioxidant activity than those of natural antioxidants such as -tocopherol and ascorbic acid , the use of these chemical compounds has begun to be restricted because of their induction of dna damage and their toxicity . thus , increasing attention has been directed to the development of safe and effective functional foods and antioxidative agents from natural sources , especially peptides derived from hydrolyzed food proteins . recently , protein hydrolysates from several fish species , such as silver carp ( hypophthalmichthys molitrix ) , brownstripe red snapper ( lutjanus vitta ) , sardinelle ( sardinella aurita ) , smooth hound ( mustelus mustelus ) , oyster ( crassostrea gigas ) , yellow stripe trevally ( selaroides leptolepis ) , round scad ( decapterus maruadsi ) , yellowfin sole ( limanda aspera ) , herring ( clupea harengus ) , and mackerel ( scomber austraasicus ) , have been reported to possess antioxidative activities . the operational conditions employed in the processing of protein isolates , the type of protease , and the degree of hydrolysis affect the antioxidant activity . especially the , proteinases used can affect both the functional properties and antioxidative activity of the protein hydrolysate obtained . protein hydrolysate from alaska pollack frame prepared by mackerel intestine crude enzyme exhibited antioxidative activity in a linoleic acid model system . prawn hydrolysate prepared using pepsin showed the most potent antioxidative activity than those prepared by other enzymes . levels and compositions of free amino acids and peptides were reported to determine the antioxidant activities of protein hydrolysates . moreover , the utilization of proteins or their hydrolysates for food and/or cosmetic applications not only presents additional advantages over other antioxidants but also confers nutritional and functional properties . recently , protein hydrolysates from cuttlefish ( sepia officinalis ) enriched in angiotensin i - converting enzyme inhibitory peptides have been produced successfully using alcalase and bacillus licheniformis nh1 proteases . nevertheless , a little information regarding the characteristic and antioxidative activity of hydrolysates prepared using both enzymes has been reported . the objective of this study was to investigate the antioxidative activity of protein hydrolysates from cuttlefish muscle prepared using alcalase and bacillus licheniformis nh1 proteases in a model system and the effect of concentration on their activities . meanwhile , solubility of hydrolysates derived from cuttlefish muscle was evaluated . 1,1-diphenyl-2-picrylhydrazyl ( dpph ) , 3-(2-pyridyl)-5,6-bis(4-phenyl - sulphonic acid)-1,2,4-triazine ( ferrozine ) , butylated hydroxyanisole ( bha ) , -tocopherol , and linoleic acid were purchased from sigma - aldrich , inc . all other chemicals , namely ammonium thiocyanate , ferric chloride , edta , tween-40 , and sodium hydroxide were of analytical grade . cuttlefish ( s. officinalis ) , in the size range of 810 cuttlefish / kg , was purchased from the fish market of sfax city , tunisia . the samples were packed in polyethylene bags , placed in ice with a sample / ice ratio of approximately 1 : 3 ( w / w ) , and transported to the research laboratory within 30 min . the mantle was cleaned , deskinned , and eviscerated and then stored in sealed plastic bags at 80c until used . the crude enzyme preparation from b. licheniformis nh1 and alcalase 2.4 l obtained from novo nordisk ( bagsverd , denmark ) was used for the production of protein hydrolysates . protease activity was determined according to the method of kembhavi et al . using casein as a substrate . one unit of protease activity was defined as the amount of enzyme required to liberate 1 g tyrosine per minute under the experimental conditions used . cuttlefish ( s. officinalis ) muscle ( 500 g ) , in 1000 ml distilled water , was first minced using a grinder ( moulinex charlotte hv3 , france ) and then cooked at 90c for 20 min to inactivate endogenous enzymes . the cooked muscle sample was then homogenized in a moulinex blender for about 2 min and hydrolyzed with enzymes under optimal conditions : the crude enzyme preparation from b. licheniformis nh1 ( ph 10.0 and 50c ) and alcalase 2.4 l ( ph 8.0 and 50c ) . the enzyme was added to the reaction at the same enzyme / substrate ratio ( e / s = 3 u / mg ) to compare hydrolytic efficiencies . during the reaction , the ph of the mixture was maintained constant by continuous addition of 4 m naoh solution . after the required digestion time , the reaction was stopped by heating the solution at 80c during 20 min to inactivate the enzyme . the cuttlefish muscle protein hydrolysates were then centrifuged at 5000 g for 20 min to separate insoluble and soluble fractions . finally , the soluble phase was freeze dried using freeze dryer ( bioblock scientific christ alpha 1 - 2 , illkrich - cedex , france ) and stored at 20c for further use . the degree of hydrolysis ( dh ) , defined as the percent ratio of the number of peptide bonds broken ( h ) to the total number of peptide bonds in the studied substrate ( htot ) , was calculated from the amount of base ( naoh ) added to keep the ph constant during the hydrolysis as given below : ( 1)dh ( % ) = hhtot100=bnbmp11htot100 , where b is the amount of naoh consumed ( ml ) to keep the ph constant during the reaction , nb is the normality of the base , mp is the mass ( g ) of protein ( n 6.25 ) , and is the average degree of dissociation of the -nh2 groups released during hydrolysis expressed as : ( 2)=10phpk1 + 10phpk , where ph and pk are the values at which the proteolysis was conducted . the total number of peptide bonds ( htot ) in a fish protein concentrate was assumed to be 8.6 meq / g . moisture and ash content were determined according to the aoac standard methods 930.15 and 942.05 , respectively . total nitrogen content of the substrate and selected hydrolysate products was determined by using the kjeldahl method . the protein and fat contents were expressed on a dry weight basis . for analysis of amino acids , the dry samples were dissolved in distilled water at 1 mg / ml , and 50 l of each sample were dried and hydrolysed in vacuum - sealed glass tube at 110c for 24 h in the presence of constant boiling 6 n hcl containing 1% ( w / v ) phenol and using norleucine as internal standard . after hydrolysis , samples were again vacuum dried , dissolved in application buffer and injected into a beckman 6300 amino acid analyzer ( beckman instruments inc . , fullerton , calif , usa ) . solubility of cuttlefish protein hydrolysates was carried out according to tsumura et al . with slight modifications . briefly , 200 mg of freeze - dried hydrolysates of cuttlefish protein were suspended in 20 ml deionized distilled water , and the ph of the mixture was adjusted to different values from 2.0 to 11.0 using either 2 n hcl or 2 n naoh solutions . the mixtures were stirred for 10 min at room temperature ( 25 1c ) and then centrifuged at 8000 g for 10 min . after appropriate dilution , the nitrogen content in the supernatant was determined by biuret method . the nitrogen solubility of the sample , defined as the amount of soluble nitrogen from the total nitrogen , was calculated as follows : ( 3)nitrogen solubility ( % ) = supernatant nitrogen concentrationsample nitrogen concentration100 . inhibition activity of in vitro lipid peroxidation of cuttlefish protein hydrolysates was determined by assessing their ability to inhibit oxidation of linoleic acid in an emulsified model system . briefly , freeze - dried hydrolysates of cuttlefish protein ( 5 mg ) were dissolved in 2.5 ml of 50 mm phosphate buffer ( ph 7.0 ) and added to a 2.5 ml of 50 mm linoleic acid in ethanol ( 95% ) . the mixture was incubated in a 10 ml tube with silicon rubber caps at 45c for 12 days in a dark , and the degree of oxidation was evaluated by measuring the ferric thiocyanate values according to the method of mitsuda et al . . aliquot ( 0.1 ml ) of reaction mixture was mixed with 4.7 ml of 75% ethanol followed by the addition of 0.1 ml of 30% ammonium thiocyanate and 0.1 ml of 20 mm ferrous chloride solution in 3.5% hcl . after stirring for 3 min , the degree of colour development , which represents the linoleic acid oxidation , was measured at 500 nm . the antioxidative capacity of the inhibition of peroxide formation in linoleic acid system was expressed as follows : ( 4)inhibition ( % ) = [ 1a500 of samplea500 of control]100 , -tocopherol , a natural antioxidant agent , and bha , a synthetic antioxidant agent , were used as reference , and distilled water as control . dpph radical - scavenging activity was measured , using the method described by bersuder et al . . a 500 l test sample was mixed with 500 l of 99.5% ethanol and 125 l of 99.5% ethanol containing 0.02% dpph . this mixture was shaken then kept in a dark at room temperature for 60 min before measuring absorbance at 517 nm . dpph radical - scavenging activity was calculated according to the following equation : ( 5)dpph radical - scavenging activity ( % ) = [ 1a517 of samplea517 of control]100 . the control was conducted in the same manner except that distilled water was used instead of sample . the chelating activity on fe was determined , using the method of decker and welch . the mixture was then reacted with 0.1 ml of 2 mm fecl2 and 0.2 ml of 5 mm 3-(2-pyridyl)-5,6-bis(4-phenyl - sulfonic acid)-1,2,4-triazine ( ferrozine ) for 20 min at room temperature . the control was prepared in the same manner except that distilled water was used instead of the sample . was then calculated as follows : ( 6)chelating activity ( % ) = [ 1a562 of samplea562 of control]100 . antiautooxidant activity was assayed using the -carotene bleaching method . in brief , 0.5 mg -carotene in 1 ml chloroform was mixed with 25 l of linoleic acid and 200 l of tween-40 . the chloroform was evaporated under vacuum at 45c ; then 100 ml distilled water was added , and the resulting mixture was vigorously stirred . an aliquot ( 2.5 ml ) of the -carotene - linoleic acid emulsion was transferred to tubes containing 0.5 ml of each sample . the tubes were immediately placed in water bath and incubated at 50c for 2 h. thereafter , the absorbance of each sample was measured at 470 nm . a control consisted of 0.5 ml of distilled water instead of the sample solution . bha was used as positive standard : ( 7)antioxidant activity ( % ) = [ 1(abssample0abssample120)(abscontrol0abscontrol120)]100 . one - way analysis of variance ( anova ) was used , and mean comparison was performed by duncan 's multiple range test . 5.1 , professional edition ( manugistics corp . , rockville , md , usa ) . differences were considered significant at p < 0.05 . it has been demonstrated that biological activities of proteins can be increased through hydrolysis with certain enzymes , and some peptides or fractions possess stronger activity than others . furthermore , the specificity of the enzyme used for the proteolysis , the conditions used during hydrolysis and the dh greatly influenced the molecular weight and amino acid composition of protein hydrolysates , and thus their biological activities . the hydrolysis of the cuttlefish proteins with nh1 proteases or alcalase was characterized by a high rate of hydrolysis for the first 1 h ( figure 1 ) . the rate of enzymatic hydrolysis was subsequently decreased , and then the enzymatic reaction reached the steady - state phase when no apparent hydrolysis took place . the shape of hydrolysis curves is similar to those previously published for many protein substrates such as fish , whey , and wheat gluten . the decrease in the reaction rate could be explained by a decrease in the concentration of peptide bonds available for hydrolysis , enzyme deactivation , and/or the inhibition of the enzyme by the products formed at high degree of hydrolysis . these products act as effective substrate competitors to the undigested or partially digested fish proteins . with the same e / s ratio , nh1 proteases showed higher dh values for cuttlefish protein hydrolysis than alcalase beyond 90 min hydrolysis period . the higher ( p < 0.05 ) level of dh by nh1 proteases treatment may be due to the fact that nh1 crude enzyme contains multiple proteases and , therefore , is a more efficient enzyme choice than alcalase for preparing cuttlefish protein hydrolysates . therefore , the susceptibility , to hydrolysis , of cuttlefish muscle proteins depends on the type of enzyme used . proximate composition of soluble fractions of freeze - dried cphs compared to that of undigested cuttlefish proteins is shown in table 1 . the proximate composition of the undigested cuttlefish muscle proteins ( ucmp ) showed that it had high protein content ( 79.15 0.48% ) . the ash and lipid contents of ucmp were 6.08 0.71% and 5.19 0.24% , respectively . alcalase and nh1 cphs powders had a white to light yellow color appearance with almost no fishy odor and taste . after 4 h of hydrolysis , alcalase - cph had the least protein content ( 75.36 0.68% ) . however , nh1-cph showed a higher protein content ( 80.11 0.75% ) . during hydrolysis , proteins were solubilised , and the insoluble nonprotein matter was removed , resulting in the high protein content in the resulting hydrolysate . the obtained results are similar to those of other studies on fish protein hydrolysates that have ranged protein from 63.4 to 90.8% [ 13 , 37 ] . generally , alkaline proteases exhibit a greater capability to solubilize fish protein compared to neutral and acidic proteases , with exception of pepsin . interestingly , both dried cuttlefish protein hydrolysates had low lipid content values 0.68 0.02% and 0.91 0.05% , in alcalase - cph and nh1-cph , respectively . the retention of a high amount of fat in the final products may limit the use of this ingredient in food applications , because fish protein hydrolysates with high lipid content can have an undesirable taste and darken owing to changes in the lipids . according to spinelli et al . the ash content was 10.12% and 12.44% in alcalase - cph , and nh1-cph respectively , representing the salt formed during ph adjustment using alkaline solution ( naoh , 4 m ) . these results are similar to those of other published studies on fish protein hydrolysates [ 39 , 43 ] . functional properties influence the usefulness of an ingredient in food and govern the physical behavior during preparation , processing , and storage . all cphs presented typical bell - shaped solubility curves with minimum solubility at ph 4 , which may correspond to the isoelectric point of protein hydrolysates , and high solubility at acidic and alkaline ph . the solubilities of cphs were quite low at ph 4 , whereas solubilities above 78% were noticeable at other phs tested . undigested cuttlefish muscle protein ( ucmp ) was less soluble than the hydrolysates , having a solubility of 7.86 0.026% and 15.86 0.056% at ph 4.0 and 9.0 , respectively ( data not shown ) . as shown in figure 2 , the solubility of alcalase - cph ( dh = 12.5% ) and nh1-cph ( dh = 15% ) reached about 93.8 0.22 and 96.33 0.7% , respectively , significantly higher ( p < 0.05 ) than that of the ucmp ( 12.05 0.017% ) . the obtained results are in line with those of klompong et al . and gbogouri et al . who reported that hydrolysates of yellow stripe trevally meat protein and salmon byproduct had an excellent solubility at high degrees of hydrolysis . from these results , we can deduce that the solubility increases with the protein fraction with lower molecular mass at higher degrees of hydrolysis . the smaller peptides are expected to have proportionally more polar residues , with the ability to form hydrogen bonds with water and increase solubility . in addition , the lowest solubility of cphs observed at ph 4.0 could be attributed to both net charge of peptides , which increase as ph moves away from pi , and surface hydrophobicity , that promotes the aggregation via hydrophobic interaction . the ph affects the charge on the weakly acidic and basic side chain groups , and hydrolysates generally show low solubility at their isoelectric points . in vitro lipid peroxidation inhibition activities of cphs were determined by assessing their ability to inhibit oxidation of linoleic acid in an emulsified model system . as shown in table 2 , all hydrolysates with different dh could act as significant retarders ( p < 0.05 ) of lipid peroxidation . for the tow protease preparations used for hydrolysis reaction of cuttlefish muscle protein , the effect of inhibiting lipid oxidation increased initially and peaked on 4.0 h of hydrolysis . according to dong et al . , the effect of inhibiting lipid oxidation of alcalase - hydrolyzed carp protein increased initially and peaked on 1.5 h of hydrolysis , followed by a slight decline during the 6 h of hydrolysis . wu et al . found that the antioxidant activity of hydrolysates derived from mackerel protein reached a maximum after 10 h of hydrolysis and then declined slightly during the 25 h of hydrolysis . the comparative study between nh1-cph ( dh = 15% ) and alcalase - cph ( dh = 12.5% ) and commercial antioxidants ( -tocopherol and bha ) on the inhibition of lipid peroxidation were conducted and illustrated in figure 3 . however , both hydrolysates have moderate protective effect on lipid peroxidation in comparison with a synthetic antioxidant bha . generally , the lack of a direct relationship between antioxidant activity and dh suggested that the specific composition ( e.g. , type of peptides , ratio of different freed amino acids ) was an important factor as well . in addition , kong and xiong reported that if the hydrolysis of zein protein with alcalase became too extensive ( time of hydrolysis > 4 h ) , the hydrolysate could reduce the peptide 's ability to act as a physical barrier to prevent oxidants from reaching the lipid fraction in the liposome . when dpph radicals encounter a proton - donating substrate such as an antioxidant , the radicals would be scavenged , and the absorbance is reduced . the decrease in absorbance thus , the dpph radicals were widely used to investigate the scavenging activity of some natural compounds . the dpph radical scavenging activity of cphs increased with increasing dh . at all designated dh , nh1-cph showed higher activity than did alcalase - cph ( p < 0.05 ) . for nh1 proteases , when dh was increased from 3.6% to 15% , the dpph radical scavenging activity markedly increased from 25.0% to 71.5% . the result suggested that the peptides in different hydrolysates might be different in terms of chain length and amino acid sequence , which contributed to varying capabilities of scavenging dpph radicals . the increase in dpph radical scavenging activity of both protein hydrolysates was in agreement with thiansilakul et al . who reported the increase in dpph radical scavenging activity as the dh of the hydrolysate from round scad muscle protein prepared using flavourzyme and alcalase increased however , klompong et al . found that dpph radical scavenging activity of protein hydrolysate prepared from the muscle of yellow stripe trevally using flavourzyme and alcalase decreased when dh increased . invert correlation between dh and dpph radical scavenging activity was obtained for protein hydrolysates prepared from alkaline - aided channel catfish protein isolates using protamex . reported that loach protein hydrolysate showed the greater dpph radical scavenging activity when dh increased . the scavenging effect of all cphs on dpph radical scavenging was concentration dependent ( figure 4 ) . the result clearly indicated that hydrolysate produced by nh proteases exhibited the highest radical scavenging activity ( 75.0 2.26% at 3 mg / ml ) . however , the hydrolysate showed a lower radical - scavenging activity than bha ( 89.87 1.5% ) at the same concentration . hydrolysate obtained by treatment with nh1 proteases showed an active radical scavenger with ic50 about 0.68 mg / ml 0.017 than alcalase protein hydrolysate ( ic50 = 0.99 mg / ml 0.024 ) . dpph is a stable free radical and can be scavenged with a proton - donating substance , such as an antioxidant . therefore , protein hydrolysates from cuttlefish muscle more likely contained peptides acting as hydrogen donors , thereby scavenging free radicals by converting them into more stable products . ferrous chelating activities of nh1-cph and alcalase - cph , determined at a sample concentration of 2.0 mg / ml , are presented in table 4 . nh1-cph showed a higher chelating activity ( p < 0.05 ) than alcalase - cph at any designated dh . ferrous ion ( fe ) is the most powerful prooxidant among metal ions , leading to the initiation and acceleration of lipid oxidation by interaction with hydrogen peroxide in a fenton reaction to produce the reactive oxygen species , hydroxyl free radical ( oh ) . therefore , chelation of metal ions by peptides in hydrolysates could retard the oxidative reaction . the result indicated that a higher dh rendered nh1-cph and alcalase - cph with higher metal chelating activities . the shorter chain of peptides might lose their ability to form the complex with fe . for both crude protease preparations used , nh1-cph ( dh = 15% ) and alcalase - cph ( dh = 12.5% ) , obtained after 4 h of hydrolysis , exhibited a higher ferrous - chelating abilities 65.0 1.77% and 55.6 1.45% , respectively . peptides in nh1-cph and alcalase - cph could effectively chelate the fe , leading to the retardation of initiation stage . the result indicated that the excessive hydrolysis of muscle protein resulted in the enhanced ferrous chelating activity , compared with the limited hydrolysis . the higher chelating activities of both hydrolysates were coincidental with the higher dpph and in vitro lipid peroxidation inhibition activity , as the dh increased . fe chelating activity of round scad protein hydrolysate prepared using alcalase showed the increase in chelating activity with increasing dh , but those treated with flavourzyme showed no difference in activity at all dh tested . with the same enzymes used , chelating activity of protein hydrolysate prepared from the muscle of yellow stripe trevally increased with increasing dh . higher ferrous chelating activity was reported for hydrolysate of silver carp using alcalase and flavourzyme when dh increased . apart from fe , other transition metals , such as cu and co , can affect the rate of lipid oxidation and decomposition of hydroperoxide . theodore et al . reported that cu chelating activity of catfish protein hydrolysate increased with increasing dh . some proteins and peptides can chelate metal ions like fe due to the presence of carboxyl and amino groups in the side chains of acidic and basic amino acids . alcalase is endopeptidase capable of hydrolyzing proteins with broad specificity for peptide bonds and is prefered for the uncharged residue , whereas nh1 proteases is a mixture of multiple proteases , which can produce both amino acids and peptides . hydrolysates showing different antioxidative activities might be attributed to the differences in the exposed side chains of peptides as governed by the specificity of different proteases towards peptide bonds in the proteins . peptides with various sizes and compositions had different capacities of scavenging or quenching free radicals [ 10 , 11 ] . nh1 proteases and alcalase more likely cleaved the peptide bonds in cuttlefish muscle at different positions , resulting in the different products with varying antioxidative activities . the ferrous chelating activity , at different concentration , was also studied with nh1-cph and alcalase - cph having , respectively , a dh of 15% and 12.5% , and compared with that of the edta ( figure 5 ) . as shown in figure 5 , both activities increased with increasing hydrolysate concentration , and reached a maximum activity with 2 mg / ml , and further increase in hydrolysate concentration did not affect the activity . the ic50 values were about 0.94 0.24 mg / ml and 1.25 0.68 mg / ml , for nh1-cph , and alcalase - cph respectively . however , both hydrolysates showed a lower metal chelating activity than did edta at all concentrations tested . for example , at 2 mg / ml the metal chelating activities of nh1-cph , alcalase - cph , and edta were 74.0% 1.55 , 61.2% 1.34 , and 98% 1.02 , respectively . the antioxidative activity of cuttlefish protein hydrolysates , nh1-cph , and alcalase - cph were studied in -carotene linoleic acid oxidation model system as presented in table 5 . when the oxidation of linoleic acid occurs , free radicals formed are able to attack the highly unsaturated -carotene molecules . as a result , -carotene is oxidized , leading to the losses in chromophore and characteristic orange colour of -carotene . the presence of antioxidant in linoleic acid emulsion system hinders -carotene bleaching , due to the chain - breaking inhibition of lipid peroxidation by neutralizing the linoleic free radical formed . as shown in table 5 , nh1-cph and alcalase - cph inhibited significantly ( p < 0.05 ) the oxidation of -carotene to different degrees . during the first hour of hydrolysis , alcalase - cph achieved rapidly a higher ability to prevent the bleaching of -carotene ( 61.5 1.66% ) than nh1-cph ( 56.5 2.31% ) . when hydrolysis reached a final dh , both hydrolysates showed a similar antioxidant activity , approximately 70% . the antioxidant activity of nh1-cph ( dh = 15% ) and alcalase - cph ( dh = 12.5% ) at different concentrations was also evaluated ( figure 6 ) . as can be seen in figure 6 , the antioxidant activity of both cuttlefish protein hydrolysates increased with increasing sample concentration . the nh1 protein hydrolysate showed the highest ability to prevent bleaching of -carotene with 81.11% inhibition at 3 mg / ml . thereafter , the lower -carotene bleaching in the systems containing these hydrolysates was observed , compared with the system added with bha . the ability of hydrolysates to prevent the bleaching of -carotene was more likely governed by their amphiphilic properties of amino acids compositions . localization / orientation at the interface of hydrophobic part of peptides to oil phase and of hydrophilic portion to aqueous phase at the interface is the major principle for emulsion stabilization . the result suggested that protein hydrolysates tested contained peptides with both hydrophilic and hydrophobic portions , in which the peptide segments with hydrophobicity in nature could adsorb at the oil droplet , while the hydrophilic domains preferably suspended in the aqueous phase . the localization of peptides with antioxidative activity at the oil - water interface could favor their antioxidative activity for oil droplet . as a result both hydrolysates contained histidine and arginine as the major amino acids and were also rich in glutamic acid / glutamine , leucine , lysine , and serine . however , both hydrolysates contained low level of cysteine and proline . from the results , both hydrolysates contained a low level of proline up to 0.29% and 0.33% . based on total amino acids , essential amino acids made up 48.85% and 50.04% of alcalase - cph and nh1-cph , respectively . therefore , they could serve as the excellent source of useful nutrients . generally , the differences in amino acid composition between both hydrolysates depended on the existing differences in enzyme specificity and hydrolysis conditions . as presented in table 6 , the total content of hydrophobic amino acids of cuttlefish protein hydrolysates obtained at dh of 12.5% and 15% with alcalase and nh1 proteases was higher , which accounted for 32.36% and 33.64% of the total amino acids , respectively . amino acids have been known to exhibit antioxidant activity ; tryptophan and histidine showed high antioxidative activity in comparison with methionine , cysteine , glycine , and alanine . antioxidative activity of histidine or a histidine containing peptide may be attributed to the chelating and lipid radical - trapping ability of the imidazole ring , whereas the tyrosine residue in the peptide may act as a potent hydrogen donor . generally , aromatic amino acids are considered to be effective radical scavengers , because they can donate protons easily to electron - deficient radicals . at the same time , their antioxidative stability can remain via resonance structures . from the results , cuttlefish protein hydrolysate had a high nutritional value , based on its amino acid profile . the objective of this work was to investigate biochemical properties and the potential antioxidant effect of cuttlefish muscle protein during hydrolysis . the cuttlefish protein hydrolysed with alkaline proteases from b. licheniformis nh1 ( nh1-cph ) and alcalase ( alcalase - cph ) resulted products with an excellent solubility over a wide ph range . moreover , nh1-cph and alcalase - cph exhibited high antioxidant activity , and the highest activities were obtained with a dh of 15% and 12.5% , respectively . although both hydrolysates were less effective than positive controls like bha , fish hydrolysates in general are considered safe products , and they are not subjected to restricted use in foods . therefore , cuttlefish muscle protein hydrolysate can be used in food systems as a natural additive possessing antioxidative properties . further works should be done to isolate and identify the specific peptides in cuttlefish protein hydrolysates that are responsible for the overall antioxidative capability .

antioxidative activities and biochemical properties of protein hydrolysates prepared from cuttlefish ( sepia officinalis ) using alcalase 2.4 l and bacillus licheniformis nh1 proteases with different degrees of hydrolysis ( dh ) were determined . for the biochemical properties , hydrolysis by both enzymes increased protein solubility to above 75% over a wide ph range . the antioxidant activities of cuttlefish protein hydrolysates ( cphs ) increase with increasing dh . in addition , all cphs exhibited antioxidative activity in a concentration - dependent manner . nh1-cphs generally showed greater antioxidative activity than alcalase protein hydrolysates ( p < 0.05 ) as indicated by the higher 1,1-diphenyl-1-picryhydrazyl ( dpph ) radical scavenging activity and ferrous chelating activity . both alcalase and nh1 protein hydrolysates were able to retard lipid peroxidation and -carotene - linoleic acid oxidation . alcalase - cph ( dh = 12.5% ) and nh1-cph ( dh = 15% ) contained 75.36% and 80.11% protein , respectively , with histidine and arginine as the major amino acids , followed by glutamic acid / glutamine , serine , lysine , and leucine . in addition , cphs have a high percentage of essential amino acids made up 48.85% and 50.04% . cuttlefish muscle protein hydrolysates had a high nutritional value and could be used as supplement to poorly balanced dietary proteins .

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

lipid oxidation is one of the major deteriorative processes in many types of foods , leading to the changes in food quality and nutritional value . recently , protein hydrolysates from several fish species , such as silver carp ( hypophthalmichthys molitrix ) , brownstripe red snapper ( lutjanus vitta ) , sardinelle ( sardinella aurita ) , smooth hound ( mustelus mustelus ) , oyster ( crassostrea gigas ) , yellow stripe trevally ( selaroides leptolepis ) , round scad ( decapterus maruadsi ) , yellowfin sole ( limanda aspera ) , herring ( clupea harengus ) , and mackerel ( scomber austraasicus ) , have been reported to possess antioxidative activities . the operational conditions employed in the processing of protein isolates , the type of protease , and the degree of hydrolysis affect the antioxidant activity . protein hydrolysate from alaska pollack frame prepared by mackerel intestine crude enzyme exhibited antioxidative activity in a linoleic acid model system . levels and compositions of free amino acids and peptides were reported to determine the antioxidant activities of protein hydrolysates . recently , protein hydrolysates from cuttlefish ( sepia officinalis ) enriched in angiotensin i - converting enzyme inhibitory peptides have been produced successfully using alcalase and bacillus licheniformis nh1 proteases . nevertheless , a little information regarding the characteristic and antioxidative activity of hydrolysates prepared using both enzymes has been reported . the objective of this study was to investigate the antioxidative activity of protein hydrolysates from cuttlefish muscle prepared using alcalase and bacillus licheniformis nh1 proteases in a model system and the effect of concentration on their activities . 1,1-diphenyl-2-picrylhydrazyl ( dpph ) , 3-(2-pyridyl)-5,6-bis(4-phenyl - sulphonic acid)-1,2,4-triazine ( ferrozine ) , butylated hydroxyanisole ( bha ) , -tocopherol , and linoleic acid were purchased from sigma - aldrich , inc . cuttlefish ( s. officinalis ) , in the size range of 810 cuttlefish / kg , was purchased from the fish market of sfax city , tunisia . the crude enzyme preparation from b. licheniformis nh1 and alcalase 2.4 l obtained from novo nordisk ( bagsverd , denmark ) was used for the production of protein hydrolysates . cuttlefish ( s. officinalis ) muscle ( 500 g ) , in 1000 ml distilled water , was first minced using a grinder ( moulinex charlotte hv3 , france ) and then cooked at 90c for 20 min to inactivate endogenous enzymes . the cooked muscle sample was then homogenized in a moulinex blender for about 2 min and hydrolyzed with enzymes under optimal conditions : the crude enzyme preparation from b. licheniformis nh1 ( ph 10.0 and 50c ) and alcalase 2.4 l ( ph 8.0 and 50c ) . the cuttlefish muscle protein hydrolysates were then centrifuged at 5000 g for 20 min to separate insoluble and soluble fractions . the degree of hydrolysis ( dh ) , defined as the percent ratio of the number of peptide bonds broken ( h ) to the total number of peptide bonds in the studied substrate ( htot ) , was calculated from the amount of base ( naoh ) added to keep the ph constant during the hydrolysis as given below : ( 1)dh ( % ) = hhtot100=bnbmp11htot100 , where b is the amount of naoh consumed ( ml ) to keep the ph constant during the reaction , nb is the normality of the base , mp is the mass ( g ) of protein ( n 6.25 ) , and is the average degree of dissociation of the -nh2 groups released during hydrolysis expressed as : ( 2)=10phpk1 + 10phpk , where ph and pk are the values at which the proteolysis was conducted . moisture and ash content were determined according to the aoac standard methods 930.15 and 942.05 , respectively . for analysis of amino acids , the dry samples were dissolved in distilled water at 1 mg / ml , and 50 l of each sample were dried and hydrolysed in vacuum - sealed glass tube at 110c for 24 h in the presence of constant boiling 6 n hcl containing 1% ( w / v ) phenol and using norleucine as internal standard . solubility of cuttlefish protein hydrolysates was carried out according to tsumura et al . briefly , 200 mg of freeze - dried hydrolysates of cuttlefish protein were suspended in 20 ml deionized distilled water , and the ph of the mixture was adjusted to different values from 2.0 to 11.0 using either 2 n hcl or 2 n naoh solutions . inhibition activity of in vitro lipid peroxidation of cuttlefish protein hydrolysates was determined by assessing their ability to inhibit oxidation of linoleic acid in an emulsified model system . briefly , freeze - dried hydrolysates of cuttlefish protein ( 5 mg ) were dissolved in 2.5 ml of 50 mm phosphate buffer ( ph 7.0 ) and added to a 2.5 ml of 50 mm linoleic acid in ethanol ( 95% ) . the antioxidative capacity of the inhibition of peroxide formation in linoleic acid system was expressed as follows : ( 4)inhibition ( % ) = [ 1a500 of samplea500 of control]100 , -tocopherol , a natural antioxidant agent , and bha , a synthetic antioxidant agent , were used as reference , and distilled water as control . an aliquot ( 2.5 ml ) of the -carotene - linoleic acid emulsion was transferred to tubes containing 0.5 ml of each sample . differences were considered significant at p < 0.05 . it has been demonstrated that biological activities of proteins can be increased through hydrolysis with certain enzymes , and some peptides or fractions possess stronger activity than others . furthermore , the specificity of the enzyme used for the proteolysis , the conditions used during hydrolysis and the dh greatly influenced the molecular weight and amino acid composition of protein hydrolysates , and thus their biological activities . the hydrolysis of the cuttlefish proteins with nh1 proteases or alcalase was characterized by a high rate of hydrolysis for the first 1 h ( figure 1 ) . the shape of hydrolysis curves is similar to those previously published for many protein substrates such as fish , whey , and wheat gluten . the decrease in the reaction rate could be explained by a decrease in the concentration of peptide bonds available for hydrolysis , enzyme deactivation , and/or the inhibition of the enzyme by the products formed at high degree of hydrolysis . with the same e / s ratio , nh1 proteases showed higher dh values for cuttlefish protein hydrolysis than alcalase beyond 90 min hydrolysis period . the higher ( p < 0.05 ) level of dh by nh1 proteases treatment may be due to the fact that nh1 crude enzyme contains multiple proteases and , therefore , is a more efficient enzyme choice than alcalase for preparing cuttlefish protein hydrolysates . alcalase and nh1 cphs powders had a white to light yellow color appearance with almost no fishy odor and taste . after 4 h of hydrolysis , alcalase - cph had the least protein content ( 75.36 0.68% ) . interestingly , both dried cuttlefish protein hydrolysates had low lipid content values 0.68 0.02% and 0.91 0.05% , in alcalase - cph and nh1-cph , respectively . the retention of a high amount of fat in the final products may limit the use of this ingredient in food applications , because fish protein hydrolysates with high lipid content can have an undesirable taste and darken owing to changes in the lipids . the ash content was 10.12% and 12.44% in alcalase - cph , and nh1-cph respectively , representing the salt formed during ph adjustment using alkaline solution ( naoh , 4 m ) . all cphs presented typical bell - shaped solubility curves with minimum solubility at ph 4 , which may correspond to the isoelectric point of protein hydrolysates , and high solubility at acidic and alkaline ph . undigested cuttlefish muscle protein ( ucmp ) was less soluble than the hydrolysates , having a solubility of 7.86 0.026% and 15.86 0.056% at ph 4.0 and 9.0 , respectively ( data not shown ) . as shown in figure 2 , the solubility of alcalase - cph ( dh = 12.5% ) and nh1-cph ( dh = 15% ) reached about 93.8 0.22 and 96.33 0.7% , respectively , significantly higher ( p < 0.05 ) than that of the ucmp ( 12.05 0.017% ) . who reported that hydrolysates of yellow stripe trevally meat protein and salmon byproduct had an excellent solubility at high degrees of hydrolysis . in addition , the lowest solubility of cphs observed at ph 4.0 could be attributed to both net charge of peptides , which increase as ph moves away from pi , and surface hydrophobicity , that promotes the aggregation via hydrophobic interaction . in vitro lipid peroxidation inhibition activities of cphs were determined by assessing their ability to inhibit oxidation of linoleic acid in an emulsified model system . as shown in table 2 , all hydrolysates with different dh could act as significant retarders ( p < 0.05 ) of lipid peroxidation . for the tow protease preparations used for hydrolysis reaction of cuttlefish muscle protein , the effect of inhibiting lipid oxidation increased initially and peaked on 4.0 h of hydrolysis . , the effect of inhibiting lipid oxidation of alcalase - hydrolyzed carp protein increased initially and peaked on 1.5 h of hydrolysis , followed by a slight decline during the 6 h of hydrolysis . found that the antioxidant activity of hydrolysates derived from mackerel protein reached a maximum after 10 h of hydrolysis and then declined slightly during the 25 h of hydrolysis . the comparative study between nh1-cph ( dh = 15% ) and alcalase - cph ( dh = 12.5% ) and commercial antioxidants ( -tocopherol and bha ) on the inhibition of lipid peroxidation were conducted and illustrated in figure 3 . in addition , kong and xiong reported that if the hydrolysis of zein protein with alcalase became too extensive ( time of hydrolysis > 4 h ) , the hydrolysate could reduce the peptide 's ability to act as a physical barrier to prevent oxidants from reaching the lipid fraction in the liposome . the dpph radical scavenging activity of cphs increased with increasing dh . at all designated dh , nh1-cph showed higher activity than did alcalase - cph ( p < 0.05 ) . for nh1 proteases , when dh was increased from 3.6% to 15% , the dpph radical scavenging activity markedly increased from 25.0% to 71.5% . the increase in dpph radical scavenging activity of both protein hydrolysates was in agreement with thiansilakul et al . who reported the increase in dpph radical scavenging activity as the dh of the hydrolysate from round scad muscle protein prepared using flavourzyme and alcalase increased however , klompong et al . found that dpph radical scavenging activity of protein hydrolysate prepared from the muscle of yellow stripe trevally using flavourzyme and alcalase decreased when dh increased . invert correlation between dh and dpph radical scavenging activity was obtained for protein hydrolysates prepared from alkaline - aided channel catfish protein isolates using protamex . however , the hydrolysate showed a lower radical - scavenging activity than bha ( 89.87 1.5% ) at the same concentration . hydrolysate obtained by treatment with nh1 proteases showed an active radical scavenger with ic50 about 0.68 mg / ml 0.017 than alcalase protein hydrolysate ( ic50 = 0.99 mg / ml 0.024 ) . therefore , protein hydrolysates from cuttlefish muscle more likely contained peptides acting as hydrogen donors , thereby scavenging free radicals by converting them into more stable products . ferrous chelating activities of nh1-cph and alcalase - cph , determined at a sample concentration of 2.0 mg / ml , are presented in table 4 . nh1-cph showed a higher chelating activity ( p < 0.05 ) than alcalase - cph at any designated dh . for both crude protease preparations used , nh1-cph ( dh = 15% ) and alcalase - cph ( dh = 12.5% ) , obtained after 4 h of hydrolysis , exhibited a higher ferrous - chelating abilities 65.0 1.77% and 55.6 1.45% , respectively . the result indicated that the excessive hydrolysis of muscle protein resulted in the enhanced ferrous chelating activity , compared with the limited hydrolysis . the higher chelating activities of both hydrolysates were coincidental with the higher dpph and in vitro lipid peroxidation inhibition activity , as the dh increased . fe chelating activity of round scad protein hydrolysate prepared using alcalase showed the increase in chelating activity with increasing dh , but those treated with flavourzyme showed no difference in activity at all dh tested . with the same enzymes used , chelating activity of protein hydrolysate prepared from the muscle of yellow stripe trevally increased with increasing dh . higher ferrous chelating activity was reported for hydrolysate of silver carp using alcalase and flavourzyme when dh increased . reported that cu chelating activity of catfish protein hydrolysate increased with increasing dh . alcalase is endopeptidase capable of hydrolyzing proteins with broad specificity for peptide bonds and is prefered for the uncharged residue , whereas nh1 proteases is a mixture of multiple proteases , which can produce both amino acids and peptides . nh1 proteases and alcalase more likely cleaved the peptide bonds in cuttlefish muscle at different positions , resulting in the different products with varying antioxidative activities . the ferrous chelating activity , at different concentration , was also studied with nh1-cph and alcalase - cph having , respectively , a dh of 15% and 12.5% , and compared with that of the edta ( figure 5 ) . the ic50 values were about 0.94 0.24 mg / ml and 1.25 0.68 mg / ml , for nh1-cph , and alcalase - cph respectively . however , both hydrolysates showed a lower metal chelating activity than did edta at all concentrations tested . for example , at 2 mg / ml the metal chelating activities of nh1-cph , alcalase - cph , and edta were 74.0% 1.55 , 61.2% 1.34 , and 98% 1.02 , respectively . the antioxidative activity of cuttlefish protein hydrolysates , nh1-cph , and alcalase - cph were studied in -carotene linoleic acid oxidation model system as presented in table 5 . as shown in table 5 , nh1-cph and alcalase - cph inhibited significantly ( p < 0.05 ) the oxidation of -carotene to different degrees . during the first hour of hydrolysis , alcalase - cph achieved rapidly a higher ability to prevent the bleaching of -carotene ( 61.5 1.66% ) than nh1-cph ( 56.5 2.31% ) . the antioxidant activity of nh1-cph ( dh = 15% ) and alcalase - cph ( dh = 12.5% ) at different concentrations was also evaluated ( figure 6 ) . as can be seen in figure 6 , the antioxidant activity of both cuttlefish protein hydrolysates increased with increasing sample concentration . as a result both hydrolysates contained histidine and arginine as the major amino acids and were also rich in glutamic acid / glutamine , leucine , lysine , and serine . based on total amino acids , essential amino acids made up 48.85% and 50.04% of alcalase - cph and nh1-cph , respectively . as presented in table 6 , the total content of hydrophobic amino acids of cuttlefish protein hydrolysates obtained at dh of 12.5% and 15% with alcalase and nh1 proteases was higher , which accounted for 32.36% and 33.64% of the total amino acids , respectively . amino acids have been known to exhibit antioxidant activity ; tryptophan and histidine showed high antioxidative activity in comparison with methionine , cysteine , glycine , and alanine . from the results , cuttlefish protein hydrolysate had a high nutritional value , based on its amino acid profile . the objective of this work was to investigate biochemical properties and the potential antioxidant effect of cuttlefish muscle protein during hydrolysis . the cuttlefish protein hydrolysed with alkaline proteases from b. licheniformis nh1 ( nh1-cph ) and alcalase ( alcalase - cph ) resulted products with an excellent solubility over a wide ph range . moreover , nh1-cph and alcalase - cph exhibited high antioxidant activity , and the highest activities were obtained with a dh of 15% and 12.5% , respectively . therefore , cuttlefish muscle protein hydrolysate can be used in food systems as a natural additive possessing antioxidative properties . further works should be done to isolate and identify the specific peptides in cuttlefish protein hydrolysates that are responsible for the overall antioxidative capability .

ocular drug delivery is challenging in terms of achieving optimum drug concentration due to unique protective mechanisms of the eye . development of a drug delivery system for attaining therapeutic concentration at the target site requires a comprehensive understanding of static and dynamic barriers of the eye . the eye has two broadly defined segments , ( a ) anterior segment , and ( b ) posterior segment . anterior segment is the front one - third of the eye that includes the optical structure in front of vitreous humor : cornea , pupil , aqueous humor , iris , lens and ciliary body . posterior segment is the back two - thirds of the eye that mainly includes sclera , choroid , retina , vitreous humor , macula , and optical nerve . the common routes of drug administration for the treatment of eye disorders are topical , systemic , periocular , and intravitreal . topical administration is the most preferred route because of highest patient compliance and least invasive nature . upon topical instillation , absorption of drugs takes place either through corneal route ( cornea , aqueous humor , intraocular tissues ) or noncorneal route ( conjunctiva , sclera , choroid / rpe ) . the cornea can be mainly divided into the epithelium , stroma and endothelium , where each layer offers a different polarity and a potential rate - limiting structure for drug permeation . the non - corneal route involves absorption across the sclera and conjunctiva into the intraocular tissues . however , a small fraction of the topically applied drugs , generally less than 5% , reaches the intraocular tissues . factors responsible for poor ocular bioavailability following topical instillation are precorneal drainage and lipoidal nature of the corneal epithelium . in addition , a major fraction of drug reaches the systemic circulation through conjunctival vessels and nasolacrimal duct , which leads to severe adverse effects . consequently , topical route has met with limited success in attaining therapeutic drug concentrations in the posterior segment . systemic administration can provide therapeutic levels in the posterior segment , but administration of high doses is necessary , which often leads to severe side effects . blood - aqueous barrier and blood - retinal barrier are the two major barriers for anterior segment and posterior segment ocular drug delivery , respectively , after systemic administration . the tight junctional complexes located in the two discrete cell layers , the endothelium of the iris / ciliary blood vessels , and the nonpigmented ciliary epithelium offer blood - aqueous barrier which prevents the entry of solutes into the aqueous humor . blood retinal barrier is composed of two types of cells , that is , retinal capillary endothelial cells and retinal pigment epithelium ( rpe ) cells which prevents the entry of solute into the retina . intravitreal administration requires frequent administration which may lead to high susceptibility for vitreous hemorrhage , retinal detachment and endophthalmitis . these side effects can be minimized by developing delivery systems which provide controlled and targeted drug delivery for prolonged periods [ 13 ] . conventional ophthalmic formulations such as solutions and suspensions exhibit poor bioavailability . over the last decade , numerous drug delivery systems have been explored to overcome the limitation of conventional dosage forms . novel formulations such as nanoparticles , liposomes , dendrimers , and niosomes were developed to enhance drug bioavailability and to minimize adverse effects [ 4 , 5 ] . among them liposomal formulations were widely explored in the last decade for drug delivery applications . in 1965 liposomes are usually within the size range of 10 nm to 1 m or greater . these vesicular systems are composed of aqueous core enclosed by phospholipid bilayers of natural or synthetic origin . liposomes are structurally classified on the basis of lipid bilayers such as small unilamellar vesicles ( suvs ) or multilamellar vesicles ( mlvs ) . furthermore , on the basis of size , liposomes are classified into small unilamellar vesicles ( suvs ) , giant unilamellar vesicles ( guvs ) , and large unilamellar vesicles ( luvs ) ( figure 1 ) . unilamellar vesicles are composed of single layer of lipid such as lecithin or phosphatidylglycerol encapsulating aqueous interior core . mlvs are metastable energy configuration having different facets depending upon the polydispersity of the liposomal formulation . drug loading capacity of liposomes depends on many factors such as size of liposomes , types of lipid utilized for preparation , and physicochemical properties of therapeutic agent itself . for example , being the smallest in size entrapping efficiency for suvs is poor in comparison to mlvs . hydrophilic drugs are entrapped in the aqueous layer , while hydrophobic drugs are stuck in the lipid bilayers . loading capacity of ionic molecules can be further improved by using cationic or anionic lipids for the preparation of liposomes . majority of liposomal formulations utilize phosphatidylcholine ( pc ) and other constituents such as cholesterol and lipid - conjugated hydrophilic polymers as the main ingredients . stability of liposomes depends upon the various properties such as surface charge , size , surface hydration , and fluidity of lipid bilayers . neutral liposomes upon systemic administration evade the elimination by reticuloendothelial system ( res ) . however , these vesicles possess higher self - aggregation tendency . in contrast , negatively and positively charged liposomes exhibit lower aggregation tendency but undergo rapid clearance by res cells due to higher interaction with serum proteins . liposomes of size less than 100 nm generally exhibit significantly higher circulation time due to decrease in opsonization of liposomes with serum protein . in general , hydration of phospholipids results in the formation of mlvs , which can be processed into suvs with proper sonication . however , addition of aqueous solution of surfactant above the critical micelle concentration results in the formation of phospholipids micelles . after the dialysis of surfactant aggregation of micelles form luvs , critical micelle concentrations of amphiphiles which can form micelles are four to five orders of magnitude higher than the phospholipids which form liposomes . most commonly solvent evaporation method , reverse phase evaporation method and detergent dialysis method are employed . the encapsulated drug from liposome can be released either through passive diffusion , vesicle erosion , or vesicle retention . in passive diffusion , drug molecules tend to penetrate through the lipid layers of liposome to reach extra vesicular layer either by diffusion or convection mechanism . the rate of diffusion depends on the size , lipid composition , and the properties of the drug itself [ 1517 ] . unilamellar liposomes exhibit faster release rate than multilamellar ones because in multilayered liposome , drug diffusion occurs through a series of barriers ; hence , the drug release is delayed . phospholipase and high - density lipoprotein present in blood plasma can damage phospholipid layers of liposome and thus results in vesicle erosion and releases the encapsulated drug into the cell . liposome - cell interactions depend on several factors like size , surface charge , composition of liposomes , targeting ligand on the surface of liposome , and biological environment . liposomes can interact with cells by four different mechanisms : adsorption , fusion , lipid exchange and endocytosis ( receptor mediated ) . liposomes can be specifically or nonspecifically adsorbed onto the cell surface or can be fused with cell membranes , and release encapsulated drug inside the cell . during adsorption , liposomes can release encapsulated drug in front of cell membrane , and released drug can enter cell via micropinocytosis . negatively charged liposomes have been found to be more efficient than neutral liposomes for internalization into the cells by endocytosis process . liposomes bind to the receptor present in the invaginations of cellular membrane and are internalized into the cell by endocytotic pathway . after endocytosis , they can fuse with the endosomal membrane to form endosome which can be delivered to lysosomes . in lysosomes , the presence of peptidase and hydrolase degrades the liposomes and their content . to avoid this degradation and thus to increase cytoplasmic bioavailability , stimuli - responsive liposomes ( such as ph or temperature ) ph - sensitive liposomes can undergo fusion with endosomal membrane and release their content directly into cytosol . in some cases liposomes become destabilized inside the endosome and release their content , or they destabilize endosomal membrane resulting in leakage of encapsulated content into cytosol [ 19 , 20 ] . in this paper we have attempted to summarize the application of liposomes in the field of ophthalmic drug delivery attempted by numerous investigators over the last decade . liposomes have been investigated for ophthalmic drug delivery since it offers advantages as a carrier system . it can enhance the permeation of poorly absorbed drug molecules by binding to the corneal surface and improving residence time . in addition , liposomes can improve pharmacokinetic profile , enhance therapeutic effect , and reduce toxicity associated with higher dose . owing to their versatile nature , liposomes have been widely investigated for the treatment of both anterior and posterior segment eye disorders . current approaches for the anterior segment drug delivery are focused on improving corneal adhesion and permeation by incorporating various bioadhesive and penetration enhancing polymers . however , in the case of posterior segment disorders , improvement of intravitreal half - life and targeted drug delivery to the retina is necessary . currently verteporfin is being used clinically in photodynamic therapy for the treatment of subfoveal choroidal neovascularization ( cnv ) , ocular histoplasmosis , or pathological myopia effectively . verteporfin is a light - activated drug which is administered by intravenous infusion . in photodynamic therapy , after the drug is injected , a low - energy laser is applied to the retina through the contact lens in order to activate verteporfin that results in closure of the abnormal blood vessels . unfortunately , photodynamic therapy usually does not permanently close the abnormal vessels and choroidal neovessels reappear after several months . another liposomal photosensitizing agent , rostaporfin , was evaluated for the treatment of age - related macular degeneration . however , there are some issues to be addressed such as formulation , and storage of liposomes is very difficult , and they are known to cause long - term side effects . intravitreal administration of liposomes has resulted in vitreal condensation , vitreal bodies in the lower part of eye , and retinal abnormalities . therefore , all these factors should be taken into account while developing liposomal formulation for ophthalmic application [ 2125 ] . in 1981 , samolin et al . investigated the role of liposomes in ophthalmic drug delivery . since then several investigators proposed strategies to enhance absorption of drugs having poor physicochemical properties . studies performed by schaeffer and krohn suggested the role of charge and size in transcorneal permeation . investigators observed four - fold higher in vitro corneal flux from penicillin g - loaded suvs . they reported corneal permeation in the order of suv+ > mlv > suv > suv > mlv free drug . these studies explored the role of vesicle type on transcorneal permeation across the excised rabbit cornea . liposomal formulation of ta produced twofold increase in drug concentration in both the cornea and aqueous humor in the rabbit model . on the contrary , liposomal formulation of hydrophilic drug , that is , dihydrostreptomycin sulfate , did not improve the corneal permeation [ 37 , 38 ] . considering these findings , it was evident that both vesicle type and physicochemical property of drug significantly affects the transcorneal flux of the formulation . earlier investigation by fitzgerald et al . was significant in exploring the clearance of liposomes by gamma scintigraphy following topical administration in the rabbit model . these investigators reported , suvs with positive charge had improved the corneal retention by interacting with negatively charged corneal surface . since then , approaches based on positively charged liposomes were explored considerably . researchers also explored immunoliposomes , lectin functionalized liposomes , and positively charged lipid analogs . among these approaches however , lectin and lipid analog - based approaches are not explored considerably in the field of ophthalmic drug delivery . approach of utilizing chitosan in the formulation was reported to be advantageous in improving the precorneal residence time due to its mucoadhesive nature . degradation of chitosan into oligosaccharides is mediated through lysozymes , and degradation products are nontoxic in nature [ 40 , 41 ] . biodegradable nature is advantageous for selecting chitosan in the formulation of ocular drug delivery systems . topical administration of chitosan - coated liposomes ( chitosomes ) improves precorneal retention and also slows down drug metabolism at the precorneal epithelial surface . chitosan - based mucoadhesive liposomal formulation of cpx was prepared and evaluated by mehanna et al . reverse phase evaporation technique was utilized for the preparation of liposomes , which were further coated with chitosan of different molecular weights . the authors reported that liposomes coated with high molecular weight chitosan were smaller in size due to complete coverage of liposomal surface , which acted as a physical barrier to inhibit aggregation . in addition , authors determined lower encapsulation efficiency ( ee ) of 49.93% for coated liposomes in comparison to uncoated negative and neutral liposomes with 71.4% and 53.2% ee , respectively , due to electrostatic repulsion between chitosan and cationic drug . negatively charged liposomes were larger in diameter due to predominantly electrostatic attraction between the positively charged chitosan and negatively charged phospholipids . rheological studies revealed ideal pseudoelastic behavior of chitosomes and higher apparent viscosity than the liposome dispersion . the author suggested that pseudoelastic property of chitosome provides prolonged retention and stability of tear film . moreover , in vitro release studies with chitosomes exhibited slower drug release rate in comparison to free liposomes due to additional diffusion barrier for drug molecule . ex vivo corneal permeation studies across isolated rabbit cornea suggested that due to absorption enhancing nature of chitosan relative permeability of chitosomes was 1.74-fold higher than free drug . furthermore , in vitro antibacterial studies revealed that chitosomes exhibited enhanced antibacterial activity than the marketed aqueous solution against reference and clinically isolated strains of pseudomonas aeruginosa and staphylococcus aureus . authors suggested the electrostatic interaction of positively charged chitosan and negatively charged bacterial cell wall enhanced the antibacterial action of liposomal formulation . comparative single dose in vivo study performed on bacterial conjunctivitis rabbit model revealed that chitosomes inhibited the growth of pseudomonas aeruginosa for 24 h. it was reported that marketed product ( clioxan ) is comparatively less effective and requires frequent administration . however , other studies suggest the advantages of water - soluble low molecular weight chitosan as potential biopolymer for coating liposomes . application of lch was advantageous in eliminating the aggregation behavior of chitosan at physiological ph that had dramatically influenced in vivo performance of the liposomal formulation . investigator reported higher ex vivo corneal penetration across excised rabbit cornea in the case of lch - coated liposomes as shown in figure 2 . however , higher concentration of lch ( 0.25% and 0.5% w / w ) did not show significant change in particle size . researchers suggested that a loose coating layer is responsible for aggregation of vesicles which resulted in higher particle size in the case of 0.1% w / v lch . moreover , the drug release at 6 h was 38.9% in noncoated liposomes whereas only 25.4% drug release was observed in liposomes coated with 0.25% w / v chitosan solution . both nontreated and treated group did not demonstrate any abnormality of the corneal and conjunctival epithelial cells . in addition , no ocular irritation and inflammatory response was observed . in vitro precorneal retention studies in rabbits showed that the elimination of chitosan - coated liposomes was slower than non - coated liposomes . authors suggested that mucin film , which primarily covers the surface of cornea and conjunctiva , is composed of negatively charged glycoprotein . in addition , hydrogen bonding interactions of lch with the ocular surface also favors precorneal retention . however , previous studies with high molecular weight chitosan - coated liposomes did not improve the precorneal retention due to enhanced intramolecular interactions . histopathological analysis of the lch - coated liposomes in rabbits after long - term irritation test revealed that the formulation was biocompatible with the ocular tissues ( figure 3 ) . application of quaternized derivatives of chitosan that is , n - trimethyl chitosan chloride ( tmc ) , with significantly higher water solubility at physiological ph , was evaluated for surface modification of coenzyme q10-loaded liposomes . in addition , surface modification with cationic polymeric film reduced particle aggregation through stearic stabilization and improved precorneal retention than uncoated liposomes due to ionic interaction with negatively charged corneal surface . investigators reported almost 4.8-fold increase in precorneal residence time measured by gamma scintigraphy after administration of 25 l of formulation . histological analysis and draize test performed on rabbits revealed that tmc was biocompatible with corneal epithelium . moreover , higher molecular weight tmc exhibited better anticataract activity in sprague dawley rats . to take the dual advantage of chitosan - based nanoparticles and liposomes , diebold et al . as mentioned earlier , chitosan nanocarriers were employed in topical drug delivery because of its mucoadhesive nature , whereas liposomes can incorporate variety of drug molecules and improve ocular drug bioavailability [ 4547 ] . these nanosystems were formulated as eye drops , which possessed combined properties of both carriers and overcome the ocular mucosal barriers . these authors evaluated the nanosystems for toxicity on spontaneously immortalized epithelial cell line from normal human conjunctiva ( ioba - nhc ) . cells pre incubated with xtt ( 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2h - tetrazolium-5-carboxyalinide ) solution ( 1 mg / ml xtt in 100 ml of phenol red - free rpmi culture medium ) were exposed to different concentrations of chitosan nanoparticles and liposome - chitosan nanoparticles complexes . cytotoxicity was determined by measuring the production of yellow color due to cleavage of xtt by mitochondrial enzymes . cell viability after exposure of liposome - chitosan nanoparticle complexes was higher in comparison to chitosan nanoparticles alone . they also performed in vivo acute tolerance test by administrating the formulations topically to the female albino new zealand rabbits . the nanosystems did not show any evidence of toxicity to the both sham - controlled and treated eyes . also , in vivo experiments have shown that nanosystems can enter the conjunctival cells without causing histological alteration to the cornea , conjunctiva , and lid tissues in the rabbit model . in addition , the complexes did not release any inflammatory mediators in cornea , conjunctiva , and eyelids . vaccination approach can successfully overcome the limitations of antiviral agents in the treatment of hsv infections . administration by conventional parenteral route has several drawbacks such as high cost , need of highly trained personnel , and needle - stick injuries . cationic liposomes containing herpes simplex virus ( hsv ) antigens were proposed as potential carriers , in the form of a periocular vaccine , to protect animals against subsequent hsv-1 ocular challenges . two different peptides , namely , dtk1 and dtk2 ( dtks ) , having antiherpetic activity were synthesized . zhang et al . utilized cytochrome - c ( cyt - c ) loaded cationic liposomes for the treatment of selenite - induced cataract in rats . this study reported improvement in the entrapment efficiency ( ee ) with increasing phosphatidylcholine component , whereas ee was lowered by incorporating stearylamine . cyt - c loaded freeze - dried liposomes were stable for one year at 4c . furthermore , these liposomes exhibited remarkable efficacy ( 28% decrease in lens opacity ) in minimizing the cataract formation . liposomal encapsulation of cyt - c has significance , but the preparation method adapted by these authors was similar to previous investigations . in another study , fluconazole liposomal formulation was evaluated in the candidal keratitis model in rabbits . in this investigation , the purpose of developing liposomal formulation was to prolong the antifungal action by increasing the contact time . in the rabbits treated with fluconazole solution , 50% healing was observed in 3 weeks , whereas 86.4% healing was observed in rabbits treated with fluconazole encapsulated liposomes . authors attributed enhanced pharmacological activity to higher viscosity and lipid solubility of fluconazole - loaded liposomes . chronic ocular infectious diseases such as conjunctivitis , bacterial keratitis need high drug concentration at the site of infection . treatment of these diseases requires frequent eye drop administrations that may cause drug resistance and also decrease patient compliance . in order to minimize precorneal drainage and increase bioavailability viscosity enhancers such as poly ( vinyl alcohol ) and polymethacrylic acid were blended with eye drop solution . many investigators evaluated the role of liposomal hydrogel formulation for the delivery of fluoroquinolone antibiotics . for example , liposomal hydrogel formulation of ciprofloxacin ( cpx ) was reported to avoid tear - driven dilution in the cul de sac . lecithin ( lec ) and -l - dipalmitoylphosphatidylcholine ( dppc ) were utilized as major ingredients in the preparation of multilamellar liposomes . poly ( vinyl alcohol ) ( pva ) and polymethacrylic acid ( pma ) derivatives were utilized for gel formulation . various formulation parameters such as viscosity and rheological property of liposomes was evaluated in relation to the in vitro release . cpx because of its negative charge electrostatically interacts with lipid head group of the phospholipid bilayers . similar electrostatic interaction between lipid bilayers and other fluoroquinolones such as ofloxacin and lomefloxacin were reported in other studies . the investigator observed that use of viscosity enhancing agents in the formulation had affected the drug release rate . the addition of gel forming agents pva and pma did not affect the rigidity of liposomal membrane , instead these polymers were adsorbed on the surface of multilamellar liposomal surface because of method of formulation . hydration of lipids with proper concentration of pva and pma results in the formation of polymer layer on the surface of the liposomes [ 54 , 55 ] . in addition , they found a remarkable difference in drug release half - time between two different lipids , that is , lec and dppc . the presence of unsaturated lipid in lec provides less rigid structure to the liposome formulation that resulted in faster drug release in comparison to dppc . hydrogel formulation has shown plastic properties ; that is , under higher shear stress condition , it remained in free flowing state , whereas it exhibited no flow state at rest . overall , the use of optimized formulation of liposomal hydrogel can sustain the release of antibacterial agents in comparison to liposomes alone , and this approach could be beneficial in the treatment of various chronic ocular infectious diseases . in another study , cpx - loaded liposomal hydrogel formulation improved transcorneal permeation in rabbit model . the investigators reported that drug entrapment efficiency was enhanced with the increase of cholesterol concentrations , which provided higher stability and lower permeability of lipid bilayers . furthermore , higher encapsulation efficiency with positively charged liposomes was observed due to favorable electrostatic attraction between cpx and cationic stearylamine . liposomes of higher size were obtained upon incorporation of charge inducing agents , which expand lipid bilayers distance . positively charged liposomes exhibited slower release rate , and cpx release was more sustained from the liposomes suspended in the carbopol gel because of additional barriers for diffusion . liposomal hydrogel displayed fivefold higher in vitro transcorneal permeation across excised rabbit cornea than the aqueous solution . although authors observed enhanced in vitro transcorneal permeation , it would be interesting to evaluate these formulations for in vivo studies , where tear dilution plays a major role . in a similar study by these researchers , transcorneal permeation of ofloxacin - loaded thermosensitive two different types of liposomes , mlv and reverse phase evaporation vesicles ( rev ) , were prepared . authors observed smaller particle size with rev relative to mlv due to differences in the method of preparation . splicing of the lipid monolayer in a more curved structure resulted in rev of smaller diameter . incorporation of liposomes in thermosensitive gels reduced the gelling time from 5 to 1 minute . transcorneal permeation studies across excised rabbit cornea revealed sevenfold higher drug permeation from the liposomal formulation than ofloxacin aqueous solution . this effect was observed due to mucoadhesive nature of the hydrogel base which prolonged the retention of formulation across the excised rabbit cornea . in addition , cationic nature of chitosan in the thermogelling system promoted corneal adherence and opened corneal epithelial tight junctions . researchers concluded that ofloxacin liposomal formulation will reduce the formation of crystalline deposit and also frequency of administration . the ocular irritation test suggests excellent tolerance of chitosan formulation evaluated with confocal laser scanning ophthalmoscope [ 56 , 57 ] . liposomal formulation was evaluated on human subjects , and effectiveness was compared to normal saline at different time points . authors reported statistically significant improvement in tear film stability and lipid layer stability in comparison to control . these studies suggest the potential of liposomal sprays in the treatment of dry eye syndrome . liposomes were also investigated for the topical delivery of intraocular pressure ( iop ) lowering agents . for example , acetazolamide was encapsulated in liposomes to enhance the solubility and corneal permeation . liposomes were formulated by reversed phase evaporation and liquid hydration methods with and without the use of positive or negative charge inducers to prepare rev and mlv . liposomes of different compositions were evaluated for entrapment efficiency , stability , in vitro release , and iop lowering efficacy in rabbit model . the entrapment efficiency of acetazolamide was found highest with positively charged liposomes followed by neutral and negatively charged liposomes because of ionic interaction between anionic drug and lipid bilayers . cationic and neutral mlvs of acetazolamide exhibited maximum effectiveness in terms of release profile for the same reason . another iop reducing agent , demeclocycline ( dem ) , was encapsulated in liposomes which enhanced ocular permeability . neutral liposomes were more effective in iop lowering effect than negatively charged liposomes or free drug . in addition , phase transition and size distribution studies showed long term stability ( 15 months ) of the liposomal formulation . shen and tu reported the application of liposomes for the delivery of ganciclovir ( gcv ) to the vitreous humor via topical administration in the rabbits . gcv liposomes were prepared by the reversed phase evaporation method utilizing pc / ch / sodium deoxycholate mixture . in vitro transcorneal permeability and in vivo ocular transcorneal permeability was 3.9-fold higher ( figure 4 ) , and ocular bioavailability of gcv liposomes was 1.7-fold greater in comparison to solution ( figure 5 ) . gcv concentrations from liposomal formulation were 2 to 10 times higher in various ocular tissues . in addition , in vivo experiments suggested that the scleral pathway contributed in the absorption of gcv liposomes , as the highest concentration of gcv was obtained in the sclera . concentrations of gcv attained in the cornea and the sclera were higher than ic50 value of gcv against cmv . the author suggested that the particle size ( i.e. , 200 nm ) and composition of the liposomes played a major role in transocular permeation . disposable contact lenses presoaked with medication solution have been utilized for continuous drug delivery . however , in presoaked contact lenses , drug molecules randomly disperse within the contact lenses and show burst release that can cause local tissue toxicity or other side effects . to avoid rapid drug release and to provide site - specific delivery , another novel strategy , liposomes loaded soft contact lenses , was proposed for the antibiotics in the treatment of ocular infections such as bacterial keratitis . contact lenses with surface - immobilized levofloxacin - loaded liposomes followed first - order release kinetics and released the drug over more than 6 days . in addition , the liposomal formulation has shown antibacterial activity against s. aureus [ 28 , 62 ] . in another study , chloramphenicol ( cap ) was encapsulated in dimyristoylphosphatidylcholine ( dmpc ) liposomes and formulated in the form of eye drops . three methods , that is , cap - part ( partitioning of cap in the vesicle bilayers ) , cap - en ( entrapment of cap via normal hydration method ) , and cap - ads ( adsorption of cap on the vesicle surface ) were employed for the preparation of liposomes . the formulation was evaluated for interaction of the drug with the phospholipid bilayers resulting in optimum efficacy against s. aureus . cap was localized in the interfacial lipid bilayers in the case of cap - en whereas entrapped deeper in the bilayers in the case of cap - part . these results showed that cap located near the interfacial region within the hydrophobic core of the liposomes had shown highest anti - bacterial activity against s. aureus for almost 5 hrs . chetoni et al . reported acyclovir ( acv ) containing positively charged unilamellar liposomes ( lipo - acv ) , administered topically into rabbit eyes . the bioavailability of lipo - acv was compared with free acv in solution ( sol ) , acv encapsulated in empty liposomes ( lipo - empty ) , and a diluted dose of commercially available acv ointment , containing same acv concentrations ( 0.12% ) . the pharmacokinetic profile of the drug in the aqueous humor of rabbits showed highest drug concentration profile for lipo - acv system with 90 minutes plateau . lipo - acv exhibited aqueous humor acv concentration in the upper range of the id50 ( 0.01 to 0.7 g / ml ) . in a separate study concentrated acv ointment ( containing 8-fold greater dose of acv ) was compared with lipo - acv . these results indicate a significant advantage of lipo - acv as an alternative to acv ointment . in order to give an insight on release mechanism of acv from liposomal vehicle , in vitro release experiments through a cellophane membrane was performed which showed lower drug release from the liposomal vehicle through cellophane membrane compared to that of sol and lipo - empty . these results sustained the concept that negatively charged corneal epithelium enhances the efficacy of positively charged liposomal formulation . pleyer et al . formulated different cationic liposomes by changing their lipid compositions in order to improve gene expressions in corneal endothelial cells . the authors reported six formulations with different cationic lipids 3[n-(n , n-dimethylaminoethane)-carbamoyl ] ( dac ) , dicarbobenzoxyspermin - carbamoyl ( sp ) , n - amidino--alanin-[2-(1,3-dioleoyloxy)propyl]amid - hydrochlorid ( dosga ) , and 1,2-dimyristyloxypropyl-3-methylhydroxethylammoniumbromide ( dmrie ) which were coupled in varying concentrations with neutral lipid dioleoylphosphatidylethanolamine ( dope ) . fixed amount of dna was entrapped in each liposome which expressed e. coli beta - galactosidase . transfection experiments on bovine corneal endothelial cells ( bcec ) indicated that sp20 ( sp / dope 20/80 ) generated highest efficiency followed by dmrie 50 ( dmrie / dope 50/50 ) ranging at approximately 3 mu per -gal per well . the researchers observed low gene expressions with dac 30 ( dac / dope 30/70 ) , and dosga 30 ( dosga / dope 30/70 ) , dosga 100 ( dosga 100 ) and no gene expressions for free dna . at a fixed dna concentration , the relative -galactosidase expressions were decreased with increasing the cationic lipid dose , which might be due to either toxic effects of cationic lipids at higher concentrations to the cells or non - optimal lipid / dna ratios . the highest efficiency of sp20 liposomes in delivering dna into bcec can be rationalized by considering its rapid and stable complexation with dna due to result of ionic interactions between the multivalent lipid and negatively charged phosphate groups of dna . sp20 was completely biodegradable compared to many synthesized lipids as it was derived from naturally occurring compounds resulting in least toxicity compared to other liposomal formulations . teshima et al . studied prednisolone- ( pls- ) incorporated liposomes to improve retention property of prednisolone . introduction of a lipophilic moiety ( palmitoyl ) to prednisolone ( pal - pls ) greatly enhanced drug retention in liposomes as lipophilic moiety increased its affinity to liposomal lipid bilayer . the investigators studied two liposomes containing two different lipids , egg phosphatidylcholine ( eggpc ) and distearoyl phosphatidylcholine ( dspc ) . ultrafiltration and gel filtration techniques were used to investigate retention properties of pls and pal - pls in liposomes . while ultrafiltration method showed high incorporation efficiency of pls into the liposomes , a significant decrease of its incorporation efficiency this result indicated that elution medium in gel filtration studies released incorporated pls from liposomes . however , incubation of liposomes with rat plasma for 1 min effectively decreased pal - pls incorporation into eggpc / chol liposomes as detected by gel filtration . the reducing effect of pal - pls incorporation into liposomes by rat - plasma was overcome by using dspc lipid in liposomal formulation . further surface modification of liposomes with a hydrophilic polymer peg resulted in the protection of the entrapped palmitoyl - pls and thus generated a stable retention property of the drug . law et al . reported topical administration of acyclovir- ( acv)- encapsulated liposomes , where in vitro corneal penetration and in vivo corneal absorption ( using male rabbits ) of acyclovir from acv - encapsulated liposomes were studied . this study reported the effect of liposomal surface charge on their corneal penetration and absorption . surface charge of liposomes plays a significant role in improving the efficiency of ocular drug delivery system . positively charged liposomes exhibited higher drug loading efficiencies as well as faster drug release rates compared to negatively charged liposomes . the penetration rate for positively charged liposomes was found to be approximately 3.6-fold lower than free acv and approximately 2-fold lower than negatively charged liposomes . similarly , acv concentration profile in aqueous humor indicated higher corneal absorption and greater corneal deposition of acv for positively charged liposomes relative to negatively charged acv and free acv . the researchers suggested that positively charged liposomes can interact electrostatically with the negatively charged surface of cornea . this interaction can result in stronger binding which leads to formation of a completely coated layer on the corneal surface . this layer may cause an increase in residence time on the cornea surface resulting in higher acv absorption and greater extent of acv deposition in the cornea compared to that of negatively charged liposomes . kawakami et al . reported o - palmitoyl prodrug of tilisolol - encapsulated liposome to improve the retention time of tilisolol in the precorneal area and vitreous body . following topical administration , the researchers observed very low retention of o - palmitoyl tilisolol in the tear fluid even when it was applied as liposomal formulation . the investigators significantly increased the retention property of liposomes by adding 2% of carmellose sodium which acted as a reservoir for liposomes . in case of intravitreal administration , o - palmitoyl tilisolol - encapsulated liposomes responded well resulting in higher drug concentration in the vitreous body compared to free tilisolol . in the last decade numerous researchers addressed the challenge of minimizing rapid clearance from precorneal site and enhancing the corneal permeation through various approaches . utilization of chitosan in the preparation of mucoadhesive and cationic formulations was widely explored for the delivery of small therapeutic molecules from different categories . other mucoadhesive polymers were also applied in the formulation of hydrogels that can regulate the drug release rate at the ocular surface . , liposomal formulation can minimize the tissue toxicity and enhance the intravitreal half - life of drugs by decreasing rapid clearance from vitreous cavity [ 67 , 68 ] . barza et al . delineated the effect of liposome size and pathological state of eye on the intravitreal elimination kinetics of carriers . recently ocular pharmacologists have utilized liposomal hydrogel and sterically ( pegylated ) stabilized liposomes to address the drawbacks associated with intravitreal administrations of liposomes . in an application , rhodamine - conjugated liposomes loaded with vasoactive intestinal peptide ( vip ) were given intravenously to healthy rats to examine efficacy in the treatment of ocular inflammation . vip is an immunomodulatory neuropeptide involved in the regulation of ocular immune response by modulating the activities of macrophages , t lymphocytes , and dendritic cells [ 19 , 71 ] . intravitreal application of vip - loaded liposomes was proposed for the treatment of endotoxin - induced uveitis . internalization of rhodamine - conjugated liposomes ( rh - lip ) alone and loaded with vip ( vip - rh - lip ) was examined in male lewis rats . the authors reported that , after single intravitreal injection , liposomes were internalized by retinal mller glial cells , resident macrophages , and rare infiltrating activated macrophages . vip - rh - lip internalized via macrophages resulted in slower release and long - term expression inside the ocular tissues and cervical lymph nodes . thus , intravenous delivery of vip by liposomes would be helpful in the treatment of uveitis and other immune - mediated eye diseases by modulating the immune microenvironment of the ocular region . evaluated the liposomal formulation dispersed in hyaluronic acid ( ha ) gel for the delivery of vip in the treatment of uveitis and uveoretinitis in lewis rats . major limitation with the vip - lp was shorter residence time in the vitreous cavity due to rapid elimination through the lymphatic circulation . investigators attempted to increase the half - life of vip - loaded liposomes ( vip - lp ) after intravitreal administration by suspending them in the hydrogel . the researchers incorporated liposomes in ha gel in order to attain sustained release of vip from the liposomes . vip - lp suspended in ha gel was retained in the vitreous cavity for 8 days after single intravitreal injection . authors reported that incorporation of liposomes in the gel had increased the viscosity of the gel due to the enhanced interaction between ha gel and phospholipids . moreover , it was reported that formulation was effective in the treatment as evident by reduced clinical score and number of polymorphonuclear cells . in a study tacrolimus - loaded liposomes the vesicles were prepared by reverse phase evaporation technique and subsequently evaluated for efficacy and safety following intravitreal injection in rats . liposomes were able to maintain the vitreous concentration of more than 50 ng / ml for 2 weeks after single administration . the formulation also reduced drug - related toxicity to inner retinal cells . in another study , abrishami et al . prepared nanoliposomes of bevacizumab . researchers attempted to reduce the clearance of bevacizumab liposomes by incorporation of cholesterol . in comparison to free drug , concentration of liposomal formulation was 5 times higher at 42 days . this study revealed that liposomal formulation of bevacizumab was proven effective in the controlled release of bevacizumab for more than 6 weeks in rabbit model . fluconazole liposomes were evaluated for the treatment of candidal endophthalmitis . in the comparative study , intravitreal injections of fluconazole solution or liposomal formulation were given at different dose levels in the rabbit eyes . administration of fluconazole solution caused photoreceptor disorientation and ultrastructural changes of the retina at the concentration of 100 g in 0.1 ml or above . in contrast , liposomal formulation of fluconazole did not show any retinal alteration up to concentration of 200 g in 0.1 ml . formulated lipid prodrug of ganciclovir ( gcv ) , 1-o - hexadecylpropanediol-3-phospho - gcv into liposomes which were injected intravitreally in rabbits . the researchers used this liposomal formulation for antiviral treatment against herpes simplex virus type 1 ( hsv-1 ) and human cytomegalovirus ( hcmv ) . nm intravitreal concentration was the most effective without causing any side effects of vitreous clarity or cataracts development in the eye . moreover , this formulation provided complete retinal protection even after simultaneous intravitreal injection . bochot et al . reported that phosphodiester oligonucleotide encapsulated sterically ( pegylated ) stabilized liposomes which were administered intravitreally in rabbits . it was the first reported use of liposomes as vehicle for intravitreal delivery of phosphodiester oligonucleotides . the investigators tried to overcome the problem of short intravitreal half - life of oligonucleotide by encapsulating [ 33p ] labeled 16-mer oligothymidylate ( pdt16 ) within liposome . after intravitreal injection liposomal formulations yielded significantly higher concentration of radiolabeled 33p within the posterior segment of the eye ( vitreous , retina , choroid , and sclera ) than the solution . a heterogeneous competitive hybridization assay revealed a significantly improved intraocular stability of pdt16 when it was administered in a liposomal formulation . the sterically stabilized hydrophilic polyethylene glycol ( peg ) chains on the liposome 's surface protected them from degradation , resulting in prolonged residence time in vitreous and sustained release of encapsulated oligonucleotide into the vitreous and the retina - choroid . controlled release of [ 33p ] pdt16 from liposomes also inhibited unwanted distribution of oligonucleotide in the nontargeted tissues ( sclera , lens ) and thus reduced overall ocular toxicity . reported cationic liposomes as nonviral gene carriers which were complexed with therapeutic dna , called lipoplexes ( lpxs ) . the authors investigated the factors responsible for inefficient vitreous diffusion of nonviral gene complexes and addressed the problems to overcome vitreous barrier for lipoplexes . fitc - dextran , fluorescent polystyrene nanospheres as models for lpxs and lpxs were mixed with vitreous gel obtained from bovine eyes , and their mobility in vitreous was studied by fluorescence recovery after photobleaching ( frap ) technique . polystyrene nanospheres can bind to collagen fibers within the vitreous due to hydrophobic interactions resulting in restricted mobility in the vitreous . to overcome this problem , hydrophilic polyethylene glycol ( peg ) chains were grafted on the surface of nanoparticles that had prevented adsorption to the collagen fibers and thus increased their mobility in the vitreous . they reported that the size of the nanospheres should be less than 500 nm to obtain good vitreous mobility ; otherwise it would be sterically hindered by vitreous network and spread nonhomogeneously throughout the vitreous resulting in accumulation near the injection site . nonpegylated cationic liposomes aggregated in the vitreous as negatively charged glycosaminoglycans ( gags ) strongly bind to the cationic lipoplexes , which neutralize positive zeta potential of lipoplexes , and thus favor aggregation . low to moderate pegylation ( 1.9 mol% dspe - peg to 9.1 mol% dspe - peg ) on cationic lipoplexes prevented their aggregation but , binding to biopolymers in the vitreous still occurred . further increase of dspe - peg to 16.7 mol% prevented both vitreous aggregation as well as binding to vitreous fibrils , resulting in homogeneous vitreous distribution and vitreous mobility . the size and zeta potential of pegylated lpxs decreased with increasing the amount of pegylated lipids ( dspe - peg ) in lpxs . the data on mobility , aggregation , and stability of lipoplexes opened up a new direction to nonviral ocular gene therapy , but some factors need to take into consideration . here transport of drugs in vitreous was assumed by diffusion mechanism only but in case of larger animal species like humans drug transport through convection plays a significant role . cortical vitreous zone containing densely packed collagen and inner limiting lamina may produce additional barriers to the diffusion of lpxs into the retina . gupta et al . evaluated fluconazole - encapsulated liposomes which were administered intravitreally in rabbit eyes . entrapping of fluconazole into liposomes significantly slowed down clearance of free fluconazole after intravitreal injection and thereby achieved higher fluconazole concentration in the vitreous . the liposomes showed longer half - life ( 23.40 h ) in comparison to free fluconazole ( 3.08 h ) . among all these investigations performed by numerous researchers , approach of entrapping bevacizumab will be advantageous for designing controlled release system for therapeutic macromolecules . another approach of using sterically stabilized liposomes for oligonucleotide delivery can be further explored for resolving the challenges in ocular gene therapy . this approach will be advantageous in minimizing the intravitreal clearance of liposomes and distribution of oligonucleotide in the non - targeted tissues . subconjunctival mode of administration has gained new momentum in delivering the drugs to both the anterior and posterior segments . subconjunctival injection of liposomes can provide retentive effect and steady - state release at the site of application . therefore , higher drug concentrations can be achieved at the target site . in addition , subconjunctival injection is better in comparison to topical application as it can improve patience compliance by avoiding repeated administrations and provide direct access of the drug to the target site [ 80 , 81 ] . absorption rate of liposome - bound low molecular weight heparin ( lmwh ) was investigated after subconjunctival injection in the treatment of subconjunctival hemorrhage ( sh ) in rabbits . low concentration of liposome - bound lmwh was observed as compared to the free lmwh in the intraocular regions ( aqueous and vitreous ) . 550 nm in size ) , liposomes remained at the site of injection and avoided lymphatic drainage . also , positively charged liposomes encapsulated higher amounts of lmwh and released the drug in a sustained manner , thus providing longer residence time and increased concentration at the targeted site . thus , subconjunctival application of liposomes is a possible strategy to avoid systemic side effects of lmwh . in a similar study , baek et al . attempted subconjunctival administration of streptokinase- ( sk- ) loaded liposomes for the treatment of sh in rabbits . the study reported that 81% of the drug was released in 48 h. higher absorption efficiency of liposomes in comparison to free drug was observed . sk - encapsulated liposomes in the early phase of sh need to be assessed . fukushima et al . reported clodronate liposomes ( cl2mdp - lip ) , which were used to inhibit infiltration of macrophages in the conjunctiva in the case of blepharo conjunctivitis ( ec ) developed in brown norway rats . they found that cl2mdp - lip effectively decreased the number of ed2-positive macrophages in the conjunctivas , where ed1-positive macrophages infiltration could only be controlled if the injection was administered just prior to ova challenge . limited investigations on subconjunctival delivery of liposomes were performed in the last decade . however , approach of utilizing liposomes of size greater than 550 nm can be explored in future for long - term delivery by minimizing the systemic clearance of liposomes through conjunctival capillaries . it would be interesting to investigate the subconjunctival clearance of liposomes of various sizes . these carriers have successfully improved the drug bioavailability by controlled and targeted delivery . in the case of topical application improvement in the precorneal retention , transcorneal permeation , and therapeutic efficacy in addition , effects of charge and composition of liposomes were explored in detail , which have provided comprehensive understanding of the interaction between liposome and ocular tissues . the applications of chitosan and hydrogel for improving the precorneal retention of liposomes were explored and shown potential for further investigation . liposomal formulations have been evaluated for encapsulation of various drug molecules of different therapeutic classes . in particular , liposomal formulation of small molecules for the treatment of bacterial conjunctivitis and glaucoma was developed . moreover , posterior segment delivery of liposomes was proven successful in enhancing the intravitreal half - life and targeted delivery to the inner retinal cells . in the case of posterior segment disorders liposomal formulation of therapeutic macromolecules was examined . however , research on targeted delivery of liposomes was limited . receptors expressed on the cornea and retina could be explored in future for targeted drug delivery utilizing surface - modified liposomes .

liposomal formulations were significantly explored over the last decade for the ophthalmic drug delivery applications . these formulations are mainly composed of phosphatidylcholine ( pc ) and other constituents such as cholesterol and lipid - conjugated hydrophilic polymers . liposomes are biodegradable and biocompatible in nature . current approaches for topical delivery of liposomes are focused on improving the corneal adhesion and permeation by incorporating various bioadhesive and penetration enhancing polymers . in the case of posterior segment disorders improvement in intravitreal half life and targeted drug delivery to the retina is achieved by liposomes . in this paper we have attempted to summarize the applications of liposomes in the field of ophthalmic drug delivery by citing numerous investigators over the last decade .

1. Introduction 2. Application of Liposomes in Ophthalmic Drug Delivery 3. Topical Applications 4. Intravitreal Applications 5. Subconjunctival Applications 6. Conclusion

consequently , topical route has met with limited success in attaining therapeutic drug concentrations in the posterior segment . systemic administration can provide therapeutic levels in the posterior segment , but administration of high doses is necessary , which often leads to severe side effects . blood - aqueous barrier and blood - retinal barrier are the two major barriers for anterior segment and posterior segment ocular drug delivery , respectively , after systemic administration . blood retinal barrier is composed of two types of cells , that is , retinal capillary endothelial cells and retinal pigment epithelium ( rpe ) cells which prevents the entry of solute into the retina . these side effects can be minimized by developing delivery systems which provide controlled and targeted drug delivery for prolonged periods [ 13 ] . over the last decade , numerous drug delivery systems have been explored to overcome the limitation of conventional dosage forms . among them liposomal formulations were widely explored in the last decade for drug delivery applications . these vesicular systems are composed of aqueous core enclosed by phospholipid bilayers of natural or synthetic origin . liposomes are structurally classified on the basis of lipid bilayers such as small unilamellar vesicles ( suvs ) or multilamellar vesicles ( mlvs ) . furthermore , on the basis of size , liposomes are classified into small unilamellar vesicles ( suvs ) , giant unilamellar vesicles ( guvs ) , and large unilamellar vesicles ( luvs ) ( figure 1 ) . unilamellar vesicles are composed of single layer of lipid such as lecithin or phosphatidylglycerol encapsulating aqueous interior core . drug loading capacity of liposomes depends on many factors such as size of liposomes , types of lipid utilized for preparation , and physicochemical properties of therapeutic agent itself . loading capacity of ionic molecules can be further improved by using cationic or anionic lipids for the preparation of liposomes . majority of liposomal formulations utilize phosphatidylcholine ( pc ) and other constituents such as cholesterol and lipid - conjugated hydrophilic polymers as the main ingredients . stability of liposomes depends upon the various properties such as surface charge , size , surface hydration , and fluidity of lipid bilayers . liposomes of size less than 100 nm generally exhibit significantly higher circulation time due to decrease in opsonization of liposomes with serum protein . however , addition of aqueous solution of surfactant above the critical micelle concentration results in the formation of phospholipids micelles . liposomes bind to the receptor present in the invaginations of cellular membrane and are internalized into the cell by endocytotic pathway . in this paper we have attempted to summarize the application of liposomes in the field of ophthalmic drug delivery attempted by numerous investigators over the last decade . liposomes have been investigated for ophthalmic drug delivery since it offers advantages as a carrier system . it can enhance the permeation of poorly absorbed drug molecules by binding to the corneal surface and improving residence time . owing to their versatile nature , liposomes have been widely investigated for the treatment of both anterior and posterior segment eye disorders . current approaches for the anterior segment drug delivery are focused on improving corneal adhesion and permeation by incorporating various bioadhesive and penetration enhancing polymers . however , in the case of posterior segment disorders , improvement of intravitreal half - life and targeted drug delivery to the retina is necessary . currently verteporfin is being used clinically in photodynamic therapy for the treatment of subfoveal choroidal neovascularization ( cnv ) , ocular histoplasmosis , or pathological myopia effectively . in photodynamic therapy , after the drug is injected , a low - energy laser is applied to the retina through the contact lens in order to activate verteporfin that results in closure of the abnormal blood vessels . however , there are some issues to be addressed such as formulation , and storage of liposomes is very difficult , and they are known to cause long - term side effects . intravitreal administration of liposomes has resulted in vitreal condensation , vitreal bodies in the lower part of eye , and retinal abnormalities . investigated the role of liposomes in ophthalmic drug delivery . they reported corneal permeation in the order of suv+ > mlv > suv > suv > mlv free drug . liposomal formulation of ta produced twofold increase in drug concentration in both the cornea and aqueous humor in the rabbit model . was significant in exploring the clearance of liposomes by gamma scintigraphy following topical administration in the rabbit model . these investigators reported , suvs with positive charge had improved the corneal retention by interacting with negatively charged corneal surface . among these approaches however , lectin and lipid analog - based approaches are not explored considerably in the field of ophthalmic drug delivery . approach of utilizing chitosan in the formulation was reported to be advantageous in improving the precorneal residence time due to its mucoadhesive nature . degradation of chitosan into oligosaccharides is mediated through lysozymes , and degradation products are nontoxic in nature [ 40 , 41 ] . biodegradable nature is advantageous for selecting chitosan in the formulation of ocular drug delivery systems . reverse phase evaporation technique was utilized for the preparation of liposomes , which were further coated with chitosan of different molecular weights . in addition , authors determined lower encapsulation efficiency ( ee ) of 49.93% for coated liposomes in comparison to uncoated negative and neutral liposomes with 71.4% and 53.2% ee , respectively , due to electrostatic repulsion between chitosan and cationic drug . investigator reported higher ex vivo corneal penetration across excised rabbit cornea in the case of lch - coated liposomes as shown in figure 2 . researchers suggested that a loose coating layer is responsible for aggregation of vesicles which resulted in higher particle size in the case of 0.1% w / v lch . histopathological analysis of the lch - coated liposomes in rabbits after long - term irritation test revealed that the formulation was biocompatible with the ocular tissues ( figure 3 ) . as mentioned earlier , chitosan nanocarriers were employed in topical drug delivery because of its mucoadhesive nature , whereas liposomes can incorporate variety of drug molecules and improve ocular drug bioavailability [ 4547 ] . they also performed in vivo acute tolerance test by administrating the formulations topically to the female albino new zealand rabbits . also , in vivo experiments have shown that nanosystems can enter the conjunctival cells without causing histological alteration to the cornea , conjunctiva , and lid tissues in the rabbit model . administration by conventional parenteral route has several drawbacks such as high cost , need of highly trained personnel , and needle - stick injuries . cationic liposomes containing herpes simplex virus ( hsv ) antigens were proposed as potential carriers , in the form of a periocular vaccine , to protect animals against subsequent hsv-1 ocular challenges . this study reported improvement in the entrapment efficiency ( ee ) with increasing phosphatidylcholine component , whereas ee was lowered by incorporating stearylamine . in another study , fluconazole liposomal formulation was evaluated in the candidal keratitis model in rabbits . in this investigation , the purpose of developing liposomal formulation was to prolong the antifungal action by increasing the contact time . in the rabbits treated with fluconazole solution , 50% healing was observed in 3 weeks , whereas 86.4% healing was observed in rabbits treated with fluconazole encapsulated liposomes . chronic ocular infectious diseases such as conjunctivitis , bacterial keratitis need high drug concentration at the site of infection . in order to minimize precorneal drainage and increase bioavailability viscosity enhancers such as poly ( vinyl alcohol ) and polymethacrylic acid were blended with eye drop solution . many investigators evaluated the role of liposomal hydrogel formulation for the delivery of fluoroquinolone antibiotics . lecithin ( lec ) and -l - dipalmitoylphosphatidylcholine ( dppc ) were utilized as major ingredients in the preparation of multilamellar liposomes . poly ( vinyl alcohol ) ( pva ) and polymethacrylic acid ( pma ) derivatives were utilized for gel formulation . various formulation parameters such as viscosity and rheological property of liposomes was evaluated in relation to the in vitro release . similar electrostatic interaction between lipid bilayers and other fluoroquinolones such as ofloxacin and lomefloxacin were reported in other studies . overall , the use of optimized formulation of liposomal hydrogel can sustain the release of antibacterial agents in comparison to liposomes alone , and this approach could be beneficial in the treatment of various chronic ocular infectious diseases . although authors observed enhanced in vitro transcorneal permeation , it would be interesting to evaluate these formulations for in vivo studies , where tear dilution plays a major role . in a similar study by these researchers , transcorneal permeation of ofloxacin - loaded thermosensitive two different types of liposomes , mlv and reverse phase evaporation vesicles ( rev ) , were prepared . authors observed smaller particle size with rev relative to mlv due to differences in the method of preparation . incorporation of liposomes in thermosensitive gels reduced the gelling time from 5 to 1 minute . in addition , cationic nature of chitosan in the thermogelling system promoted corneal adherence and opened corneal epithelial tight junctions . authors reported statistically significant improvement in tear film stability and lipid layer stability in comparison to control . these studies suggest the potential of liposomal sprays in the treatment of dry eye syndrome . liposomes were also investigated for the topical delivery of intraocular pressure ( iop ) lowering agents . cationic and neutral mlvs of acetazolamide exhibited maximum effectiveness in terms of release profile for the same reason . shen and tu reported the application of liposomes for the delivery of ganciclovir ( gcv ) to the vitreous humor via topical administration in the rabbits . to avoid rapid drug release and to provide site - specific delivery , another novel strategy , liposomes loaded soft contact lenses , was proposed for the antibiotics in the treatment of ocular infections such as bacterial keratitis . three methods , that is , cap - part ( partitioning of cap in the vesicle bilayers ) , cap - en ( entrapment of cap via normal hydration method ) , and cap - ads ( adsorption of cap on the vesicle surface ) were employed for the preparation of liposomes . cap was localized in the interfacial lipid bilayers in the case of cap - en whereas entrapped deeper in the bilayers in the case of cap - part . sp20 was completely biodegradable compared to many synthesized lipids as it was derived from naturally occurring compounds resulting in least toxicity compared to other liposomal formulations . the investigators studied two liposomes containing two different lipids , egg phosphatidylcholine ( eggpc ) and distearoyl phosphatidylcholine ( dspc ) . however , incubation of liposomes with rat plasma for 1 min effectively decreased pal - pls incorporation into eggpc / chol liposomes as detected by gel filtration . further surface modification of liposomes with a hydrophilic polymer peg resulted in the protection of the entrapped palmitoyl - pls and thus generated a stable retention property of the drug . surface charge of liposomes plays a significant role in improving the efficiency of ocular drug delivery system . this interaction can result in stronger binding which leads to formation of a completely coated layer on the corneal surface . reported o - palmitoyl prodrug of tilisolol - encapsulated liposome to improve the retention time of tilisolol in the precorneal area and vitreous body . in case of intravitreal administration , o - palmitoyl tilisolol - encapsulated liposomes responded well resulting in higher drug concentration in the vitreous body compared to free tilisolol . in the last decade numerous researchers addressed the challenge of minimizing rapid clearance from precorneal site and enhancing the corneal permeation through various approaches . utilization of chitosan in the preparation of mucoadhesive and cationic formulations was widely explored for the delivery of small therapeutic molecules from different categories . in an application , rhodamine - conjugated liposomes loaded with vasoactive intestinal peptide ( vip ) were given intravenously to healthy rats to examine efficacy in the treatment of ocular inflammation . vip is an immunomodulatory neuropeptide involved in the regulation of ocular immune response by modulating the activities of macrophages , t lymphocytes , and dendritic cells [ 19 , 71 ] . internalization of rhodamine - conjugated liposomes ( rh - lip ) alone and loaded with vip ( vip - rh - lip ) was examined in male lewis rats . thus , intravenous delivery of vip by liposomes would be helpful in the treatment of uveitis and other immune - mediated eye diseases by modulating the immune microenvironment of the ocular region . evaluated the liposomal formulation dispersed in hyaluronic acid ( ha ) gel for the delivery of vip in the treatment of uveitis and uveoretinitis in lewis rats . investigators attempted to increase the half - life of vip - loaded liposomes ( vip - lp ) after intravitreal administration by suspending them in the hydrogel . the researchers incorporated liposomes in ha gel in order to attain sustained release of vip from the liposomes . authors reported that incorporation of liposomes in the gel had increased the viscosity of the gel due to the enhanced interaction between ha gel and phospholipids . this study revealed that liposomal formulation of bevacizumab was proven effective in the controlled release of bevacizumab for more than 6 weeks in rabbit model . in the comparative study , intravitreal injections of fluconazole solution or liposomal formulation were given at different dose levels in the rabbit eyes . administration of fluconazole solution caused photoreceptor disorientation and ultrastructural changes of the retina at the concentration of 100 g in 0.1 ml or above . the researchers used this liposomal formulation for antiviral treatment against herpes simplex virus type 1 ( hsv-1 ) and human cytomegalovirus ( hcmv ) . it was the first reported use of liposomes as vehicle for intravitreal delivery of phosphodiester oligonucleotides . after intravitreal injection liposomal formulations yielded significantly higher concentration of radiolabeled 33p within the posterior segment of the eye ( vitreous , retina , choroid , and sclera ) than the solution . controlled release of [ 33p ] pdt16 from liposomes also inhibited unwanted distribution of oligonucleotide in the nontargeted tissues ( sclera , lens ) and thus reduced overall ocular toxicity . to overcome this problem , hydrophilic polyethylene glycol ( peg ) chains were grafted on the surface of nanoparticles that had prevented adsorption to the collagen fibers and thus increased their mobility in the vitreous . nonpegylated cationic liposomes aggregated in the vitreous as negatively charged glycosaminoglycans ( gags ) strongly bind to the cationic lipoplexes , which neutralize positive zeta potential of lipoplexes , and thus favor aggregation . cortical vitreous zone containing densely packed collagen and inner limiting lamina may produce additional barriers to the diffusion of lpxs into the retina . entrapping of fluconazole into liposomes significantly slowed down clearance of free fluconazole after intravitreal injection and thereby achieved higher fluconazole concentration in the vitreous . this approach will be advantageous in minimizing the intravitreal clearance of liposomes and distribution of oligonucleotide in the non - targeted tissues . subconjunctival injection of liposomes can provide retentive effect and steady - state release at the site of application . low concentration of liposome - bound lmwh was observed as compared to the free lmwh in the intraocular regions ( aqueous and vitreous ) . thus , subconjunctival application of liposomes is a possible strategy to avoid systemic side effects of lmwh . attempted subconjunctival administration of streptokinase- ( sk- ) loaded liposomes for the treatment of sh in rabbits . the study reported that 81% of the drug was released in 48 h. higher absorption efficiency of liposomes in comparison to free drug was observed . sk - encapsulated liposomes in the early phase of sh need to be assessed . reported clodronate liposomes ( cl2mdp - lip ) , which were used to inhibit infiltration of macrophages in the conjunctiva in the case of blepharo conjunctivitis ( ec ) developed in brown norway rats . they found that cl2mdp - lip effectively decreased the number of ed2-positive macrophages in the conjunctivas , where ed1-positive macrophages infiltration could only be controlled if the injection was administered just prior to ova challenge . limited investigations on subconjunctival delivery of liposomes were performed in the last decade . however , approach of utilizing liposomes of size greater than 550 nm can be explored in future for long - term delivery by minimizing the systemic clearance of liposomes through conjunctival capillaries . in the case of topical application improvement in the precorneal retention , transcorneal permeation , and therapeutic efficacy in addition , effects of charge and composition of liposomes were explored in detail , which have provided comprehensive understanding of the interaction between liposome and ocular tissues . the applications of chitosan and hydrogel for improving the precorneal retention of liposomes were explored and shown potential for further investigation . liposomal formulations have been evaluated for encapsulation of various drug molecules of different therapeutic classes . moreover , posterior segment delivery of liposomes was proven successful in enhancing the intravitreal half - life and targeted delivery to the inner retinal cells . in the case of posterior segment disorders liposomal formulation of therapeutic macromolecules was examined . however , research on targeted delivery of liposomes was limited . receptors expressed on the cornea and retina could be explored in future for targeted drug delivery utilizing surface - modified liposomes .

leptin , an adipocyte - derived hormone , affects glucose homeostasis in part by directly modulating pancreatic -cell function ( 13 ) . systemic leptin infusion acutely decreases insulin secretion and impairs glucose tolerance in rodents ( 4 ) . loss of these suppressive actions of leptin in mice with selective absence of leptin receptors in the pancreas leads to fasting hyperinsulinemia and increased responsiveness to glucose- and incretin - stimulated insulin secretion ( 57 ) . these findings have led some to speculate that the diminished responsiveness of pancreatic -cells to the negative effects of leptin partly contributes to the hyperinsulinemia typically observed in obesity ( 4,7,8 ) . however , whether leptin negatively affects -cell function in humans is unclear . lipodystrophy , characterized by selective deficiency of adipose tissue , is a hypoleptinemic state associated with hyperinsulinemia , severe insulin resistance , glucose intolerance , and dyslipidemia ( 1 ) . leptin replacement in patients with lipodystrophy attenuates insulin resistance , hyperinsulinemia , and hyperglycemia ( 1,9,10 ) . although insulin secretion was not assessed in these studies , reductions in insulin levels ( fasting and glucose induced ) were attributed to improvements in insulin sensitivity ( 11,12 ) . a solitary study has reported the effects of leptin replacement on insulin secretion in a genetically leptin - deficient adult male ( 13 ) . in this patient , except for this single report , we are unaware of studies examining the short - term effects of leptin replacement on -cell function . to that end , we report the early effects of short - term ( 1620 weeks ) metreleptin ( an analog of human leptin ) administration on -cell function in patients with lipodystrophy . the study protocol ( initiated in july 2000 ) was approved by the institutional review board of the national institute of diabetes and digestive and kidney diseases . written informed consent was obtained from all subjects or their legal guardian , and assent was obtained from participants under 18 years of age . patients were from the u.s . and a number of other countries in europe , asia , the middle east , and south america . study subjects reported here are from an open - label , prospective , currently ongoing study examining the long - term safety and clinical effects of metreleptin treatment in patients with congenital or acquired lipodystrophy ( 9,11,12 ) . the rationale , inclusion criteria , and study design have been described previously ( 9,11 ) . inclusion criteria for leptin replacement included hypoleptinemia ( < 12 ng / ml ) , metabolic abnormalities such as hypertriglyceridemia and/or lipoatrophic diabetes , and the ability to adhere to the leptin replacement protocol . metreleptin was provided by amgen ( thousand oaks , ca ) initially and amylin pharmaceuticals ( san diego , ca ) subsequently ( 9,11 ) . leptin therapy was provided as a self - administered once- or twice - daily subcutaneous injection as previously described ( 9,11 ) . patients were seen at the clinical research center of the national institutes of health every 46 months for the first year and then every 612 months thereafter . the current study examined the early effects of leptin replacement on -cell function derived from oral glucose tolerance test ( ogtt ) data . in this report , we include a subgroup of patients ( n = 13 ) who were on stable doses of oral hypoglycemic agents prior to and at 1620 weeks of leptin therapy . ogtts were performed at baseline and after 1620 weeks of leptin administration . during each visit , after an overnight fast , patients underwent an ogtt with plasma sampling at baseline and 30 , 60 , 90 , 120 , and 180 min after glucose ingestion for the measurement of glucose , insulin , and c - peptide concentrations . all oral hypoglycemic agents were held on the day of the ogtt . the -cell model used in the current study , describing the relationship between insulin secretion and glucose concentration , has been described ( 14 ) . insulin secretory rates ( isrs ) were calculated from deconvolution of the plasma c - peptide concentrations . the relationship between insulin secretion and plasma glucose concentrations is modeled as the sum of two components . the first component represents the dependence of insulin secretion on absolute glucose concentration at any time point during the ogtt and is characterized by a dose - response function relating these two variables . the dose response is modulated by various factors ( e.g. , incretins , neurotransmitters , and degree and duration of hyperglycemia ) , which are collectively modeled as a potentiation factor . the second component represents the dependence of insulin secretion on the rate of change of plasma glucose and depends on the first derivative of plasma glucose concentration against time and a parameter denoted as rate sensitivity . consequently , this parameter reflects the changes in insulin secretion in response to rapid changes in glucose concentration . oral glucose insulin sensitivity ( ogis ) and insulin sensitivity index ( isi ) were used as surrogate measures of glucose insulin sensitivity / resistance ( 14,15 ) . this method provides an isi that is an estimate of the glucose clearance during a euglycemic - hyperglycemic clamp and is expressed as milliliters per minute per square meter of body surface area ( 14 ) . isi ( matsuda ) was calculated as previously described ( 15 ) . after testing for normality significance of changes in measures of -cell function ( postleptin minus baseline values ) was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . data are presented as means sd or median ( interquartile range [ iqr ] ) . the study protocol ( initiated in july 2000 ) was approved by the institutional review board of the national institute of diabetes and digestive and kidney diseases . written informed consent was obtained from all subjects or their legal guardian , and assent was obtained from participants under 18 years of age . patients were from the u.s . and a number of other countries in europe , asia , the middle east , and south america . study subjects reported here are from an open - label , prospective , currently ongoing study examining the long - term safety and clinical effects of metreleptin treatment in patients with congenital or acquired lipodystrophy ( 9,11,12 ) . the rationale , inclusion criteria , and study design have been described previously ( 9,11 ) . inclusion criteria for leptin replacement included hypoleptinemia ( < 12 ng / ml ) , metabolic abnormalities such as hypertriglyceridemia and/or lipoatrophic diabetes , and the ability to adhere to the leptin replacement protocol . metreleptin was provided by amgen ( thousand oaks , ca ) initially and amylin pharmaceuticals ( san diego , ca ) subsequently ( 9,11 ) . leptin therapy was provided as a self - administered once- or twice - daily subcutaneous injection as previously described ( 9,11 ) . patients were seen at the clinical research center of the national institutes of health every 46 months for the first year and then every 612 months thereafter . the current study examined the early effects of leptin replacement on -cell function derived from oral glucose tolerance test ( ogtt ) data . in this report , we include a subgroup of patients ( n = 13 ) who were on stable doses of oral hypoglycemic agents prior to and at 1620 weeks of leptin therapy . ogtts were performed at baseline and after 1620 weeks of leptin administration . during each visit , after an overnight fast , patients underwent an ogtt with plasma sampling at baseline and 30 , 60 , 90 , 120 , and 180 min after glucose ingestion for the measurement of glucose , insulin , and c - peptide concentrations . the -cell model used in the current study , describing the relationship between insulin secretion and glucose concentration , has been described ( 14 ) . insulin secretory rates ( isrs ) were calculated from deconvolution of the plasma c - peptide concentrations . the relationship between insulin secretion and plasma glucose concentrations is modeled as the sum of two components . the first component represents the dependence of insulin secretion on absolute glucose concentration at any time point during the ogtt and is characterized by a dose - response function relating these two variables . the dose response is modulated by various factors ( e.g. , incretins , neurotransmitters , and degree and duration of hyperglycemia ) , which are collectively modeled as a potentiation factor . the second component represents the dependence of insulin secretion on the rate of change of plasma glucose and depends on the first derivative of plasma glucose concentration against time and a parameter denoted as rate sensitivity . consequently , this parameter reflects the changes in insulin secretion in response to rapid changes in glucose concentration . oral glucose insulin sensitivity ( ogis ) and insulin sensitivity index ( isi ) were used as surrogate measures of glucose insulin sensitivity / resistance ( 14,15 ) . this method provides an isi that is an estimate of the glucose clearance during a euglycemic - hyperglycemic clamp and is expressed as milliliters per minute per square meter of body surface area ( 14 ) . significance of changes in measures of -cell function ( postleptin minus baseline values ) was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . data are presented as means sd or median ( interquartile range [ iqr ] ) . thirteen patients with lipodystrophy treated with leptin are included in this study ( congenital generalized lipodystrophy , n = 5 ; acquired generalized lipodystrophy , n = 2 ; familial partial lipodystrophy , n = 5 ; acquired partial lipodystrophy , n = 1 ) . the median baseline age was 15 years with a range of 854 years . patients were on stable doses of metformin ( n = 12 ) and/or thiazolidinedione ( n = 3 ) prior to and at the end of 1620 weeks of leptin therapy . dyslipidemia was treated with stable doses of lipid - lowering agents ( fenofibrate , n = 3 ; omega-3-acid ethyl esters , n = 1 ) as previously described ( 9 ) . prior to leptin therapy , median serum leptin levels ( trough ) patients with partial lipodystrophy had higher levels of leptin compared with generalized lipodystrophy ( mean sd 4.1 1.2 vs. 0.7 0.3 , p = 0.002 ) . all of our patients were diabetic with an average glycosylated hemoglobin level ( a1c ) of 7.2% with mean fasting plasma glucose levels < 6.6 mmol / l , suggesting that glycemic control was good prior to initiation of leptin . as previously reported , these patients had high levels of triglycerides and free fatty acids ( ffas ) ( table 1 ) , and various metabolic parameters were similar in partial and generalized lipodystrophy patients ( data not shown ) . clinical characteristics and metabolic parameters before and after leptin replacement in patients with lipodystrophy the initial dose of metreleptin was based on weight , sex , and age , and subsequent dose adjustments were based on individual metabolic responses of patients . the average daily dose of metreleptin at the end of 1620 weeks was 4.19 1.87 mg ( mean sd ) . as expected , leptin levels increased significantly with therapy ( table 1 ) . leptin replacement was associated with a significant decrease in total cholesterol , ldl , triglyceride , and ffa levels with no change in hdl levels ( table 1 ) . after leptin therapy , a1c levels were significantly lower , but fasting glucose and insulin and 2-h post - ogtt values were not different from baseline ( table 1 ) . however , area under the curve for glucose ( aucg ) during ogtt was lower after leptin therapy ( 1,816 731 vs. 2,063 598 mmol l 180 min , p = 0.02 ) . surrogate measures of insulin sensitivity , ogis and isi , were not significantly modulated , but tended to increase ( 25% ) with leptin replacement ( table 1 ) . these changes in insulin sensitivity and the lower glucose auc partially explain the effects of leptin on a1c . 1 . mean basal insulin secretion rate prior to leptin initiation was 324 153 pmol / m / min ( sd ) , and basal insulin secretion rate adjusted for age and insulin sensitivity ( ogis ) was not significantly different after leptin replacement ( 345 254 pmol / m / min , p = 0.10 ) . similarly , leptin administration had no significant effect on total insulin secretion ( post vs. pre : 111 42 vs. 170 123 nmol / m , p = 0.13 ) . model - derived -cell insulin secretion and glucose dose responses obtained from ogtts administered pre- and post - leptin therapy are shown in fig . the slopes of these curves represent the ability of the -cell to respond to an increment in plasma glucose concentration termed glucose sensitivity . leptin treatment had no significant effect on glucose sensitivity ( post vs. pre ) : 65 ( 112 ) vs. 48 ( 167 ) pmol / min / m / mm , p = 0.39 . likewise , rate sensitivity , a measure of the insulin secretory component that responds to rapid changes in plasma glucose concentrations , was also unaltered with treatment : 206 ( 1,449 ) vs. 1,215 ( 6,915 ) pmol / min / m / mm , p = 0.13 . age , duration of diabetes , race , sex , and type of lipodystrophy may potentially modify the effects of leptin . at baseline , age was negatively related to basal isr ( r = 0.72 , p = 0.005 ) and total isr ( r = 0.47 , p = 0.09 ) . changes in measures of -cell function ( post - leptin minus baseline values ) were adjusted for age . the median duration of diabetes in this study was 9.5 years ( range 429 ) . however , duration of diabetes was unrelated to basal isr ( r = 0.14 , p = 0.68 ) or total isr ( r = 0.41 , p = 0.18 ) , and the significance of changes in measures of -cell function after leptin therapy remained unaltered after adjusting for duration of diabetes . the effect of leptin treatment on parameters of -cell function was not significantly different between partial and generalized lipodystrophy patients ( pgroup time interaction > 0.5 ) . thus , these results suggest that leptin replacement in the short - term had no significant effect on insulin secretion or -cell glucose responsiveness . insulin secretion and -cell glucose sensitivity in patients with lipodystrophy before and after leptin replacement . fasting isr ( a ) , total insulin output after an oral glucose load ( b ) , and model - derived dose response between isr and plasma glucose concentration ( c ) . significance of changes in measures of -cell function ( post - leptin minus baseline values ) in panels a and b was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . , congenital generalized lipodystrophy ; , acquired generalized lipodystrophy ; , familial partial lipodystrophy ; , acquired partial lipodystrophy . basal insulin clearance ( fasting isrs / fasting insulin ) and ogtt - insulin clearance ( incremental aucs of isrs / incremental aucs of insulin concentrations ) rates were measured prior to and after leptin therapy . before leptin therapy , basal insulin clearance rates were similar in partial ( 1.99 0.76 l / min / m ) and generalized ( 1.66 0.33 l / min / m ) lipodystrophy patients . however , ogtt - insulin clearance rates were significantly higher in patients with partial lipodystrophy ( 1.25 0.33 vs. 0.80 0.25 l / min / m , p = 0.02 ) . in the overall cohort , leptin treatment had no significant effect on basal ( post vs. pre : 1.97 1.04 vs. 1.81 0.57 l / min / m , p = 0.62 ) or ogtt - insulin clearance ( 1.12 0.39 vs. 1.00 0.36 l / min / m , p = 0.39 ) . the lack of leptin effects on insulin clearance was similar in partial and generalized lipodystrophy patients . thirteen patients with lipodystrophy treated with leptin are included in this study ( congenital generalized lipodystrophy , n = 5 ; acquired generalized lipodystrophy , n = 2 ; familial partial lipodystrophy , n = 5 ; acquired partial lipodystrophy , n = 1 ) . the median baseline age was 15 years with a range of 854 years . patients were on stable doses of metformin ( n = 12 ) and/or thiazolidinedione ( n = 3 ) prior to and at the end of 1620 weeks of leptin therapy . dyslipidemia was treated with stable doses of lipid - lowering agents ( fenofibrate , n = 3 ; omega-3-acid ethyl esters , n = 1 ) as previously described ( 9 ) . prior to leptin therapy , median serum leptin levels ( trough ) patients with partial lipodystrophy had higher levels of leptin compared with generalized lipodystrophy ( mean sd 4.1 1.2 vs. 0.7 0.3 , p = 0.002 ) . all of our patients were diabetic with an average glycosylated hemoglobin level ( a1c ) of 7.2% with mean fasting plasma glucose levels < 6.6 mmol / l , suggesting that glycemic control was good prior to initiation of leptin . as previously reported , these patients had high levels of triglycerides and free fatty acids ( ffas ) ( table 1 ) , and various metabolic parameters were similar in partial and generalized lipodystrophy patients ( data not shown ) . the initial dose of metreleptin was based on weight , sex , and age , and subsequent dose adjustments were based on individual metabolic responses of patients . the average daily dose of metreleptin at the end of 1620 weeks was 4.19 1.87 mg ( mean sd ) . leptin replacement was associated with a significant decrease in total cholesterol , ldl , triglyceride , and ffa levels with no change in hdl levels ( table 1 ) . after leptin therapy , a1c levels were significantly lower , but fasting glucose and insulin and 2-h post - ogtt values were not different from baseline ( table 1 ) . however , area under the curve for glucose ( aucg ) during ogtt was lower after leptin therapy ( 1,816 731 vs. 2,063 598 mmol l 180 min , p = 0.02 ) . surrogate measures of insulin sensitivity , ogis and isi , were not significantly modulated , but tended to increase ( 25% ) with leptin replacement ( table 1 ) . these changes in insulin sensitivity and the lower glucose auc partially explain the effects of leptin on a1c . 1 . mean basal insulin secretion rate prior to leptin initiation was 324 153 pmol / m / min ( sd ) , and basal insulin secretion rate adjusted for age and insulin sensitivity ( ogis ) was not significantly different after leptin replacement ( 345 254 pmol / m / min , p = 0.10 ) . similarly , leptin administration had no significant effect on total insulin secretion ( post vs. pre : 111 42 vs. 170 123 nmol / m , p = 0.13 ) . model - derived -cell insulin secretion and glucose dose responses obtained from ogtts administered pre- and post - leptin therapy are shown in fig . the slopes of these curves represent the ability of the -cell to respond to an increment in plasma glucose concentration termed glucose sensitivity . leptin treatment had no significant effect on glucose sensitivity ( post vs. pre ) : 65 ( 112 ) vs. 48 ( 167 ) pmol / min / m / mm , p = 0.39 . likewise , rate sensitivity , a measure of the insulin secretory component that responds to rapid changes in plasma glucose concentrations , was also unaltered with treatment : 206 ( 1,449 ) vs. 1,215 ( 6,915 ) pmol / min / m / mm , p = 0.13 . age , duration of diabetes , race , sex , and type of lipodystrophy may potentially modify the effects of leptin . at baseline , age was negatively related to basal isr ( r = 0.72 , p = 0.005 ) and total isr ( r = 0.47 , p = 0.09 ) . changes in measures of -cell function ( post - leptin minus baseline values ) were adjusted for age . the median duration of diabetes in this study was 9.5 years ( range 429 ) . however , duration of diabetes was unrelated to basal isr ( r = 0.14 , p = 0.68 ) or total isr ( r = 0.41 , p = 0.18 ) , and the significance of changes in measures of -cell function after leptin therapy remained unaltered after adjusting for duration of diabetes . the effect of leptin treatment on parameters of -cell function was not significantly different between partial and generalized lipodystrophy patients ( pgroup time interaction > 0.5 ) . thus , these results suggest that leptin replacement in the short - term had no significant effect on insulin secretion or -cell glucose responsiveness . insulin secretion and -cell glucose sensitivity in patients with lipodystrophy before and after leptin replacement . fasting isr ( a ) , total insulin output after an oral glucose load ( b ) , and model - derived dose response between isr and plasma glucose concentration ( c ) . significance of changes in measures of -cell function ( post - leptin minus baseline values ) in panels a and b was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . , congenital generalized lipodystrophy ; , acquired generalized lipodystrophy ; , familial partial lipodystrophy ; , acquired partial lipodystrophy . basal insulin clearance ( fasting isrs / fasting insulin ) and ogtt - insulin clearance ( incremental aucs of isrs / incremental aucs of insulin concentrations ) rates were measured prior to and after leptin therapy . before leptin therapy , basal insulin clearance rates were similar in partial ( 1.99 0.76 l / min / m ) and generalized ( 1.66 0.33 l / min / m ) lipodystrophy patients . however , ogtt - insulin clearance rates were significantly higher in patients with partial lipodystrophy ( 1.25 0.33 vs. 0.80 0.25 l / min / m , p = 0.02 ) . in the overall cohort , leptin treatment had no significant effect on basal ( post vs. pre : 1.97 1.04 vs. 1.81 0.57 l / min / m , p = 0.62 ) or ogtt - insulin clearance ( 1.12 0.39 vs. 1.00 0.36 l / min / m , p = 0.39 ) . the lack of leptin effects on insulin clearance was similar in partial and generalized lipodystrophy patients . in the current study , leptin replacement therapy had no significant impact on -cell function in patients with lipodystrophy treated for 1620 weeks . the effect of leptin on human -cell function and insulin secretion has not been thoroughly investigated . in that context , our study results suggest that , contrary to the suppressive effects of leptin on insulin secretion consistently observed in animal and in vitro studies , leptin replacement does not attenuate the increased insulin secretion observed in these patients . leptin replacement in lipodystrophy patients is associated with reductions in fasting insulin levels and after an oral glucose load after 12 months ( 11,12 ) . in contrast , in another study , mean serum levels of insulin after an ogtt remained unaltered in patients with congenital generalized lipodystrophy ( n = 5 ) and increased in acquired generalized lipodystrophy ( n = 2 ) despite improvements in insulin sensitivity and glycemia after 4 months of leptin replacement ( 16 ) . insulin secretion in one genetically leptin - deficient adult male was deduced by deconvolution of c - peptide levels obtained during a meal ( 13 ) . in the first week after leptin replacement , however , hepatic extraction of insulin was increased simultaneously , resulting in lower peripheral insulin levels ( by 20% ) . after 18 and 24 months of leptin therapy , insulin secretion was reduced along with improvements in insulin sensitivity . we evaluated -cell function by using a mathematical model that describes the dynamic relationship between plasma glucose concentration and -cell insulin secretion . in order to account for changes in insulin sensitivity , we used surrogate indices derived from an ogtt , ogis and isi ( matsuda ) . patients in this study were hypoleptinemic and severely insulin resistant with hyperinsulinemia ( table 1 ) . fasting isrs in these patients were approximately twofold higher than those reported in morbidly obese , insulin - resistant , type 2 diabetic individuals ( 140 pmol / m / min ) ( 17 ) . this degree of hyperinsulinemia afforded us the opportunity to evaluate the potential inhibitory effects of leptin on insulin secretion . in fact , our sample size ( n = 13 ) was sufficient to provide 80% power with = 0.05 to detect a 20% decrease in fasting isr . nonetheless , we did not observe any significant effects of leptin replacement on fasting insulin secretion or total insulin output during an ogtt ( fig . 1 ) . glucose sensitivity measures the ability of the -cell to respond to a glycemic stimulus and is a determinant of insulin secretion ( 18 ) . -cell glucose sensitivity is impaired in individuals with diabetes and glucose intolerance ( 17,18 ) . in our patients , median values of -cell glucose sensitivity are in the range typically observed in diabetic individuals ( 17,18 ) . even though a1c and ffa levels were significantly better after leptin treatment ( table 1 ) , 1 ) . our results are inconsistent with findings from studies in cell culture and animals . leptin receptors are expressed in human islets and mediate the inhibitory actions of leptin on insulin secretion in -cells in vitro ( 4,8 ) . leptin hyperpolarizes -cells by opening katp channels , reduces intracellular calcium levels , and accelerates the hydrolysis of camp to attenuate glucose- and agonist - induced insulin secretion ( 8,19 ) . systemic infusion of leptin in normal or leptin - deficient hyperinsulinemic ob / ob mice , but not leptin receptor deficient db / db mice , acutely ( within minutes ) decreases plasma insulin levels ( 4 ) . in ob / ob mice , leptin administration for 24 these direct insulin - suppressing effects were also observed during short - term ( 4-week ) leptin replacement in ob / ob mice prior to any significant changes in body weight or food intake ( 21 ) . consistent with these findings , selective knockout of leptin receptors in the pancreas of mice leads to hyperinsulinemia ( 57 ) . the reasons for the discrepancy in the results from our study and leptin - deficient rodent models are unclear . it is possible that the hypoleptinemic state and the accompanying significant elevations in ffa and ectopic lipid deposition may have altered the insulin secretory dynamics in human -cells differently than in rodents . in fact , in obese rodents ( due to leptin deficiency or diet induced ) , leptin treatment reduces pancreatic fat accumulation ( 22,23 ) . we have previously shown that leptin replacement for 1620 weeks decreases hepatic steatosis in individuals with lipodystrophy ( 24,25 ) . leptin , similar to its effects on hepatic steatosis , may also decrease pancreatic steatosis in humans . however , we are not aware of any human studies that have examined the effects of leptin on pancreatic fat content in patients with non - hiv lipodystrophy . although hepatic steatosis is frequently observed in individuals with lipodystrophy , postmortem studies in few patients do not report pancreatic steatosis ( 2629 ) . although these findings need further confirmation , it appears that islet amyloidosis may be the characteristic pathologic feature in these patients ( 2628 ) . thus , species - dependent and tissue - specific antisteatotic effects of leptin may explain the discrepancy in the effects of leptin in rodents and humans . leptin inhibits insulin secretion from human islets in vitro ( 4,8 ) ; however , studies examining the acute effects of agonist and/or glucose - stimulated insulin secretion in healthy individuals are necessary to rule out species - dependent in vivo effects of leptin . we also did not observe any significant improvements in insulin sensitivity as measured by ogis and isi after leptin replacement ( table 1 ) . although there was a trend toward higher ogis and isi after treatment ( p = 0.09 ) , it is possible that a more sensitive method , such as the euglycemic - hyperinsulinemic clamp , would have detected improvements in insulin sensitivity as reported in prior studies ( 1,24,30 ) . one of the early effects of leptin treatment is an increase in insulin clearance ( 13 ) . however , 1620 weeks of leptin replacement did not modulate insulin clearance in this study . in this study , we chose to report the early effects of leptin replacement on -cell function . we have previously reported on the long - term effects of leptin therapy ( 911,25 ) . leptin therapy decreases fasting glucose levels , improves glucose tolerance and insulin sensitivity , reduces a1c , lowers triglycerides , and attenuates steatohepatitis in lipodystrophy ( 911,25 ) . improvements in insulin sensitivity and changes in ectopic lipid accumulation have been suggested to mediate these effects of leptin . however , a recent study suggests that modest glycemic benefits of long - term leptin therapy extend to patients with insulin receptor mutations ( 31 ) . considering that patients with insulin receptor mutations have a fixed defect in insulin sensitivity and do not manifest ectopic lipid accumulation , leptin favorably affects glucose metabolism through other unidentified mechanisms . future studies examining the short- and long - term effects on -cell function and pancreatic fat content are warranted to confirm the long - term effects of leptin on pancreatic function . first , we derived measures of -cell function by mathematically modeling plasma glucose and c - peptide levels measured during ogtt and used surrogate indices ( ogis and isi ) to assess insulin sensitivity . although the model is widely used , our findings need to be confirmed using gold - standard techniques such as euglycemic - hyperinsulinemic and hyperglycemic clamp techniques . these techniques will also provide additional information regarding the different phases of insulin secretion . whether leptin has phase - specific effects on insulin secretion is not known . nonetheless , the effects of leptin on -cell glucose sensitivity to first - phase insulin response can not be adequately assessed during an ogtt . it is likely that glucose clamp technique would have had sufficient sensitivity to detect significant changes in insulin sensitivity after leptin therapy . second , our study is open - label and nonrandomized and lacks a control treatment arm . third , because of our study design , we did not examine the initial effects of leptin therapy prior to 16 weeks . finally , the lack of leptin effects on -cell function in this group of hypoleptinemic individuals can not be generalized to normal healthy individuals . in summary , we report that short - term leptin replacement did not significantly modulate insulin secretion and -cell function in patients with lipodystrophy . our study results do not support the notion that hyperinsulinemia in leptin - deficient or leptin - resistant states is partly due to the lack of tonic and direct insulin - suppressive effects of leptin on the pancreas . it is likely that improvements in fasting hyperinsulinemia and -cell function after prolonged leptin therapy are secondary to changes in insulin sensitivity , glycemia , and dyslipidemia .

objectiveleptin administration is known to directly modulate pancreatic -cell function in leptin - deficient rodent models . however , human studies examining the effects of leptin administration on -cell function are lacking . in this study , we examined the effects ( 1620 weeks ) of leptin replacement on -cell function in patients with lipodystrophy.research design and methodsin a prospective , open - label , currently ongoing study , we studied the effects of leptin replacement on -cell function in 13 patients with congenital or acquired lipodystrophy . insulin secretory rate ( isr ) was calculated by c - peptide deconvolution from plasma glucose and c - peptide levels measured during oral glucose tolerance tests ( ogtts ) performed at baseline and after 1620 weeks of leptin replacement . -cell glucose sensitivity and rate sensitivity were assessed by mathematical modeling of ogtt.resultsthere was a significant decrease in triglycerides , free fatty acids , and glycosylated hemoglobin levels ( a1c ) after leptin therapy . patients with lipodystrophy have high fasting and glucose - stimulated isr . however , leptin therapy had no significant effect on fasting isr , total insulin secretion during ogtt , -cell glucose sensitivity , rate sensitivity , or insulin clearance.conclusionsin contrast to the suppressive effects of leptin on -cell function in rodents , 1620-week treatment with leptin in lipodystrophy patients did not significantly affect insulin secretion or -cell function in leptin - deficient individuals with lipodystrophy .

Introduction Research Design and Methods Study Design and Study Subjects Study Procedures Measures of Insulin Secretion and Resistance Statistical Analyses Results Clinical Characteristics of Study Subjects Leptin Replacement Therapy Effects of Leptin Replacement on Metabolic Parameters, Insulin Sensitivity, and Insulin Secretion Effects of Leptin Replacement on Insulin Clearance Conclusions

loss of these suppressive actions of leptin in mice with selective absence of leptin receptors in the pancreas leads to fasting hyperinsulinemia and increased responsiveness to glucose- and incretin - stimulated insulin secretion ( 57 ) . leptin replacement in patients with lipodystrophy attenuates insulin resistance , hyperinsulinemia , and hyperglycemia ( 1,9,10 ) . although insulin secretion was not assessed in these studies , reductions in insulin levels ( fasting and glucose induced ) were attributed to improvements in insulin sensitivity ( 11,12 ) . a solitary study has reported the effects of leptin replacement on insulin secretion in a genetically leptin - deficient adult male ( 13 ) . in this patient , except for this single report , we are unaware of studies examining the short - term effects of leptin replacement on -cell function . to that end , we report the early effects of short - term ( 1620 weeks ) metreleptin ( an analog of human leptin ) administration on -cell function in patients with lipodystrophy . study subjects reported here are from an open - label , prospective , currently ongoing study examining the long - term safety and clinical effects of metreleptin treatment in patients with congenital or acquired lipodystrophy ( 9,11,12 ) . the current study examined the early effects of leptin replacement on -cell function derived from oral glucose tolerance test ( ogtt ) data . in this report , we include a subgroup of patients ( n = 13 ) who were on stable doses of oral hypoglycemic agents prior to and at 1620 weeks of leptin therapy . ogtts were performed at baseline and after 1620 weeks of leptin administration . during each visit , after an overnight fast , patients underwent an ogtt with plasma sampling at baseline and 30 , 60 , 90 , 120 , and 180 min after glucose ingestion for the measurement of glucose , insulin , and c - peptide concentrations . the -cell model used in the current study , describing the relationship between insulin secretion and glucose concentration , has been described ( 14 ) . the second component represents the dependence of insulin secretion on the rate of change of plasma glucose and depends on the first derivative of plasma glucose concentration against time and a parameter denoted as rate sensitivity . after testing for normality significance of changes in measures of -cell function ( postleptin minus baseline values ) was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . study subjects reported here are from an open - label , prospective , currently ongoing study examining the long - term safety and clinical effects of metreleptin treatment in patients with congenital or acquired lipodystrophy ( 9,11,12 ) . inclusion criteria for leptin replacement included hypoleptinemia ( < 12 ng / ml ) , metabolic abnormalities such as hypertriglyceridemia and/or lipoatrophic diabetes , and the ability to adhere to the leptin replacement protocol . the current study examined the early effects of leptin replacement on -cell function derived from oral glucose tolerance test ( ogtt ) data . in this report , we include a subgroup of patients ( n = 13 ) who were on stable doses of oral hypoglycemic agents prior to and at 1620 weeks of leptin therapy . ogtts were performed at baseline and after 1620 weeks of leptin administration . during each visit , after an overnight fast , patients underwent an ogtt with plasma sampling at baseline and 30 , 60 , 90 , 120 , and 180 min after glucose ingestion for the measurement of glucose , insulin , and c - peptide concentrations . insulin secretory rates ( isrs ) were calculated from deconvolution of the plasma c - peptide concentrations . the second component represents the dependence of insulin secretion on the rate of change of plasma glucose and depends on the first derivative of plasma glucose concentration against time and a parameter denoted as rate sensitivity . significance of changes in measures of -cell function ( postleptin minus baseline values ) was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . thirteen patients with lipodystrophy treated with leptin are included in this study ( congenital generalized lipodystrophy , n = 5 ; acquired generalized lipodystrophy , n = 2 ; familial partial lipodystrophy , n = 5 ; acquired partial lipodystrophy , n = 1 ) . patients were on stable doses of metformin ( n = 12 ) and/or thiazolidinedione ( n = 3 ) prior to and at the end of 1620 weeks of leptin therapy . prior to leptin therapy , median serum leptin levels ( trough ) patients with partial lipodystrophy had higher levels of leptin compared with generalized lipodystrophy ( mean sd 4.1 1.2 vs. 0.7 0.3 , p = 0.002 ) . all of our patients were diabetic with an average glycosylated hemoglobin level ( a1c ) of 7.2% with mean fasting plasma glucose levels < 6.6 mmol / l , suggesting that glycemic control was good prior to initiation of leptin . as previously reported , these patients had high levels of triglycerides and free fatty acids ( ffas ) ( table 1 ) , and various metabolic parameters were similar in partial and generalized lipodystrophy patients ( data not shown ) . clinical characteristics and metabolic parameters before and after leptin replacement in patients with lipodystrophy the initial dose of metreleptin was based on weight , sex , and age , and subsequent dose adjustments were based on individual metabolic responses of patients . leptin replacement was associated with a significant decrease in total cholesterol , ldl , triglyceride , and ffa levels with no change in hdl levels ( table 1 ) . however , area under the curve for glucose ( aucg ) during ogtt was lower after leptin therapy ( 1,816 731 vs. 2,063 598 mmol l 180 min , p = 0.02 ) . surrogate measures of insulin sensitivity , ogis and isi , were not significantly modulated , but tended to increase ( 25% ) with leptin replacement ( table 1 ) . these changes in insulin sensitivity and the lower glucose auc partially explain the effects of leptin on a1c . mean basal insulin secretion rate prior to leptin initiation was 324 153 pmol / m / min ( sd ) , and basal insulin secretion rate adjusted for age and insulin sensitivity ( ogis ) was not significantly different after leptin replacement ( 345 254 pmol / m / min , p = 0.10 ) . similarly , leptin administration had no significant effect on total insulin secretion ( post vs. pre : 111 42 vs. 170 123 nmol / m , p = 0.13 ) . leptin treatment had no significant effect on glucose sensitivity ( post vs. pre ) : 65 ( 112 ) vs. 48 ( 167 ) pmol / min / m / mm , p = 0.39 . likewise , rate sensitivity , a measure of the insulin secretory component that responds to rapid changes in plasma glucose concentrations , was also unaltered with treatment : 206 ( 1,449 ) vs. 1,215 ( 6,915 ) pmol / min / m / mm , p = 0.13 . age , duration of diabetes , race , sex , and type of lipodystrophy may potentially modify the effects of leptin . however , duration of diabetes was unrelated to basal isr ( r = 0.14 , p = 0.68 ) or total isr ( r = 0.41 , p = 0.18 ) , and the significance of changes in measures of -cell function after leptin therapy remained unaltered after adjusting for duration of diabetes . the effect of leptin treatment on parameters of -cell function was not significantly different between partial and generalized lipodystrophy patients ( pgroup time interaction > 0.5 ) . thus , these results suggest that leptin replacement in the short - term had no significant effect on insulin secretion or -cell glucose responsiveness . insulin secretion and -cell glucose sensitivity in patients with lipodystrophy before and after leptin replacement . fasting isr ( a ) , total insulin output after an oral glucose load ( b ) , and model - derived dose response between isr and plasma glucose concentration ( c ) . significance of changes in measures of -cell function ( post - leptin minus baseline values ) in panels a and b was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . basal insulin clearance ( fasting isrs / fasting insulin ) and ogtt - insulin clearance ( incremental aucs of isrs / incremental aucs of insulin concentrations ) rates were measured prior to and after leptin therapy . however , ogtt - insulin clearance rates were significantly higher in patients with partial lipodystrophy ( 1.25 0.33 vs. 0.80 0.25 l / min / m , p = 0.02 ) . in the overall cohort , leptin treatment had no significant effect on basal ( post vs. pre : 1.97 1.04 vs. 1.81 0.57 l / min / m , p = 0.62 ) or ogtt - insulin clearance ( 1.12 0.39 vs. 1.00 0.36 l / min / m , p = 0.39 ) . thirteen patients with lipodystrophy treated with leptin are included in this study ( congenital generalized lipodystrophy , n = 5 ; acquired generalized lipodystrophy , n = 2 ; familial partial lipodystrophy , n = 5 ; acquired partial lipodystrophy , n = 1 ) . patients were on stable doses of metformin ( n = 12 ) and/or thiazolidinedione ( n = 3 ) prior to and at the end of 1620 weeks of leptin therapy . prior to leptin therapy , median serum leptin levels ( trough ) patients with partial lipodystrophy had higher levels of leptin compared with generalized lipodystrophy ( mean sd 4.1 1.2 vs. 0.7 0.3 , p = 0.002 ) . all of our patients were diabetic with an average glycosylated hemoglobin level ( a1c ) of 7.2% with mean fasting plasma glucose levels < 6.6 mmol / l , suggesting that glycemic control was good prior to initiation of leptin . as previously reported , these patients had high levels of triglycerides and free fatty acids ( ffas ) ( table 1 ) , and various metabolic parameters were similar in partial and generalized lipodystrophy patients ( data not shown ) . leptin replacement was associated with a significant decrease in total cholesterol , ldl , triglyceride , and ffa levels with no change in hdl levels ( table 1 ) . however , area under the curve for glucose ( aucg ) during ogtt was lower after leptin therapy ( 1,816 731 vs. 2,063 598 mmol l 180 min , p = 0.02 ) . surrogate measures of insulin sensitivity , ogis and isi , were not significantly modulated , but tended to increase ( 25% ) with leptin replacement ( table 1 ) . these changes in insulin sensitivity and the lower glucose auc partially explain the effects of leptin on a1c . mean basal insulin secretion rate prior to leptin initiation was 324 153 pmol / m / min ( sd ) , and basal insulin secretion rate adjusted for age and insulin sensitivity ( ogis ) was not significantly different after leptin replacement ( 345 254 pmol / m / min , p = 0.10 ) . similarly , leptin administration had no significant effect on total insulin secretion ( post vs. pre : 111 42 vs. 170 123 nmol / m , p = 0.13 ) . model - derived -cell insulin secretion and glucose dose responses obtained from ogtts administered pre- and post - leptin therapy are shown in fig . leptin treatment had no significant effect on glucose sensitivity ( post vs. pre ) : 65 ( 112 ) vs. 48 ( 167 ) pmol / min / m / mm , p = 0.39 . likewise , rate sensitivity , a measure of the insulin secretory component that responds to rapid changes in plasma glucose concentrations , was also unaltered with treatment : 206 ( 1,449 ) vs. 1,215 ( 6,915 ) pmol / min / m / mm , p = 0.13 . age , duration of diabetes , race , sex , and type of lipodystrophy may potentially modify the effects of leptin . however , duration of diabetes was unrelated to basal isr ( r = 0.14 , p = 0.68 ) or total isr ( r = 0.41 , p = 0.18 ) , and the significance of changes in measures of -cell function after leptin therapy remained unaltered after adjusting for duration of diabetes . the effect of leptin treatment on parameters of -cell function was not significantly different between partial and generalized lipodystrophy patients ( pgroup time interaction > 0.5 ) . thus , these results suggest that leptin replacement in the short - term had no significant effect on insulin secretion or -cell glucose responsiveness . insulin secretion and -cell glucose sensitivity in patients with lipodystrophy before and after leptin replacement . fasting isr ( a ) , total insulin output after an oral glucose load ( b ) , and model - derived dose response between isr and plasma glucose concentration ( c ) . significance of changes in measures of -cell function ( post - leptin minus baseline values ) in panels a and b was calculated by one - way ancova adjusted for age , value of the dependent variable at baseline , and changes in ogis . basal insulin clearance ( fasting isrs / fasting insulin ) and ogtt - insulin clearance ( incremental aucs of isrs / incremental aucs of insulin concentrations ) rates were measured prior to and after leptin therapy . however , ogtt - insulin clearance rates were significantly higher in patients with partial lipodystrophy ( 1.25 0.33 vs. 0.80 0.25 l / min / m , p = 0.02 ) . in the overall cohort , leptin treatment had no significant effect on basal ( post vs. pre : 1.97 1.04 vs. 1.81 0.57 l / min / m , p = 0.62 ) or ogtt - insulin clearance ( 1.12 0.39 vs. 1.00 0.36 l / min / m , p = 0.39 ) . in the current study , leptin replacement therapy had no significant impact on -cell function in patients with lipodystrophy treated for 1620 weeks . the effect of leptin on human -cell function and insulin secretion has not been thoroughly investigated . in that context , our study results suggest that , contrary to the suppressive effects of leptin on insulin secretion consistently observed in animal and in vitro studies , leptin replacement does not attenuate the increased insulin secretion observed in these patients . leptin replacement in lipodystrophy patients is associated with reductions in fasting insulin levels and after an oral glucose load after 12 months ( 11,12 ) . in contrast , in another study , mean serum levels of insulin after an ogtt remained unaltered in patients with congenital generalized lipodystrophy ( n = 5 ) and increased in acquired generalized lipodystrophy ( n = 2 ) despite improvements in insulin sensitivity and glycemia after 4 months of leptin replacement ( 16 ) . insulin secretion in one genetically leptin - deficient adult male was deduced by deconvolution of c - peptide levels obtained during a meal ( 13 ) . in the first week after leptin replacement , however , hepatic extraction of insulin was increased simultaneously , resulting in lower peripheral insulin levels ( by 20% ) . after 18 and 24 months of leptin therapy , insulin secretion was reduced along with improvements in insulin sensitivity . this degree of hyperinsulinemia afforded us the opportunity to evaluate the potential inhibitory effects of leptin on insulin secretion . nonetheless , we did not observe any significant effects of leptin replacement on fasting insulin secretion or total insulin output during an ogtt ( fig . -cell glucose sensitivity is impaired in individuals with diabetes and glucose intolerance ( 17,18 ) . systemic infusion of leptin in normal or leptin - deficient hyperinsulinemic ob / ob mice , but not leptin receptor deficient db / db mice , acutely ( within minutes ) decreases plasma insulin levels ( 4 ) . in ob / ob mice , leptin administration for 24 these direct insulin - suppressing effects were also observed during short - term ( 4-week ) leptin replacement in ob / ob mice prior to any significant changes in body weight or food intake ( 21 ) . the reasons for the discrepancy in the results from our study and leptin - deficient rodent models are unclear . we have previously shown that leptin replacement for 1620 weeks decreases hepatic steatosis in individuals with lipodystrophy ( 24,25 ) . however , we are not aware of any human studies that have examined the effects of leptin on pancreatic fat content in patients with non - hiv lipodystrophy . thus , species - dependent and tissue - specific antisteatotic effects of leptin may explain the discrepancy in the effects of leptin in rodents and humans . leptin inhibits insulin secretion from human islets in vitro ( 4,8 ) ; however , studies examining the acute effects of agonist and/or glucose - stimulated insulin secretion in healthy individuals are necessary to rule out species - dependent in vivo effects of leptin . we also did not observe any significant improvements in insulin sensitivity as measured by ogis and isi after leptin replacement ( table 1 ) . one of the early effects of leptin treatment is an increase in insulin clearance ( 13 ) . however , 1620 weeks of leptin replacement did not modulate insulin clearance in this study . in this study , we chose to report the early effects of leptin replacement on -cell function . leptin therapy decreases fasting glucose levels , improves glucose tolerance and insulin sensitivity , reduces a1c , lowers triglycerides , and attenuates steatohepatitis in lipodystrophy ( 911,25 ) . improvements in insulin sensitivity and changes in ectopic lipid accumulation have been suggested to mediate these effects of leptin . future studies examining the short- and long - term effects on -cell function and pancreatic fat content are warranted to confirm the long - term effects of leptin on pancreatic function . first , we derived measures of -cell function by mathematically modeling plasma glucose and c - peptide levels measured during ogtt and used surrogate indices ( ogis and isi ) to assess insulin sensitivity . nonetheless , the effects of leptin on -cell glucose sensitivity to first - phase insulin response can not be adequately assessed during an ogtt . third , because of our study design , we did not examine the initial effects of leptin therapy prior to 16 weeks . finally , the lack of leptin effects on -cell function in this group of hypoleptinemic individuals can not be generalized to normal healthy individuals . in summary , we report that short - term leptin replacement did not significantly modulate insulin secretion and -cell function in patients with lipodystrophy . our study results do not support the notion that hyperinsulinemia in leptin - deficient or leptin - resistant states is partly due to the lack of tonic and direct insulin - suppressive effects of leptin on the pancreas . it is likely that improvements in fasting hyperinsulinemia and -cell function after prolonged leptin therapy are secondary to changes in insulin sensitivity , glycemia , and dyslipidemia .

survival of biological systems crucially depends on robustness to harmful genetic mutations , i.e. genetic robustness , and to changes in environmental conditions ( 13 ) first , alternative signaling and metabolic pathways provide an important mechanism for rerouting in many molecular networks ( 4,5 ) . second , a major role in genetic robustness is attributed to gene duplicates ( 1,6 ) . gene duplications are frequent in evolution and range in size from small - scale ( ssd ) to whole - genome events ( wgd ) ( 7,8 ) . while in 90% of the cases one duplicate is eventually lost in evolution ( 6 ) , duplicated genes that remain in the genome can , at least partially , backup each other 's functions . importantly , functional compensation by duplicates plays a significant role in buffering deleterious human mutations ( 9 ) . duplicates that acquire distinct molecular functions ( mfs ) are naturally unable to compensate for one another . in addition , even if mf is conserved , incomplete compensation between duplicates is possible owing to different expression patterns or dosage effects . gene duplications are the major source of new genes ( 10 ) and several conceptual models of duplicates evolution have been proposed ( 11,12 ) . in the neofunctionalization model one duplicate gains new functions , i.e. functions not associated with the ancestral gene , while the other duplicate retains the ancestral functions ( 10,13,14 ) . in contrast , in the subfunctionalization model both duplicates become indispensable and are retained in evolution by partitioning the ancestral gene functions ( 15,16 ) . both these models imply an eventual loss of the ability of duplicates to fully substitute for each other . it is also likely that a significant fraction of duplicates are fixed and retained in genomes owing to selective advantages , such as dosage effects or condition - specific expression patterns , present from the moment of duplication ( 17,18 ) . in cases of fixation due to a selective advantage even though full compensation between duplicates is not expected in the long term , the ability of duplicates to buffer deleterious mutations of their paralogs has been now demonstrated by several independent observations . these include a lower than expected fraction of essential genes with close duplicates ( 1 ) , a paucity of pairwise epistatic interactions involving duplicated genes ( 19 ) , and an excess of aggravating genetic interactions between paralogs ( 20,21 ) . the contribution of duplicates to robustness has been primarily considered in the context of qualitative or quantitative growth phenotypes either in nutrient rich or in a small number of laboratory conditions ( 1,22,23 ) . although popular in experiments , these conditions are unlikely to approximate well a natural milieu of living systems , which are constantly bombarded by a diverse array of environmental stresses and stimuli . perhaps more importantly , even if there is a strong compensatory interaction between a pair of duplicates , an evolutionary relevant decrease in fitness can still persist due to an incomplete buffering after a damaging mutation in one of the duplicates ( 24 ) . in the context of long - term evolution , there may not be much difference between mutations leading to the lethal phenotype and mutations associated with a fitness decrease substantially larger than the inverse of the effective population size ( 25,26 ) . given that typical population sizes of free - living microbial species are large ( > 1010 ) ( 27 ) , even a small fitness decrease can be effectively lethal for these organisms . consequently , quantitative analyses of growth phenotypes , preferably in multiple environmental conditions , are necessary to understand the extent to which compensation between duplicates plays an important role in natural biological populations . here we perform such an analysis and show that in the context of natural populations , genetic buffering mediated by duplicates is likely to be rare and , surprisingly , it is not a monotonic function of duplicates divergence . gene and protein sequences for saccharomyces cerevisiae , saccharomyces paradoxus , saccharomyces bayanus , saccharomyces castelli , saccharomyces mikatae , saccharomyces kudriavzevii and saccharomyces kluyveri were obtained from the saccharomyces genome database ( sgd ; http://downloads.yeastgenome.org/ ) and the study by kellis et al . pairs of gene duplicates were identified by sequence homology between proteins within each genome using blastp ( 29 ) . only duplicates that were bidirectional best hits and could be aligned by > 80% of each open reading frame s sequence length were considered in our analysis ( 30 ) . following previous studies ( 1 ) , we excluded ribosomal genes from the analysis owing to their high expression , dominant impact on growth and strong codon adaptation bias . evolutionary distances between duplicated genes were estimated using the method of yang and nielsen ( 31 ) implemented in the paml package ( 32 ) ; the use of other methods , such as maximum likelihood , to estimate ka and ks did not significantly change the observed patterns ( supplementary figure s1a ) . ( 33 ) to measure the fitness contribution of duplicates across multiple environmental conditions and chemical perturbations . using a p - value cutoff of 0.01 , we obtained the number of experimental conditions for which a growth defect was observed for every single gene deletion mutant . we also analyzed quantitative growth measurements for double and single deletion yeast strains obtained from deluna et al . ( 36 ) . to functionally characterize duplicated genes , gene ontology ( go ) ( 37 ) annotations were collected from sgd and enzyme commission ( ec ) annotations from the comprehensive yeast genome database ( cygd ) ( 38 ) . ( 40 ) , codon adaptation index ( cai ) calculations based on the data set by lu et al . hillenmeyer et al . ( 33 ) quantified growth phenotypes of single - gene yeast deletion strains in a large collection of environmental conditions . the assembled data set contains 5.5 million phenotypes of heterozygous and homozygous mutants in 400 conditions . the sampled conditions represent 27 different environmental stresses and hundreds of perturbations with diverse chemical compounds . environmental stresses comprised different growth media , media lacking specific vitamins or amino acids , as well as different ph and temperature regimes . this comprehensive collection of phenotypes allowed us to investigate in detail the diversification of duplicates functions and their contribution to genetic robustness in multiple conditions . we first investigated how the average number of sensitive conditions , i.e. conditions with a significant growth decrease due to deletion of one duplicate , depends on sequence divergence ( ka ) between the duplicated genes ( figure 1a and b ) . we considered the fraction of different conditions with a growth phenotype as a quantitative measure of compensation capacity for duplicates at various divergence distances . for close duplicates the average number of sensitive conditions is not significantly different from that of a random pair of yeast singletons ( figure 1b , horizontal line ) . importantly , this result does not imply that random gene pairs and close duplicates are equivalent in terms of the similarity of their mf . as we demonstrate below , the observed pattern is likely due to a higher overall functional load of close duplicates . here and throughout the article we use the term functional load of a gene to characterize the average fitness decrease across considered conditions due to the gene deletion ; we note that , based on the definition above , the functional load is not a measure of the total number of mfs a gene has , but it reflects the gene s overall fitness contribution . figure 1.compensation patterns between yeast duplicates as a function of their evolutionary divergence , ka , the number of nonsynonymous substitutions per site . ( a ) scatterplot of the fraction of sensitive conditions , i.e. conditions with detectable growth phenotypes resulting from duplicate gene deletion , versus ka . the horizontal lines in a and b indicate the average fraction of sensitive conditions for a random pair of yeast singletons . ( c ) the average fraction of essential duplicates , i.e. duplicates with a lethal phenotype on deletion , as a function of ka . ( d ) fraction of conditions with a significant growth decrease for deletion of yeast duplicates arising from small - scale ( ssd ) and whole - genome duplications ( wgd ) . the duplicates were classified as ssd or wgd based on the study by kellis et al . ( 8) the horizontal line shows the average fraction of sensitive conditions for a random pair of yeast singletons . in the figures , compensation patterns between yeast duplicates as a function of their evolutionary divergence , ka , the number of nonsynonymous substitutions per site . ( a ) scatterplot of the fraction of sensitive conditions , i.e. conditions with detectable growth phenotypes resulting from duplicate gene deletion , versus ka . the horizontal lines in a and b indicate the average fraction of sensitive conditions for a random pair of yeast singletons . ( c ) the average fraction of essential duplicates , i.e. duplicates with a lethal phenotype on deletion , as a function of ka . ( d ) fraction of conditions with a significant growth decrease for deletion of yeast duplicates arising from small - scale ( ssd ) and whole - genome duplications ( wgd ) . the duplicates were classified as ssd or wgd based on the study by kellis et al . ( 8) the horizontal line shows the average fraction of sensitive conditions for a random pair of yeast singletons . in the figures , interestingly , the number of sensitive conditions initially drops as duplicates diverge , decreasing about 30% at the distances corresponding to ka 0.1 ( ks 1 , see supplementary figure s2a and b ) . as duplicates diverge further , the average number of sensitive conditions increases again , reaching the average for a random pair of yeast singletons at ka 0.25 . the trend shown in figure 1b is not sensitive to the p - value cutoff used to determine the significance of the growth decrease observed in mutant strains ( supplementary figure s3 ) . a similar trend was also observed for the average growth decrease ( functional load ) , measured either by log ratios or z - scores across all tested conditions ( supplementary figure s4a and b ) . bin - free analyses of the data ( supplementary figures s1b and c and s2b ) also revealed a smaller fitness cost due to the loss of duplicates at intermediate distances ( ka 0.1 ) . because most actively growing wild - type yeast populations are diploid ( 42 ) , we mainly focused our analysis on heterozygous mutant strains . the patterns of functional compensation for heterozygous and homozygous mutants are similar when multiple - drug resistance genes , as defined by hillenmeyer et al . the trends also remain similar when only environmental perturbations are analyzed in the homozygous experiments ( supplementary figure s4d ) . we also checked that the observed compensation patterns due to closest duplicates are not significantly influenced by additional , i.e. more diverged , paralogs ( supplementary figure s4e ) . this lack of significant compensation by diverged duplicates results in an approximately linear relationship between the number of sensitive conditions per yeast protein family and the family size ( supplementary figure s5 ) . finally , the observed compensation patterns were not affected by removal of gene pairs with a high cai ( supplementary figure s6a ) , suggesting that the observed trend can not be explained by expression - based constraints on the rate of duplicate sequence evolution ( ka ) ( 43 ) or high expression levels of certain duplicates . it is interesting to compare the ability of duplicates to buffer mutations leading to any detectable growth decrease beyond a given fitness threshold ( figure 1b ) and their role in protecting against the no - growth phenotype , i.e. the likelihood to observe essential genes in duplicate pairs . in figure 1c , using data from the study by giaever et al . ( 36 ) , we show the fraction of essential duplicates as a function of their divergence . in agreement with previous studies ( 1,22,23 ) we found that the fraction of essential genes remains low and approximately constant for close duplicates , and increases substantially only at divergence distances corresponding to ka > 0.4 . notably , this pattern is qualitatively different from the compensation for quantitative growth phenotypes ( figure 1b ) , demonstrating the aforementioned impact of using quantitative phenotypes to assess the evolutionarily relevant consequences of mutations . also in contrast to patterns obtained in studies based on essential genes ( 22 ) , we observed similar compensation profiles for gene pairs originating from small - scale and genome - wide duplications ( figure 1d ) . because all wgd duplicates have the same age , this result suggests that the ability of duplicates to buffer each other s function across multiple conditions depends more strongly on their sequence divergence than on the time since duplication . it is likely that the observed decrease in the number of sensitive conditions at intermediate divergence distances ( ka 0.1 ) is due to a decrease of the functional load carried at these distances by the union of duplicate genes . to explore this possibility , we considered the quantitative fitness data from deluna et al . ( 34 ) and the synthetic genetic array ( sga ) data from costanzo et al . ( 35 ) . in these studies , the authors performed quantitative growth measurements of yeast strains with individual and simultaneous deletions of duplicates . using the single deletion phenotypes from the deluna et al . . studies ( figure 2b ) , we observed fitness profiles similar to the one obtained based on the data from hillenmeyer et al . ( figure 1b ) as a function of ka , with smaller phenotypic effects at intermediate distances . interestingly , the overall functional load of duplicate pairs , measured by the phenotype of double deletions , indeed substantially decreases with their divergence ( figure 2c and d ) . this result suggests that while close duplicates are more likely to have similar functions , their higher functional load makes complete compensation less likely . because the overall functional load of duplicates remains approximately constant for ka > 0.15 , the higher fraction of detectable growth phenotypes at these distances is likely due to a decreased ability for functional compensation as duplicates diverge . compensation between duplicates quantified by the presence of aggravating interactions between duplicate pairs decreases as a function of sequence divergence ( figure 2e and f ) [ see ( 21 ) ] . figure 2.growth phenotypes for individual and simultaneous deletion of duplicates as a function of their sequence divergence ( ka ) . the results in the first column ( a , c , e ) are based on the competition experiments by deluna et al . ( 34 ) , and in the second column ( b , d , f ) on the synthetic genetic arrays ( sga ) by costanzo et al . ( 35 ) . ( a , b ) fractions of single duplicate deletions with a significant growth decrease . ( c , d ) fractions of simultaneous ( double ) duplicate deletions with a significant growth decrease . due to different measurement sensitivities of the two studies , different cutoffs were used to determine a significant growth decrease : 1% for deluna et al . ( b , d ) ; the presented results are not sensitive to the exact cutoff values ( see supplementary figure s7 ) . ( e , f ) fraction of paralogs with a significant negative epistatic interaction from the studies of deluna et al . and growth phenotypes for individual and simultaneous deletion of duplicates as a function of their sequence divergence ( ka ) . the results in the first column ( a , c , e ) are based on the competition experiments by deluna et al . ( 34 ) , and in the second column ( b , d , f ) on the synthetic genetic arrays ( sga ) by costanzo et al . ( a , b ) fractions of single duplicate deletions with a significant growth decrease . ( c , d ) fractions of simultaneous ( double ) duplicate deletions with a significant growth decrease . due to different measurement sensitivities of the two studies , different cutoffs were used to determine a significant growth decrease : 1% for deluna et al . ( b , d ) ; the presented results are not sensitive to the exact cutoff values ( see supplementary figure s7 ) . ( e , f ) fraction of paralogs with a significant negative epistatic interaction from the studies of deluna et al . and besides a smaller overall functional load , it is possible that duplicates at intermediate distances have other properties that favor genetic robustness . to explore this possibility , for each duplicate pair , we looked at the gene with the largest and the gene with the smallest number of sensitive conditions ( figure 3a ) . notably , while the duplicate with more conditions ( figure 3a , more sensitive duplicate ) follows the average trend for all duplicates ( figure 1b ) , the duplicate with fewer conditions ( figure 3a , less sensitive duplicate ) shows a steady gain in the number of conditions as a function of ka . consequently , the functional load of close duplicates , measured by the number of sensitive conditions , is different , and this difference becomes significantly smaller as the genes diverge ( figure 3b . r = 0.64 , p = 7 10 , see also supplementary figure s2c ) . close duplicates with the larger number of sensitive conditions also show a higher evolutionary constraint , evaluated by the normalized ratio of nonsynonymous to synonymous substitutions per nucleotide site , ka / ks ( wilcoxon signed rank test p = 7 10 , figure 2c ) . this result agrees with previous reports of asymmetric evolution of duplicates in the context of co - expression , genetic interaction and protein for example , if the less sensitive duplicate is expressed only under specific environmental conditions . ( a ) the average fraction of sensitive conditions for the duplicates with the higher and lower number of sensitive conditions in each pair ; ka values represent sequence divergence between duplicates . ( b ) the relative difference in the number of sensitive conditions between duplicates as a function of their initial divergence ; ka values represent sequence divergence between duplicates . the relative difference was calculated as the absolute difference in the number of sensitive conditions between duplicates normalized to the total number of sensitive conditions for the pair ( spearman s r = 0.60 , p = 2 10 ; pearson s r = 0.64 , p = 7 10 ) . ( c ) the average ka / ks ratio for the paralogs with the largest ( more sensitive ) and smallest ( less sensitive ) number of conditions with a significant growth decrease . ka / ks ratios were calculated relative to orthologous sequences in s. bayanus . only duplicates with ka ( a ) the average fraction of sensitive conditions for the duplicates with the higher and lower number of sensitive conditions in each pair ; ka values represent sequence divergence between duplicates . ( b ) the relative difference in the number of sensitive conditions between duplicates as a function of their initial divergence ; ka values represent sequence divergence between duplicates . the relative difference was calculated as the absolute difference in the number of sensitive conditions between duplicates normalized to the total number of sensitive conditions for the pair ( spearman s r = 0.60 , p = 2 10 ; pearson s r = 0.64 , p = 7 10 ) . ( c ) the average ka / ks ratio for the paralogs with the largest ( more sensitive ) and smallest ( less sensitive ) number of conditions with a significant growth decrease . ka / ks ratios were calculated relative to orthologous sequences in s. bayanus . only duplicates with ka the p - value is for the wilcoxon signed rank test . to further explore the mechanism behind the observed backup patterns , we analyzed the functional diversification of yeast duplicates as a function of their sequence divergence ( ka ) . first , for genes encoding metabolic enzymes we calculated the fraction of gene pairs with conserved ec numbers ( figure 4a ) ; the conservation of ec numbers indicates that corresponding proteins catalyze identical biochemical reactions . second , we calculated the fraction of shared go terms describing protein mf for all duplicates ( figure 4b ) . both measures showed that the mf of yeast duplicates typically starts to substantially diverge only at about ka > 0.4 . the timing of this divergence approximately coincides with a significant increase in the fraction of essential duplicates ( figure 1c ) . on the other hand , the significant changes in the number of quantitative growth phenotypes are observed when the mf of duplicates is usually still conserved . ( a ) fraction of metabolic duplicates sharing the same ec numbers ; conservation of ec numbers indicates catalysis of identical biochemical reactions . ( c ) fraction of go biological process ( bp ) terms shared between duplicates . in panels b and c we considered only go terms with a distance of three or more to the corresponding go root hierarchy term . ( d ) dashed line , the average number of different transcription factor binding motifs per duplicate pair . transcription factor ( tf ) binding motifs were compiled from the studies of kafri et al . solid line , the average number of transcription factor deletions in s. cerevisiae that significantly affect the expression of duplicate genes . the data were obtained from the study by hu et al . ( 47 ) . for comparison we also show the average number of motifs and tf mutants affecting expression for random pairs of yeast singletons ( horizontal dashed and solid lines ) ; the p - values were calculated using the mann ( e ) the average dosage compensation ( responsiveness ) of duplicates as a function of sequence divergence ( ka ) . the data for the average expression responsiveness was obtained from the work of springer et al . responsiveness was measured in diploid yeast strains as the log2 ratio ( perturbed versus normal ) of expression changes for the remaining gene copy following deletion of the equivalent gene copy on a sister chromosome . ( a ) fraction of metabolic duplicates sharing the same ec numbers ; conservation of ec numbers indicates catalysis of identical biochemical reactions . ( c ) fraction of go biological process ( bp ) terms shared between duplicates . in panels b and c we considered only go terms with a distance of three or more to the corresponding go root hierarchy term . ( d ) dashed line , the average number of different transcription factor binding motifs per duplicate pair . transcription factor ( tf ) binding motifs were compiled from the studies of kafri et al . solid line , the average number of transcription factor deletions in s. cerevisiae that significantly affect the expression of duplicate genes . the data were obtained from the study by hu et al . ( 47 ) . for comparison we also show the average number of motifs and tf mutants affecting expression for random pairs of yeast singletons ( horizontal dashed and solid lines ) ; the p - values were calculated using the mann ( e ) the average dosage compensation ( responsiveness ) of duplicates as a function of sequence divergence ( ka ) . the data for the average expression responsiveness was obtained from the work of springer et al . responsiveness was measured in diploid yeast strains as the log2 ratio ( perturbed versus normal ) of expression changes for the remaining gene copy following deletion of the equivalent gene copy on a sister chromosome . a complementary analysis of transcription factor binding sites suggests that gene regulation plays an important role in establishing the observed compensation patterns . it was previously demonstrated that duplicated yeast genes have , on average , a higher number of cis - regulatory motifs than singleton genes ( 46 ) . using a comprehensive data set of 150 known and predicted dna binding motifs in yeast ( 28,39 ) , we found that the average number of different motifs regulating a duplicate pair increases significantly at ka 0.1 ( figure 4d , dashed line , mann the average number of different motifs per duplicate pair is more than twice the number of motifs for a pair of yeast singletons ( figure 4d , dashed horizontal line ) . the number of regulatory motifs increases both for the duplicate with the highest and the duplicate with the smallest number of sensitive conditions ( supplementary figure s8a and b ) . the increase in complexity of the duplicates regulation at ka 0.1 is also confirmed by a significant increase ( mann whitney u test , p = 1 10 ) at these distances of the number of transcription factor mutants ( 47 ) affecting duplicate gene expression ( figure 4d , solid line ) . while the total number of dna motifs regulating duplicates initially increases with divergence , the fraction of shared motifs [ supplementary figure s9a , see also ( 48 ) ] , the overlap in go terms describing biological processes ( figure 4c ) and the overlap in cellular localization observed in fluorescence - tagging experiments ( 40 ) decrease ( supplementary figure s8b ) . such a pattern suggests that the increase in regulatory complexity allows duplicates to specialize for different biological processes while mostly preserving common mfs . the ability of duplicates with partially diverged regulatory regions to compensate for each other through expression changes of the intact gene was previously described by kafri et al . ( 50 ) showed that on deletion of one duplicate , expression changes of the remaining paralog are often need - based , i.e. they happen primarily when the corresponding function is required . such regulatory backup circuits should , at least in some cases , enable functional compensation between homologs with different expression patterns in wild type . ( 51 ) , who measured the expression changes of yeast genes when one of two genomic copies was deleted in diploid cells , we observed a significant dosage response only for genes forming recently duplicated pairs ( ka < 0.15 , figure 4e ) . finally , the patterns of diversification and functional compensation described above should correlate with the process of duplicate loss in evolution . we investigated the retention of yeast duplicates using the complete genomic sequences of seven species : s. cerevisiae , s. paradoxus , s. bayanus , s. castelli , s. mikatae , s. kudriavzevii and s. kluyveri . we calculated the number of remaining duplicates as a function of their sequence divergence ( figure 5 , see also supplementary figure s10a for the corresponding relationships in individual yeast species ) . this analysis suggests that a relatively brief initial period of high duplicate loss ( 6 ) is followed by a long evolutionary period ( ka > 0.1 ) during which the average loss rate decreases > 10-fold ( red in figure 5 ) . interestingly , the loss rate significantly decreases approximately at the divergence distance when duplicates become more similar in terms of their functional load ( figure 2b ) and when their regulatory complexity significantly increases ( figure 5d ) . it is likely that the duplicates surviving the initial loss stage develop independent functionalities and are preserved for long times in the genomes of yeast species . figure 5.the average number of duplicates retained in the genomes of yeast species as a function of the duplicates divergence ka , the number of nonsynonymous substitutions per site . the number of remaining duplicates was averaged over the genomes of seven yeast species : s. cerevisiae , s. paradoxus , s. bayanus , s. castelli , s. mikatae , s. kudriavzevii and s. kluyveri . see also supplementary figure s10 for the number of remaining duplicates in the individual species and for the number of remaining duplicates as a function of ks . the rate of duplicate loss in evolution is > 10 times lower for the distances corresponding to ka > 0.1 compared with the distances at ka < 0.1 . in the figure , error bars represent the sem . the average number of duplicates retained in the genomes of yeast species as a function of the duplicates divergence ka , the number of nonsynonymous substitutions per site . the number of remaining duplicates was averaged over the genomes of seven yeast species : s. cerevisiae , s. paradoxus , s. bayanus , s. castelli , s. mikatae , s. kudriavzevii and s. kluyveri . see also supplementary figure s10 for the number of remaining duplicates in the individual species and for the number of remaining duplicates as a function of ks . the rate of duplicate loss in evolution is > 10 times lower for the distances corresponding to ka > 0.1 compared with the distances at ka < 0.1 . in the figure , in the present study , we analyzed genetic robustness due to duplicates in the context of quantitative growth phenotypes and sensitivities to gene deletions in multiple environmental conditions . such robustness is important for understanding the buffering of deleterious mutations in large natural biological populations . our results demonstrate that , contrary to commonly held view , close gene duplicates are unlikely to provide a high level of backup in the context of large natural populations . consequently , it is unlikely that many duplicates are fixed in natural populations specifically due to selection for robustness . our analysis also suggests that duplicate redundancies described in genomics databases , and frequently observed in laboratory experiments , should be considered with caution , at least with respect to their functions in natural biological populations . to investigate this point further ( 52 ) , of 112 yeast duplicates reported to be at least partially redundant in research publications . these duplicates have been described as redundant based on their functional overlap and compensatory interactions observed in small - scale experimental studies . interestingly , based on the number of conditions with quantitative growth phenotypes from the study by hillenmeyer et al . ( 35 ) , the duplicates annotated as redundant are not significantly different from all other yeast duplicates ( mann whitney u , p = 0.13 and 0.35 , respectively , supplementary figure s11 ) . this demonstrates that , although many yeast duplicates indeed may show functional overlap in some laboratory conditions , their compensation properties will probably be significantly less important in large natural populations due to the ability of purifying selection to efficiently prune mutations causing even a small fitness decrease . it is likely that several different factors contribute to the relative paucity of functional compensation between paralogs at small divergence distances . a significant fraction of duplications are likely to be fixed owing to dosage effects ( 17 ) , and functional compensation between such duplicates in the context of natural populations is unlikely . for example , the lack of significant compensation between histone pairs , hta1-hta2 and hht1-hht2 , is likely to be a consequence of their role in maintaining proper histone levels in yeast cells . gene dosage may explain the inability of some duplicates to backup each other , but it is unlikely to be the only explanation . we showed that even when all duplicate pairs with a high cai ( supplementary figure s6a ) or pairs forming known protein complexes ( supplementary figure s6b ) are removed from the analysis , the patterns of functional compensation remain similar . notably , genes with a high cai have been also associated with higher frequencies of interlocus gene conversion ( igc ) ( 53,54 ) . while igc can slow down the rate of duplicates sequence divergence ( 55 ) , analyses based only on wgds with no evidence of igc [ using data recently reported by casola et al . close duplicates are also less likely to compensate for each other probably owing to the aforementioned dichotomy in their functional loads ( figure 3a and b ) . many close duplicates can be classified , based on their activity and breadth of expression , into a major and a minor functional isoforms . for example , the glyceraldehyde-3-phosphate dehydrogenase tdh1 is active under various stress conditions , while its isoenzyme tdh2 is used primarily during exponential growth ( 57 ) . similarly , the ubiquitin conjugating enzyme ubc4 is expressed during exponential growth , while its duplicate ubc5 is active during stationary phase ( 58 ) . the difference in functional load for close yeast duplicates is also consistent with the asymmetric partition of functions , interactions and gene expression , observed between close duplicates in other organisms , for example , arabidopsis and human ( 45,59,60 ) . this suggests that duplicate - dependent compensation in the context of natural populations may be limited in other species as well . our analysis suggests that a typical lifecycle of gene duplicates in yeast consists of several distinct evolutionary stages ( 11,12 ) . in the first stage ( at duplicate distances corresponding to ka < 0.05 ) , duplicates tend to have high overall functional loads and significant asymmetry in the number of sensitive conditions ; both of these factors make complete compensation unlikely . the high functional load of close duplicates suggests that adaptive selection plays an important role in their fixation . in the second stage ( 0.05 < ka < 0.25 ) , as duplicates diverge further , their overall functional load usually decreases . this may happen , for example , due to relaxation of the environmental conditions , which facilitated the original duplicate fixation . the vast majority of duplicates , likely the paralogs with relatively smaller functional loads ( figure 3c ) , are lost at this stage ( figure 5 ) . gene pairs that survive the period of high duplicate loss display more balanced functional loads and complex regulation ; these gene pairs are usually retained for long evolutionary times in yeast genomes ( figure 5 ) . surviving duplicates can provide at least partial compensation at intermediate divergence distances and also serve as an important source of new protein functions . in the third stage ( ka > 0.3 or 70% sequence identity ) , the lifecycle of duplicates is completed when their functional roles diverge , and their quantitative compensation properties become indistinguishable from those of random pairs of yeast singletons . national institutes of health ( nih ) [ gm079759 ] and the national centers for biomedical computing [ u54ca121852 ] .

gene duplications are a major source of evolutionary innovations . understanding the functional divergence of duplicates and their role in genetic robustness is an important challenge in biology . previously , analyses of genetic robustness were primarily focused on duplicates essentiality and epistasis in several laboratory conditions . in this study , we use several quantitative data sets to understand compensatory interactions between saccharomyces cerevisiae duplicates that are likely to be relevant in natural biological populations . we find that , owing to their high functional load , close duplicates are unlikely to provide substantial backup in the context of large natural populations . interestingly , as duplicates diverge from each other , their overall functional load is reduced . at intermediate divergence distances the quantitative decrease in fitness due to removal of one duplicate becomes smaller . at these distances , yeast duplicates display more balanced functional loads and their transcriptional control becomes significantly more complex . as yeast duplicates diverge beyond 70% sequence identity , their ability to compensate for each other becomes similar to that of random pairs of singletons .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING Supplementary Material

genetic robustness , and to changes in environmental conditions ( 13 ) first , alternative signaling and metabolic pathways provide an important mechanism for rerouting in many molecular networks ( 4,5 ) . second , a major role in genetic robustness is attributed to gene duplicates ( 1,6 ) . gene duplications are frequent in evolution and range in size from small - scale ( ssd ) to whole - genome events ( wgd ) ( 7,8 ) . while in 90% of the cases one duplicate is eventually lost in evolution ( 6 ) , duplicated genes that remain in the genome can , at least partially , backup each other 's functions . importantly , functional compensation by duplicates plays a significant role in buffering deleterious human mutations ( 9 ) . duplicates that acquire distinct molecular functions ( mfs ) are naturally unable to compensate for one another . gene duplications are the major source of new genes ( 10 ) and several conceptual models of duplicates evolution have been proposed ( 11,12 ) . in the neofunctionalization model one duplicate gains new functions , i.e. both these models imply an eventual loss of the ability of duplicates to fully substitute for each other . it is also likely that a significant fraction of duplicates are fixed and retained in genomes owing to selective advantages , such as dosage effects or condition - specific expression patterns , present from the moment of duplication ( 17,18 ) . in cases of fixation due to a selective advantage even though full compensation between duplicates is not expected in the long term , the ability of duplicates to buffer deleterious mutations of their paralogs has been now demonstrated by several independent observations . these include a lower than expected fraction of essential genes with close duplicates ( 1 ) , a paucity of pairwise epistatic interactions involving duplicated genes ( 19 ) , and an excess of aggravating genetic interactions between paralogs ( 20,21 ) . the contribution of duplicates to robustness has been primarily considered in the context of qualitative or quantitative growth phenotypes either in nutrient rich or in a small number of laboratory conditions ( 1,22,23 ) . although popular in experiments , these conditions are unlikely to approximate well a natural milieu of living systems , which are constantly bombarded by a diverse array of environmental stresses and stimuli . perhaps more importantly , even if there is a strong compensatory interaction between a pair of duplicates , an evolutionary relevant decrease in fitness can still persist due to an incomplete buffering after a damaging mutation in one of the duplicates ( 24 ) . in the context of long - term evolution , there may not be much difference between mutations leading to the lethal phenotype and mutations associated with a fitness decrease substantially larger than the inverse of the effective population size ( 25,26 ) . consequently , quantitative analyses of growth phenotypes , preferably in multiple environmental conditions , are necessary to understand the extent to which compensation between duplicates plays an important role in natural biological populations . here we perform such an analysis and show that in the context of natural populations , genetic buffering mediated by duplicates is likely to be rare and , surprisingly , it is not a monotonic function of duplicates divergence . pairs of gene duplicates were identified by sequence homology between proteins within each genome using blastp ( 29 ) . following previous studies ( 1 ) , we excluded ribosomal genes from the analysis owing to their high expression , dominant impact on growth and strong codon adaptation bias . evolutionary distances between duplicated genes were estimated using the method of yang and nielsen ( 31 ) implemented in the paml package ( 32 ) ; the use of other methods , such as maximum likelihood , to estimate ka and ks did not significantly change the observed patterns ( supplementary figure s1a ) . ( 33 ) to measure the fitness contribution of duplicates across multiple environmental conditions and chemical perturbations . using a p - value cutoff of 0.01 , we obtained the number of experimental conditions for which a growth defect was observed for every single gene deletion mutant . environmental stresses comprised different growth media , media lacking specific vitamins or amino acids , as well as different ph and temperature regimes . this comprehensive collection of phenotypes allowed us to investigate in detail the diversification of duplicates functions and their contribution to genetic robustness in multiple conditions . conditions with a significant growth decrease due to deletion of one duplicate , depends on sequence divergence ( ka ) between the duplicated genes ( figure 1a and b ) . for close duplicates the average number of sensitive conditions is not significantly different from that of a random pair of yeast singletons ( figure 1b , horizontal line ) . importantly , this result does not imply that random gene pairs and close duplicates are equivalent in terms of the similarity of their mf . as we demonstrate below , the observed pattern is likely due to a higher overall functional load of close duplicates . here and throughout the article we use the term functional load of a gene to characterize the average fitness decrease across considered conditions due to the gene deletion ; we note that , based on the definition above , the functional load is not a measure of the total number of mfs a gene has , but it reflects the gene s overall fitness contribution . figure 1.compensation patterns between yeast duplicates as a function of their evolutionary divergence , ka , the number of nonsynonymous substitutions per site . duplicates with a lethal phenotype on deletion , as a function of ka . in the figures , compensation patterns between yeast duplicates as a function of their evolutionary divergence , ka , the number of nonsynonymous substitutions per site . ( d ) fraction of conditions with a significant growth decrease for deletion of yeast duplicates arising from small - scale ( ssd ) and whole - genome duplications ( wgd ) . in the figures , interestingly , the number of sensitive conditions initially drops as duplicates diverge , decreasing about 30% at the distances corresponding to ka 0.1 ( ks 1 , see supplementary figure s2a and b ) . as duplicates diverge further , the average number of sensitive conditions increases again , reaching the average for a random pair of yeast singletons at ka 0.25 . a similar trend was also observed for the average growth decrease ( functional load ) , measured either by log ratios or z - scores across all tested conditions ( supplementary figure s4a and b ) . bin - free analyses of the data ( supplementary figures s1b and c and s2b ) also revealed a smaller fitness cost due to the loss of duplicates at intermediate distances ( ka 0.1 ) . because most actively growing wild - type yeast populations are diploid ( 42 ) , we mainly focused our analysis on heterozygous mutant strains . the patterns of functional compensation for heterozygous and homozygous mutants are similar when multiple - drug resistance genes , as defined by hillenmeyer et al . the trends also remain similar when only environmental perturbations are analyzed in the homozygous experiments ( supplementary figure s4d ) . we also checked that the observed compensation patterns due to closest duplicates are not significantly influenced by additional , i.e. it is interesting to compare the ability of duplicates to buffer mutations leading to any detectable growth decrease beyond a given fitness threshold ( figure 1b ) and their role in protecting against the no - growth phenotype , i.e. in agreement with previous studies ( 1,22,23 ) we found that the fraction of essential genes remains low and approximately constant for close duplicates , and increases substantially only at divergence distances corresponding to ka > 0.4 . because all wgd duplicates have the same age , this result suggests that the ability of duplicates to buffer each other s function across multiple conditions depends more strongly on their sequence divergence than on the time since duplication . it is likely that the observed decrease in the number of sensitive conditions at intermediate divergence distances ( ka 0.1 ) is due to a decrease of the functional load carried at these distances by the union of duplicate genes . to explore this possibility , we considered the quantitative fitness data from deluna et al . studies ( figure 2b ) , we observed fitness profiles similar to the one obtained based on the data from hillenmeyer et al . interestingly , the overall functional load of duplicate pairs , measured by the phenotype of double deletions , indeed substantially decreases with their divergence ( figure 2c and d ) . this result suggests that while close duplicates are more likely to have similar functions , their higher functional load makes complete compensation less likely . because the overall functional load of duplicates remains approximately constant for ka > 0.15 , the higher fraction of detectable growth phenotypes at these distances is likely due to a decreased ability for functional compensation as duplicates diverge . compensation between duplicates quantified by the presence of aggravating interactions between duplicate pairs decreases as a function of sequence divergence ( figure 2e and f ) [ see ( 21 ) ] . figure 2.growth phenotypes for individual and simultaneous deletion of duplicates as a function of their sequence divergence ( ka ) . the results in the first column ( a , c , e ) are based on the competition experiments by deluna et al . due to different measurement sensitivities of the two studies , different cutoffs were used to determine a significant growth decrease : 1% for deluna et al . and growth phenotypes for individual and simultaneous deletion of duplicates as a function of their sequence divergence ( ka ) . the results in the first column ( a , c , e ) are based on the competition experiments by deluna et al . ( 34 ) , and in the second column ( b , d , f ) on the synthetic genetic arrays ( sga ) by costanzo et al . and besides a smaller overall functional load , it is possible that duplicates at intermediate distances have other properties that favor genetic robustness . to explore this possibility , for each duplicate pair , we looked at the gene with the largest and the gene with the smallest number of sensitive conditions ( figure 3a ) . notably , while the duplicate with more conditions ( figure 3a , more sensitive duplicate ) follows the average trend for all duplicates ( figure 1b ) , the duplicate with fewer conditions ( figure 3a , less sensitive duplicate ) shows a steady gain in the number of conditions as a function of ka . consequently , the functional load of close duplicates , measured by the number of sensitive conditions , is different , and this difference becomes significantly smaller as the genes diverge ( figure 3b . close duplicates with the larger number of sensitive conditions also show a higher evolutionary constraint , evaluated by the normalized ratio of nonsynonymous to synonymous substitutions per nucleotide site , ka / ks ( wilcoxon signed rank test p = 7 10 , figure 2c ) . this result agrees with previous reports of asymmetric evolution of duplicates in the context of co - expression , genetic interaction and protein for example , if the less sensitive duplicate is expressed only under specific environmental conditions . ( a ) the average fraction of sensitive conditions for the duplicates with the higher and lower number of sensitive conditions in each pair ; ka values represent sequence divergence between duplicates . ( b ) the relative difference in the number of sensitive conditions between duplicates as a function of their initial divergence ; ka values represent sequence divergence between duplicates . the relative difference was calculated as the absolute difference in the number of sensitive conditions between duplicates normalized to the total number of sensitive conditions for the pair ( spearman s r = 0.60 , p = 2 10 ; pearson s r = 0.64 , p = 7 10 ) . only duplicates with ka ( a ) the average fraction of sensitive conditions for the duplicates with the higher and lower number of sensitive conditions in each pair ; ka values represent sequence divergence between duplicates . ( b ) the relative difference in the number of sensitive conditions between duplicates as a function of their initial divergence ; ka values represent sequence divergence between duplicates . the relative difference was calculated as the absolute difference in the number of sensitive conditions between duplicates normalized to the total number of sensitive conditions for the pair ( spearman s r = 0.60 , p = 2 10 ; pearson s r = 0.64 , p = 7 10 ) . to further explore the mechanism behind the observed backup patterns , we analyzed the functional diversification of yeast duplicates as a function of their sequence divergence ( ka ) . second , we calculated the fraction of shared go terms describing protein mf for all duplicates ( figure 4b ) . the timing of this divergence approximately coincides with a significant increase in the fraction of essential duplicates ( figure 1c ) . on the other hand , the significant changes in the number of quantitative growth phenotypes are observed when the mf of duplicates is usually still conserved . for comparison we also show the average number of motifs and tf mutants affecting expression for random pairs of yeast singletons ( horizontal dashed and solid lines ) ; the p - values were calculated using the mann ( e ) the average dosage compensation ( responsiveness ) of duplicates as a function of sequence divergence ( ka ) . for comparison we also show the average number of motifs and tf mutants affecting expression for random pairs of yeast singletons ( horizontal dashed and solid lines ) ; the p - values were calculated using the mann ( e ) the average dosage compensation ( responsiveness ) of duplicates as a function of sequence divergence ( ka ) . a complementary analysis of transcription factor binding sites suggests that gene regulation plays an important role in establishing the observed compensation patterns . using a comprehensive data set of 150 known and predicted dna binding motifs in yeast ( 28,39 ) , we found that the average number of different motifs regulating a duplicate pair increases significantly at ka 0.1 ( figure 4d , dashed line , mann the average number of different motifs per duplicate pair is more than twice the number of motifs for a pair of yeast singletons ( figure 4d , dashed horizontal line ) . the increase in complexity of the duplicates regulation at ka 0.1 is also confirmed by a significant increase ( mann whitney u test , p = 1 10 ) at these distances of the number of transcription factor mutants ( 47 ) affecting duplicate gene expression ( figure 4d , solid line ) . the ability of duplicates with partially diverged regulatory regions to compensate for each other through expression changes of the intact gene was previously described by kafri et al . ( 50 ) showed that on deletion of one duplicate , expression changes of the remaining paralog are often need - based , i.e. we investigated the retention of yeast duplicates using the complete genomic sequences of seven species : s. cerevisiae , s. paradoxus , s. bayanus , s. castelli , s. mikatae , s. kudriavzevii and s. kluyveri . interestingly , the loss rate significantly decreases approximately at the divergence distance when duplicates become more similar in terms of their functional load ( figure 2b ) and when their regulatory complexity significantly increases ( figure 5d ) . it is likely that the duplicates surviving the initial loss stage develop independent functionalities and are preserved for long times in the genomes of yeast species . figure 5.the average number of duplicates retained in the genomes of yeast species as a function of the duplicates divergence ka , the number of nonsynonymous substitutions per site . see also supplementary figure s10 for the number of remaining duplicates in the individual species and for the number of remaining duplicates as a function of ks . the average number of duplicates retained in the genomes of yeast species as a function of the duplicates divergence ka , the number of nonsynonymous substitutions per site . in the figure , in the present study , we analyzed genetic robustness due to duplicates in the context of quantitative growth phenotypes and sensitivities to gene deletions in multiple environmental conditions . such robustness is important for understanding the buffering of deleterious mutations in large natural biological populations . our results demonstrate that , contrary to commonly held view , close gene duplicates are unlikely to provide a high level of backup in the context of large natural populations . consequently , it is unlikely that many duplicates are fixed in natural populations specifically due to selection for robustness . our analysis also suggests that duplicate redundancies described in genomics databases , and frequently observed in laboratory experiments , should be considered with caution , at least with respect to their functions in natural biological populations . to investigate this point further ( 52 ) , of 112 yeast duplicates reported to be at least partially redundant in research publications . these duplicates have been described as redundant based on their functional overlap and compensatory interactions observed in small - scale experimental studies . interestingly , based on the number of conditions with quantitative growth phenotypes from the study by hillenmeyer et al . this demonstrates that , although many yeast duplicates indeed may show functional overlap in some laboratory conditions , their compensation properties will probably be significantly less important in large natural populations due to the ability of purifying selection to efficiently prune mutations causing even a small fitness decrease . it is likely that several different factors contribute to the relative paucity of functional compensation between paralogs at small divergence distances . a significant fraction of duplications are likely to be fixed owing to dosage effects ( 17 ) , and functional compensation between such duplicates in the context of natural populations is unlikely . for example , the lack of significant compensation between histone pairs , hta1-hta2 and hht1-hht2 , is likely to be a consequence of their role in maintaining proper histone levels in yeast cells . gene dosage may explain the inability of some duplicates to backup each other , but it is unlikely to be the only explanation . while igc can slow down the rate of duplicates sequence divergence ( 55 ) , analyses based only on wgds with no evidence of igc [ using data recently reported by casola et al . close duplicates are also less likely to compensate for each other probably owing to the aforementioned dichotomy in their functional loads ( figure 3a and b ) . many close duplicates can be classified , based on their activity and breadth of expression , into a major and a minor functional isoforms . the difference in functional load for close yeast duplicates is also consistent with the asymmetric partition of functions , interactions and gene expression , observed between close duplicates in other organisms , for example , arabidopsis and human ( 45,59,60 ) . this suggests that duplicate - dependent compensation in the context of natural populations may be limited in other species as well . in the first stage ( at duplicate distances corresponding to ka < 0.05 ) , duplicates tend to have high overall functional loads and significant asymmetry in the number of sensitive conditions ; both of these factors make complete compensation unlikely . the high functional load of close duplicates suggests that adaptive selection plays an important role in their fixation . in the second stage ( 0.05 < ka < 0.25 ) , as duplicates diverge further , their overall functional load usually decreases . this may happen , for example , due to relaxation of the environmental conditions , which facilitated the original duplicate fixation . the vast majority of duplicates , likely the paralogs with relatively smaller functional loads ( figure 3c ) , are lost at this stage ( figure 5 ) . gene pairs that survive the period of high duplicate loss display more balanced functional loads and complex regulation ; these gene pairs are usually retained for long evolutionary times in yeast genomes ( figure 5 ) . surviving duplicates can provide at least partial compensation at intermediate divergence distances and also serve as an important source of new protein functions . in the third stage ( ka > 0.3 or 70% sequence identity ) , the lifecycle of duplicates is completed when their functional roles diverge , and their quantitative compensation properties become indistinguishable from those of random pairs of yeast singletons .

periodontal disease is characterized by chronic inflammatory processes caused by the presence of specific pathogen microorganisms , which trigger host response , progressive destruction of alveolar bone and apical migration of connective and epithelial attachments over time . removal of the endotoxins , bacteria and calculus in affected tissues is recommended to control the inflammation and help the healing of gingival structures . the main treatment modality for periodontal health is scaling and root planing either during surgical intervention or in a non - surgical approach with the endpoint of producing a root that is biologically acceptable for healing processes . these mechanical procedures may be accomplished by using sonic , ultrasonic and manual instruments with the intention of removing the subgingival biofilm , calculus and endotoxins from the root surface and also to accomplish a maintainable shift in microbiota . it is frequently necessary that even infected cementum and dentin needs to be removed when the toxic products and bacteria have penetrated into tissue . moreover , calcification of the bacterial biofilm left on the root surface during scaling and root - planing , which is composed primarily of calcium phosphate mineral salts deposited between and within remnants of formerly viable microorganisms , may increase the risk for plaque retention and attachment loss . it is well known that scaling and root planing creates a layer of organic and mineralized debris known as the smear layer that covers the surface of the instrumented roots and occludes the dentin tubules . the thickness of this smear layer usually ranges from 2 - 15 m and it might be considered a contaminated physical barrier between the periodontal tissues and the root surface . ideally the procedures of scaling and root planning should leave smooth root surfaces , relatively free of smear layer and any associated calculus , bacteria or endotoxin - contaminated root cementum 9 , 22 . the objective of this study was to investigate the ultramorphology of the root surfaces induced by mechanical instrumentation performed using conventional curettes or piezoelectric scalers when used single - handedly or with a combined technique , using scanning electron microscopy ( sem ) and confocal scanning laser microscopy ( clsm ) . the hypothesis tested was that the periodontal instrumentation of scaling and root planing performed with a combined curette / ultrasonic technique would be able to remove the cementum and create a root surface characterized by reduced presence of smear layer when compared to singlehanded instrumentation . thirty single - rooted healthy human teeth , freshly extracted for orthodontic or surgical reasons , were used in this study . the teeth were extracted after informed consent was obtained by participants according to a treatment protocol approved by the human assurance committee of the department of oral surgery of the university politecnica delle marche , ancona , italy . the selection of the teeth was performed in order to have the maximum standardization of the root surface and according to the following criteria : 1 : absence of calculus and debris ; 2 : absence of dental caries ; 3 : absence of any type of dental restoration ; 4 : no history of non - surgical periodontal treatment including scaling and root - planing prior the date of extraction . after extraction , teeth were stored in a sterile saline solution at 4c for no longer than 1 month . the rationale behind the above mentioned criteria of tooth selection is based on the fact that , since the main objective of this study was focused on the morphological characteristics of the smear layer , we thought that using periodontally - affected root surfaces may present many variables that could influence the standardization of the study . the crowns of the extracted teeth were cut approximately 1 mm below the cementoenamel junction ( cej ) using a high - speed diamond saw under copious water cooling . a second cut was made 3 mm away from the apex of the root . subsequently , a longitudinal cut was made in order to obtain root fragments with the following dimensions of : length : 6 mm ; width : 4 mm ; depth : 2 mm . the specimens were washed and cleaned in an ultrasonic bath for 15 min and examined at 30 by stereoscopic microscopy in order to assure that no residual periodontal or gingival tissue remained on the root surface . in case of any remnant presence of periodontal tissue on the surface , it was gently removed using a gracey - curette hand scaler ( hu- friedy mfg . the specimens were then stored for no longer than 5 days in distilled water buffered to ph 7.4 using 28% nh 4 the external root surface of each root fragment was left uncovered , while the remaining sides were covered by dental wax . all specimens were randomly divided into 3 groups ( n=10/group ) and were instrumented by 2 operators , previously trained and calibrated during a pilot study , who performed the planing with standard angulations , the same stroking pressure during instrumentation of the specimens and adopting a stable lateral force using the method of kishida , et al . each experimental surface was instrumented by applying 30 working strokes in vertical direction using a new and sharpened gracey s curette 7 - 8 ( hu - friedy mfg . co. , inc . ) by 2 different operators who performed an effective planing with a 60 - 70 working angle and applying an appropriate amount of pressure during the strokes . 2 . a coated titanium nitride periodontal tip mounted on a piezoelectric ultrasonic handpiece ( piezolight 5 ; castellini spa , castel maggiore , bologna , italy ) working at 25 khz for 30 s ( about 30 strokes ) was used in a vertical direction under copious and constant water irrigation with a 10 - 15 working angle and applying an appropriate amount of pressure [ ~5n ] . each experimental surface was at first instrumented by applying 15 working strokes in a vertical direction using a gracey s curette 7 - 8 ( hu - friedy mfg . subsequently , the root specimens were treated using a coated titanium nitride periodontal tip mounted on an ultrasonic handpiece ( piezolight 5 , castellini spa ) working at 25 khz for 15 s ( about 15 strokes ) in a vertical direction under copious water irrigation . the pressure and the angulations of working strokes used in this combined technique were the same as those used for each single handed technique . the specimens were imaged using a confocal laser scanning microscopy ( leica sp2 clsm , heidelberg , germany ) equipped with a 63 , 1.4 na oil immersion lens using 514 nm argon / helium ion laser illumination to evaluate the modifications induced by the experimental treatments on dentin surfaces devoid of artifacts usually induced by specimen preparation required for sem . reflected light from the dentin surface was detected with a photomultiplier tube using reflection filters . a z - step of 1 m was used to optically section the specimens to depths of up to 30 m below surface . the z - axis scan of the dentin surface was converted into pseudo - colour for better visualization , and compiled into both single and topographic projections using leica sp2 clsm image - processing software ( leica sp2 clsm ) . the configuration of the system was standardized and used at the same level for the entire investigation . after the clsm investigation , all specimens from groups a , b and c were fixed with 4% formaldehyde in 0.2 m phosphate buffer ( ph=7.2 ) at room temperature for 24 h. following fixation , the specimens were dehydrated using an ascending series of graded ethyl alcohol solutions at the concentrations of 50 , 80 , 95 and 100% for 10 min at each concentration . prior to scanning electron microscopy examination , the specimens were mounted on sem stubs with a silver paint , dried and then sputter coated with gold . the microscopy investigation ( sem ) was performed using a scanning electron microscope ( jeol 5200 , joel corp . each root specimen was considered as a sample - unit and 3 sem micrographs were randomly obtained from each specimen , with different standardized magnifications ( 1,000 , 2,000 and 5,000 ) resulting in a total number of 9 images per specimen ; these images were intended to be representative of the most common features observed in each sample . the micrographs were assessed by 2 examiners blinded to the experimental procedures but previously instructed during a pilot study to use the the following index of root surface and smear layer morphology characteristics , as follows : grade 1 - thick and compact smear layer , no dentin tubules open ; grade 2 - thin smear layer , no presence of dentin tubules ; grade 3 - residues of smear debris partially occluding dentin tubules ; grade 4 - absence of smear layer on the dentin surface with exposed collagen fibrils . for each specimen , a single value was assigned after the evaluation of the 9 representative images ( 1,000 , 2,000 5,000 ) , resulting in 10 values per group . the distribution of smear layer morphology grades was tested using pearson s test and significance was established at p=0.001 and adjusted residuals were used to identify significant treatment grade interactions . thirty single - rooted healthy human teeth , freshly extracted for orthodontic or surgical reasons , were used in this study . the teeth were extracted after informed consent was obtained by participants according to a treatment protocol approved by the human assurance committee of the department of oral surgery of the university politecnica delle marche , ancona , italy . the selection of the teeth was performed in order to have the maximum standardization of the root surface and according to the following criteria : 1 : absence of calculus and debris ; 2 : absence of dental caries ; 3 : absence of any type of dental restoration ; 4 : no history of non - surgical periodontal treatment including scaling and root - planing prior the date of extraction . after extraction , teeth were stored in a sterile saline solution at 4c for no longer than 1 month . the rationale behind the above mentioned criteria of tooth selection is based on the fact that , since the main objective of this study was focused on the morphological characteristics of the smear layer , we thought that using periodontally - affected root surfaces may present many variables that could influence the standardization of the study . the crowns of the extracted teeth were cut approximately 1 mm below the cementoenamel junction ( cej ) using a high - speed diamond saw under copious water cooling . a second cut was made 3 mm away from the apex of the root . subsequently , a longitudinal cut was made in order to obtain root fragments with the following dimensions of : length : 6 mm ; width : 4 mm ; depth : 2 mm . the specimens were washed and cleaned in an ultrasonic bath for 15 min and examined at 30 by stereoscopic microscopy in order to assure that no residual periodontal or gingival tissue remained on the root surface . in case of any remnant presence of periodontal tissue on the surface , it was gently removed using a gracey - curette hand scaler ( hu- friedy mfg . the specimens were then stored for no longer than 5 days in distilled water buffered to ph 7.4 using 28% nh 4 the external root surface of each root fragment was left uncovered , while the remaining sides were covered by dental wax . all specimens were randomly divided into 3 groups ( n=10/group ) and were instrumented by 2 operators , previously trained and calibrated during a pilot study , who performed the planing with standard angulations , the same stroking pressure during instrumentation of the specimens and adopting a stable lateral force using the method of kishida , et al . each experimental surface was instrumented by applying 30 working strokes in vertical direction using a new and sharpened gracey s curette 7 - 8 ( hu - friedy mfg . co. , inc . ) by 2 different operators who performed an effective planing with a 60 - 70 working angle and applying an appropriate amount of pressure during the strokes . 2 . a coated titanium nitride periodontal tip mounted on a piezoelectric ultrasonic handpiece ( piezolight 5 ; castellini spa , castel maggiore , bologna , italy ) working at 25 khz for 30 s ( about 30 strokes ) was used in a vertical direction under copious and constant water irrigation with a 10 - 15 working angle and applying an appropriate amount of pressure [ ~5n ] . each experimental surface was at first instrumented by applying 15 working strokes in a vertical direction using a gracey s curette 7 - 8 ( hu - friedy mfg . subsequently , the root specimens were treated using a coated titanium nitride periodontal tip mounted on an ultrasonic handpiece ( piezolight 5 , castellini spa ) working at 25 khz for 15 s ( about 15 strokes ) in a vertical direction under copious water irrigation . the pressure and the angulations of working strokes used in this combined technique were the same as those used for each single handed technique . the specimens were imaged using a confocal laser scanning microscopy ( leica sp2 clsm , heidelberg , germany ) equipped with a 63 , 1.4 na oil immersion lens using 514 nm argon / helium ion laser illumination to evaluate the modifications induced by the experimental treatments on dentin surfaces devoid of artifacts usually induced by specimen preparation required for sem . reflected light from the dentin surface was detected with a photomultiplier tube using reflection filters . a z - step of 1 m was used to optically section the specimens to depths of up to 30 m below surface . the z - axis scan of the dentin surface was converted into pseudo - colour for better visualization , and compiled into both single and topographic projections using leica sp2 clsm image - processing software ( leica sp2 clsm ) . the configuration of the system was standardized and used at the same level for the entire investigation . after the clsm investigation , all specimens from groups a , b and c were fixed with 4% formaldehyde in 0.2 m phosphate buffer ( ph=7.2 ) at room temperature for 24 h. following fixation , the specimens were dehydrated using an ascending series of graded ethyl alcohol solutions at the concentrations of 50 , 80 , 95 and 100% for 10 min at each concentration . prior to scanning electron microscopy examination , the specimens were mounted on sem stubs with a silver paint , dried and then sputter coated with gold . the microscopy investigation ( sem ) each root specimen was considered as a sample - unit and 3 sem micrographs were randomly obtained from each specimen , with different standardized magnifications ( 1,000 , 2,000 and 5,000 ) resulting in a total number of 9 images per specimen ; these images were intended to be representative of the most common features observed in each sample . the micrographs were assessed by 2 examiners blinded to the experimental procedures but previously instructed during a pilot study to use the the following index of root surface and smear layer morphology characteristics , as follows : grade 1 - thick and compact smear layer , no dentin tubules open ; grade 2 - thin smear layer , no presence of dentin tubules ; grade 3 - residues of smear debris partially occluding dentin tubules ; grade 4 - absence of smear layer on the dentin surface with exposed collagen fibrils . for each specimen , a single value was assigned after the evaluation of the 9 representative images ( 1,000 , 2,000 5,000 ) , resulting in 10 values per group . the distribution of smear layer morphology grades was tested using pearson s test and significance was established at p=0.001 and adjusted residuals were used to identify significant treatment grade interactions . periodontal treatments of scaling and root planing performed with manual , sonic or combined instrumentation induced several morphological changes on the root surface . the scanning electron micrographic appearance of the morphology of the root surface treated by hand curette ( group a ) revealed a compact and multilayered smear layer ( figure 1a ) characterized by the presence of superficial furrows with a distinguishing morphology similar to that of tree - bark the morphology of the surfaces of the root fragments treated with the ultrasonic scaler only ( group b ) appeared irregular with few grooves on the root surfaces ( figure 2a ) . higher magnification images showed that those root surfaces were covered by a porous and thin layer of debris ( figures 2b and c ) . clsm showed that the root surface treated by curette presented a smoother surface compare to those treated using the ultrasonic tip ( figures 3a , b ) . the combined manual / ultrasonic instrumentation ( group c ) showed a peculiar morphology to the root surface with many differences when compared to the other treatments . the morphology of the root surfaces revealed that the cementum was completely removed with the exposure of the root dentin and dentin tubules ( figure 4a ) . the morphology of the root surfaces was characterized by the presence of very few grooves and a slender remnant smear layer which only partially covered the root dentin ( figure 4b ) . higher magnification images showed that the remnant smear layer produced by this combined manual / ultrasonic technique partially obliterated the dentin tubules giving them a characteristic conformation similar to that of a flute - mouthpiece ( figure 4c ) . in some case , it was also possible to observe areas with exposed collagen fibrils ( figures 5a and 4b ) . the clsm confirmed the results observed with sem showing only a slender smear layer that partially obliterated the dentin tubules ( figures 6a , b ) . the statistical analysis performed on the values obtained using the index of root surface and smear layer morphology characteristics showed statistical differences between the groups ( p<0.001 ) ( table 1 ) . scanning electron microscopy showing the morphology of the root surfaces treated with hand curettes . a : mechanical periodontal treatment with curettes creates a compact and multilayered smear layer characterized by the presence of superficial furrows ; b ( 2,000 ) and c ( 5,000 ) : at higher magnification it is possible to clearly observe a distinguished morphology similar to that of tree - bark ( pointer ) induced by the working strokes of the curette . no exposed dentin tubules were observed subsequent to the mechanical periodontal treatment with curettes scanning electron microscopy showing the morphology of the root surfaces treated with the piezoelectric scaler . a : non - surgical periodontal treatment performed with this method creates an irregular root surface with few shallow grooves ; b : higher magnification ( 2,000 ) showing that the root cementum was not removed by the ultrasonic instrumentation but was only altered and characterized by pits and tips ( pointer ) ; c : ( 5,000 ) presence of a porous and thin layer of debris with no exposed dentin tubules ( pointer ) confocal laser scanning microscopy topographical images . a : root surface treated with hand curettes showing a compact smear layer characterized by the presence of superficial furrows . no exposed dentin tubules were observed subsequent to the mechanical periodontal treatment with the curettes ; b : root surfaces treated using the piezoelectric scaler , showing an irregular and coarse root surface with few shallow grooves pits and tips ( pointer ) scanning electron microscopy appearance of the morphology of the root surfaces treated with the simultaneous manual and ultrasonic instrumentation . image a shows the morphology of the root surfaces characterized by very few grooves and slender remnant smear layer which only partially covered the root dentin . image b : at higher magnification it is possible to observe that the root - cementum was completely removed with the exposure of root dentin and dentin tubules ( pointer ) . image c shows that the remnant smear layer produced by this combined manual / sonic technique obliterated the dentin tubules ( pointer ) and many of them had a characteristic conformation similar to the shape of a flute - mouthpiece scanning electron microscopy ( a and b ) of the morphology of root surfaces treated with simultaneous manual and ultrasonic instrumentation reveal that some areas with exposed collagen fibrils ( pointer ) confocal laser scanning microscopy topographical ( a ) and single projection ( b ) images of the root surfaces treated using the simultaneous curette / ultrasonic instrumentation technique showing the morphology of the root surfaces characterized by slender remnant smear layer that only partially covered the root dentin ( b ) . it is possible to observe that the root cementum was completely removed with the exposure of root dentin and dentin tubules distribution of the scores for morphological characteristics of the root surface * different lower case letters indicate statistically significant differences ( p<0.001 ) . the specimens were assessed using the following index of smear layer removal : ( grade 1 : thick and compact smear layer , no dentinal tubules open ; grade 2 : thin smear layer , no presence of dentinal tubules ; grade 3 : residues of smear debris partially occluding dentinal tubules ; grade 4 : absence of smear layer on the dentine surface with exposed collagen fibrils ) periodontally - affected root surfaces treated with conventional non - surgical periodontal treatment of scaling and root planing might be not completely free from contaminants due to the presence of a residual smear layer created during instrumentations . the presence of the smear layer on the instrumented root surfaces has been shown to act as a physical barrier between the periodontal tissues and the root surface unsuitable for reintegration in periodontal connective tissue . thus , this study investigated the ultramorphology of the smear layer created on the root surface by conventional curettes and periodontal ultrasonic scalers when used singlehandedly or with a combined instrumentation technique . the results of this study indicated that manual and ultrasonic instrumentation induced several morphological changes on the root surface both when used single - handedly and when these instruments were used simultaneously with a combined instrumentation technique . the clsm and sem - ultramorphology analysis showed that manual periodontal instrumentation with a curette created a compact and multilayered smear layer on the root surface . this smear layer was characterized by the presence of superficial grooves with a tree - bark-like appearance , probably induced by the working strokes . conversely , the piezoelectric instrumentation created cementum - root surfaces were rich with irregularities characterized by several pits and partially covered by a thin and porous layer of debris . no dentin tubules were exposed after this type of periodontal instrumentation indicating that the cementum was not removed from the root surface . it has been shown that some ultrasonic scalers used on root dentin produced only some residual smear debris in relation to dentin tubules . moreover , the use of ultrasonic scalers in nonsurgical periodontal treatment creates a smooth root surface with minimal damage and tight attachment of fibroblasts 26 , 27 , 33 . ( 2006 ) have shown that the diamond - coated tip with sonic scaler instrumentation and ultrasonic instrumentation produce similar root surface roughness , higher than curette instrumentation . however , it is important to consider that patients who have been affected by chronic periodontal diseases for a long time and had never been treated before with any surgical or non - surgical periodontal treatments might need further and more incisive instrumentation . indeed , rohanizadeh and legeros ( 2005 ) have demonstrated , using transmission electron microscopy ( tem ) , that the old calculus is in direct connection or in fusion with the tooth apatite crystals of the cement . it was shown that a very strong attachment of calculus to the tooth surface existed , more so than the cohesive strength within the calculus itself . fractographic analysis also showed occurrence of fracture within the calculus but not at the calculus - tooth surface interface suggesting that clinical calculus removal may efficiently remove areas of the calculus prone to fracture but may still leave a part of the calculus strongly attached to the tooth surface . the remaining calculus ( calcium phosphate crystals ) could promote further calculus formation by attracting plaque colonization and eventual plaque calcification and/or serve as nucleating centers for calcium phosphate crystal growth . hence , the ideal instrument should remove all the calculus from the root surfaces and leave behind a surface as smooth as possible without any iatrogenic effects . although , drisko ( 2001 ) clearly stated that ultrasonic or sonic instrumentations have similar effects to that of hand scaling and root planing when analyzing removal of biofilm , calculus and endotoxins . many clinicians have advocated gentle treatment of the root surface , based on observations that endotoxin does not penetrate the exposed root cementum , but forms a loosely attached superficial layer on its surface . on the other hand , under sem observation , the curette proved to be more effective in removing the calculus - associated cementum from periodontally - affected root surfaces , when compared with the ultrasonic device , using sem . moreover , under tem observation , curetted cementum exhibited newly synthesized fibrilar material and collagen fibrils produced by healthy , functional fibroblasts attached to the instrumented surface , and apparently oriented towards the curetted cemental surface . these results strongly suggested that improved cellular attachment can be promoted if superficial cementum is first removed by mechanical curettage . in this study , it was shown that when the two types of periodontal instrumentation were used simultaneously with a combined curette / ultrasonic instrumentation , the root morphology was totally different from that observed when they were used single - handedly . in the evaluation of the morphology of these root surfaces , it was revealed that the cementum was completely removed and the root dentin tubules were exposed . the root surfaces were characterized by few grooves and a very small amount of remnant smear debris , which only partially covered the dentin tubules . moreover , at higher magnification ( sem ) it was often observed that the remnant smear layer produced by this combined manual / sonic technique obliterated the dentin tubules , giving them a characteristic flutemouthpiece conformation . the combined manual / ultrasonic instrumentation induced in some cases the complete removal of the smear layer and the exposure of collagen fibrils ( figure 4 ) . it has been shown that exposure of the dentin matrix of the root surface allows formation of a proper fibrinclot , which is a determinant factor for the positive outcome of early wound healing events , thus facilitating the integration between the root surface and the healing connective tissue favoring migration and attachment of gingival fibroblasts . the combined curette / ultrasonic instrumentation , used with a shorter working time than singlehanded instrumentation , completely removed the cementum from the root surfaces and , at the same time , the use of the ultrasonic scalers allowed the decontamination of the root surfaces from the smear layer created by the curettes . thus , as the objective of this study was the evaluation of the ultramorphology of the root surfaces and of the smear layer created after non - surgical periodontal treatments using manual and sonic instruments , it is possible to assume that the use of curettes followed by ultrasonic periodontal instruments may create root surfaces relatively free from any gross smear layer compared to the root surfaces singlehandedly instrumented by curette or ultrasonic scalers . further studies should investigate the effects of instrumentation on the lost substance and the roughness of the root surfaces after this alternative non - surgical periodontal technique of instrumentation during scaling and root - planing . the hypothesis tested in this laboratory study that scaling and root planing performed with a combined curette / ultrasonic technique would be able to create a smooth root surface with a reduced smear layer presence when compared to singlehandedly instrumentation was accepted . future research should be also conducted to evaluate the clinical effectiveness of this alternative technique in the healing processes and in the regeneration of connective tissues with relative reduction of pocket depth .

objectivethe purpose of this study was to investigate the ultramorphology of the root surfaces induced by mechanical instrumentation performed using conventional curettes or piezoelectric scalers when used single - handedly or with a combined technique.material and methodsthirty single - rooted teeth were selected and divided into 3 groups : group a , instrumentation with curettes ; group b instrumentation with titanium nitride coated periodontal tip mounted in a piezoelectric handpiece ; group c , combined technique with curette / ultrasonic piezoelectric instrumentation . the specimens were processed and analyzed using confocal and scanning electron microscopy . differences between the different groups of instrumentation were determined using pearson s 2 with significance predetermined at =0.001.resultsperiodontal scaling and root planing performed with curettes , ultrasonic or combined instrumentation induced several morphological changes on the root surface . the curettes produced a compact and thick multilayered smear layer , while the morphology of the root surfaces after ultrasonic scaler treatment appeared irregular with few grooves and a thin smear layer . the combination of curette / ultrasonic instrumentation showed exposed root dentin tubules with a surface morphology characterized by the presence of very few grooves and slender remnants of smear layer which only partially covered the root dentin . in some cases , it was also possible to observe areas with exposed collagen fibrils.conclusionthe curette - ultrasonic simultaneous instrumentation may combine the beneficial effects of each instrument in a single technique creating a root surface relatively free from the physical barrier of smear layer and dentin tubules orifices partial occlusion .

INTRODUCTION MATERIAL AND METHODS Specimen Preparation Experimental Design Confocal Laser Scanning Microscopy Scanning Electron Microscopy (SEM) RESULTS DISCUSSION CONCLUSION

periodontal disease is characterized by chronic inflammatory processes caused by the presence of specific pathogen microorganisms , which trigger host response , progressive destruction of alveolar bone and apical migration of connective and epithelial attachments over time . the main treatment modality for periodontal health is scaling and root planing either during surgical intervention or in a non - surgical approach with the endpoint of producing a root that is biologically acceptable for healing processes . these mechanical procedures may be accomplished by using sonic , ultrasonic and manual instruments with the intention of removing the subgingival biofilm , calculus and endotoxins from the root surface and also to accomplish a maintainable shift in microbiota . moreover , calcification of the bacterial biofilm left on the root surface during scaling and root - planing , which is composed primarily of calcium phosphate mineral salts deposited between and within remnants of formerly viable microorganisms , may increase the risk for plaque retention and attachment loss . it is well known that scaling and root planing creates a layer of organic and mineralized debris known as the smear layer that covers the surface of the instrumented roots and occludes the dentin tubules . the thickness of this smear layer usually ranges from 2 - 15 m and it might be considered a contaminated physical barrier between the periodontal tissues and the root surface . ideally the procedures of scaling and root planning should leave smooth root surfaces , relatively free of smear layer and any associated calculus , bacteria or endotoxin - contaminated root cementum 9 , 22 . the objective of this study was to investigate the ultramorphology of the root surfaces induced by mechanical instrumentation performed using conventional curettes or piezoelectric scalers when used single - handedly or with a combined technique , using scanning electron microscopy ( sem ) and confocal scanning laser microscopy ( clsm ) . the hypothesis tested was that the periodontal instrumentation of scaling and root planing performed with a combined curette / ultrasonic technique would be able to remove the cementum and create a root surface characterized by reduced presence of smear layer when compared to singlehanded instrumentation . the selection of the teeth was performed in order to have the maximum standardization of the root surface and according to the following criteria : 1 : absence of calculus and debris ; 2 : absence of dental caries ; 3 : absence of any type of dental restoration ; 4 : no history of non - surgical periodontal treatment including scaling and root - planing prior the date of extraction . the rationale behind the above mentioned criteria of tooth selection is based on the fact that , since the main objective of this study was focused on the morphological characteristics of the smear layer , we thought that using periodontally - affected root surfaces may present many variables that could influence the standardization of the study . the specimens were washed and cleaned in an ultrasonic bath for 15 min and examined at 30 by stereoscopic microscopy in order to assure that no residual periodontal or gingival tissue remained on the root surface . in case of any remnant presence of periodontal tissue on the surface , it was gently removed using a gracey - curette hand scaler ( hu- friedy mfg . the specimens were then stored for no longer than 5 days in distilled water buffered to ph 7.4 using 28% nh 4 the external root surface of each root fragment was left uncovered , while the remaining sides were covered by dental wax . all specimens were randomly divided into 3 groups ( n=10/group ) and were instrumented by 2 operators , previously trained and calibrated during a pilot study , who performed the planing with standard angulations , the same stroking pressure during instrumentation of the specimens and adopting a stable lateral force using the method of kishida , et al . a coated titanium nitride periodontal tip mounted on a piezoelectric ultrasonic handpiece ( piezolight 5 ; castellini spa , castel maggiore , bologna , italy ) working at 25 khz for 30 s ( about 30 strokes ) was used in a vertical direction under copious and constant water irrigation with a 10 - 15 working angle and applying an appropriate amount of pressure [ ~5n ] . subsequently , the root specimens were treated using a coated titanium nitride periodontal tip mounted on an ultrasonic handpiece ( piezolight 5 , castellini spa ) working at 25 khz for 15 s ( about 15 strokes ) in a vertical direction under copious water irrigation . the specimens were imaged using a confocal laser scanning microscopy ( leica sp2 clsm , heidelberg , germany ) equipped with a 63 , 1.4 na oil immersion lens using 514 nm argon / helium ion laser illumination to evaluate the modifications induced by the experimental treatments on dentin surfaces devoid of artifacts usually induced by specimen preparation required for sem . prior to scanning electron microscopy examination , the specimens were mounted on sem stubs with a silver paint , dried and then sputter coated with gold . the micrographs were assessed by 2 examiners blinded to the experimental procedures but previously instructed during a pilot study to use the the following index of root surface and smear layer morphology characteristics , as follows : grade 1 - thick and compact smear layer , no dentin tubules open ; grade 2 - thin smear layer , no presence of dentin tubules ; grade 3 - residues of smear debris partially occluding dentin tubules ; grade 4 - absence of smear layer on the dentin surface with exposed collagen fibrils . the distribution of smear layer morphology grades was tested using pearson s test and significance was established at p=0.001 and adjusted residuals were used to identify significant treatment grade interactions . the selection of the teeth was performed in order to have the maximum standardization of the root surface and according to the following criteria : 1 : absence of calculus and debris ; 2 : absence of dental caries ; 3 : absence of any type of dental restoration ; 4 : no history of non - surgical periodontal treatment including scaling and root - planing prior the date of extraction . the rationale behind the above mentioned criteria of tooth selection is based on the fact that , since the main objective of this study was focused on the morphological characteristics of the smear layer , we thought that using periodontally - affected root surfaces may present many variables that could influence the standardization of the study . the specimens were washed and cleaned in an ultrasonic bath for 15 min and examined at 30 by stereoscopic microscopy in order to assure that no residual periodontal or gingival tissue remained on the root surface . in case of any remnant presence of periodontal tissue on the surface , it was gently removed using a gracey - curette hand scaler ( hu- friedy mfg . the specimens were then stored for no longer than 5 days in distilled water buffered to ph 7.4 using 28% nh 4 the external root surface of each root fragment was left uncovered , while the remaining sides were covered by dental wax . all specimens were randomly divided into 3 groups ( n=10/group ) and were instrumented by 2 operators , previously trained and calibrated during a pilot study , who performed the planing with standard angulations , the same stroking pressure during instrumentation of the specimens and adopting a stable lateral force using the method of kishida , et al . a coated titanium nitride periodontal tip mounted on a piezoelectric ultrasonic handpiece ( piezolight 5 ; castellini spa , castel maggiore , bologna , italy ) working at 25 khz for 30 s ( about 30 strokes ) was used in a vertical direction under copious and constant water irrigation with a 10 - 15 working angle and applying an appropriate amount of pressure [ ~5n ] . subsequently , the root specimens were treated using a coated titanium nitride periodontal tip mounted on an ultrasonic handpiece ( piezolight 5 , castellini spa ) working at 25 khz for 15 s ( about 15 strokes ) in a vertical direction under copious water irrigation . the specimens were imaged using a confocal laser scanning microscopy ( leica sp2 clsm , heidelberg , germany ) equipped with a 63 , 1.4 na oil immersion lens using 514 nm argon / helium ion laser illumination to evaluate the modifications induced by the experimental treatments on dentin surfaces devoid of artifacts usually induced by specimen preparation required for sem . prior to scanning electron microscopy examination , the specimens were mounted on sem stubs with a silver paint , dried and then sputter coated with gold . the micrographs were assessed by 2 examiners blinded to the experimental procedures but previously instructed during a pilot study to use the the following index of root surface and smear layer morphology characteristics , as follows : grade 1 - thick and compact smear layer , no dentin tubules open ; grade 2 - thin smear layer , no presence of dentin tubules ; grade 3 - residues of smear debris partially occluding dentin tubules ; grade 4 - absence of smear layer on the dentin surface with exposed collagen fibrils . the distribution of smear layer morphology grades was tested using pearson s test and significance was established at p=0.001 and adjusted residuals were used to identify significant treatment grade interactions . periodontal treatments of scaling and root planing performed with manual , sonic or combined instrumentation induced several morphological changes on the root surface . the scanning electron micrographic appearance of the morphology of the root surface treated by hand curette ( group a ) revealed a compact and multilayered smear layer ( figure 1a ) characterized by the presence of superficial furrows with a distinguishing morphology similar to that of tree - bark the morphology of the surfaces of the root fragments treated with the ultrasonic scaler only ( group b ) appeared irregular with few grooves on the root surfaces ( figure 2a ) . the combined manual / ultrasonic instrumentation ( group c ) showed a peculiar morphology to the root surface with many differences when compared to the other treatments . the morphology of the root surfaces revealed that the cementum was completely removed with the exposure of the root dentin and dentin tubules ( figure 4a ) . the morphology of the root surfaces was characterized by the presence of very few grooves and a slender remnant smear layer which only partially covered the root dentin ( figure 4b ) . in some case , it was also possible to observe areas with exposed collagen fibrils ( figures 5a and 4b ) . the statistical analysis performed on the values obtained using the index of root surface and smear layer morphology characteristics showed statistical differences between the groups ( p<0.001 ) ( table 1 ) . scanning electron microscopy showing the morphology of the root surfaces treated with hand curettes . a : mechanical periodontal treatment with curettes creates a compact and multilayered smear layer characterized by the presence of superficial furrows ; b ( 2,000 ) and c ( 5,000 ) : at higher magnification it is possible to clearly observe a distinguished morphology similar to that of tree - bark ( pointer ) induced by the working strokes of the curette . no exposed dentin tubules were observed subsequent to the mechanical periodontal treatment with curettes scanning electron microscopy showing the morphology of the root surfaces treated with the piezoelectric scaler . a : non - surgical periodontal treatment performed with this method creates an irregular root surface with few shallow grooves ; b : higher magnification ( 2,000 ) showing that the root cementum was not removed by the ultrasonic instrumentation but was only altered and characterized by pits and tips ( pointer ) ; c : ( 5,000 ) presence of a porous and thin layer of debris with no exposed dentin tubules ( pointer ) confocal laser scanning microscopy topographical images . a : root surface treated with hand curettes showing a compact smear layer characterized by the presence of superficial furrows . no exposed dentin tubules were observed subsequent to the mechanical periodontal treatment with the curettes ; b : root surfaces treated using the piezoelectric scaler , showing an irregular and coarse root surface with few shallow grooves pits and tips ( pointer ) scanning electron microscopy appearance of the morphology of the root surfaces treated with the simultaneous manual and ultrasonic instrumentation . image a shows the morphology of the root surfaces characterized by very few grooves and slender remnant smear layer which only partially covered the root dentin . image b : at higher magnification it is possible to observe that the root - cementum was completely removed with the exposure of root dentin and dentin tubules ( pointer ) . image c shows that the remnant smear layer produced by this combined manual / sonic technique obliterated the dentin tubules ( pointer ) and many of them had a characteristic conformation similar to the shape of a flute - mouthpiece scanning electron microscopy ( a and b ) of the morphology of root surfaces treated with simultaneous manual and ultrasonic instrumentation reveal that some areas with exposed collagen fibrils ( pointer ) confocal laser scanning microscopy topographical ( a ) and single projection ( b ) images of the root surfaces treated using the simultaneous curette / ultrasonic instrumentation technique showing the morphology of the root surfaces characterized by slender remnant smear layer that only partially covered the root dentin ( b ) . it is possible to observe that the root cementum was completely removed with the exposure of root dentin and dentin tubules distribution of the scores for morphological characteristics of the root surface * different lower case letters indicate statistically significant differences ( p<0.001 ) . the specimens were assessed using the following index of smear layer removal : ( grade 1 : thick and compact smear layer , no dentinal tubules open ; grade 2 : thin smear layer , no presence of dentinal tubules ; grade 3 : residues of smear debris partially occluding dentinal tubules ; grade 4 : absence of smear layer on the dentine surface with exposed collagen fibrils ) periodontally - affected root surfaces treated with conventional non - surgical periodontal treatment of scaling and root planing might be not completely free from contaminants due to the presence of a residual smear layer created during instrumentations . the presence of the smear layer on the instrumented root surfaces has been shown to act as a physical barrier between the periodontal tissues and the root surface unsuitable for reintegration in periodontal connective tissue . thus , this study investigated the ultramorphology of the smear layer created on the root surface by conventional curettes and periodontal ultrasonic scalers when used singlehandedly or with a combined instrumentation technique . the results of this study indicated that manual and ultrasonic instrumentation induced several morphological changes on the root surface both when used single - handedly and when these instruments were used simultaneously with a combined instrumentation technique . the clsm and sem - ultramorphology analysis showed that manual periodontal instrumentation with a curette created a compact and multilayered smear layer on the root surface . this smear layer was characterized by the presence of superficial grooves with a tree - bark-like appearance , probably induced by the working strokes . conversely , the piezoelectric instrumentation created cementum - root surfaces were rich with irregularities characterized by several pits and partially covered by a thin and porous layer of debris . hence , the ideal instrument should remove all the calculus from the root surfaces and leave behind a surface as smooth as possible without any iatrogenic effects . many clinicians have advocated gentle treatment of the root surface , based on observations that endotoxin does not penetrate the exposed root cementum , but forms a loosely attached superficial layer on its surface . in this study , it was shown that when the two types of periodontal instrumentation were used simultaneously with a combined curette / ultrasonic instrumentation , the root morphology was totally different from that observed when they were used single - handedly . in the evaluation of the morphology of these root surfaces , it was revealed that the cementum was completely removed and the root dentin tubules were exposed . the root surfaces were characterized by few grooves and a very small amount of remnant smear debris , which only partially covered the dentin tubules . the combined manual / ultrasonic instrumentation induced in some cases the complete removal of the smear layer and the exposure of collagen fibrils ( figure 4 ) . it has been shown that exposure of the dentin matrix of the root surface allows formation of a proper fibrinclot , which is a determinant factor for the positive outcome of early wound healing events , thus facilitating the integration between the root surface and the healing connective tissue favoring migration and attachment of gingival fibroblasts . the combined curette / ultrasonic instrumentation , used with a shorter working time than singlehanded instrumentation , completely removed the cementum from the root surfaces and , at the same time , the use of the ultrasonic scalers allowed the decontamination of the root surfaces from the smear layer created by the curettes . thus , as the objective of this study was the evaluation of the ultramorphology of the root surfaces and of the smear layer created after non - surgical periodontal treatments using manual and sonic instruments , it is possible to assume that the use of curettes followed by ultrasonic periodontal instruments may create root surfaces relatively free from any gross smear layer compared to the root surfaces singlehandedly instrumented by curette or ultrasonic scalers . further studies should investigate the effects of instrumentation on the lost substance and the roughness of the root surfaces after this alternative non - surgical periodontal technique of instrumentation during scaling and root - planing . the hypothesis tested in this laboratory study that scaling and root planing performed with a combined curette / ultrasonic technique would be able to create a smooth root surface with a reduced smear layer presence when compared to singlehandedly instrumentation was accepted .

the nurses health study ii ( nhs ii ) is a prospective cohort of 116,671 female registered nurses aged 2444 years at baseline in 1989 . this cohort is followed using biennial mailed questionnaires on lifestyle , diet , and medical history . the study has maintained a response rate of 90% for every 2-year period ( 7 ) . in 1997 , participants were asked if they would be willing to complete a questionnaire about diet during high school ( hs - ffq [ high school food frequency questionnaire ] ) , at which time they were 3453 years of age . comparison of these women with the entire cohort suggests that they do not differ with regard to baseline dietary intake or t2 dm risk factors . this analysis is restricted to the 47,355 women who returned the hs - ffq in 1998 ( 73% of those sent the questionnaire ) . we excluded participants if they reported implausible daily caloric intakes of < 500 or 3,500 kcal / day for current intake levels ( n = 2,920 ) and < 500 or 5,000 kcal / day for high school intake levels ( n = 1,181 ) , or left 10 items blank ( n = 225 ) . in addition , we excluded participants if they had a confirmed diagnosis of diabetes ( n = 502 t2 dm , n = 42 type 1 diabetes ) or death ( n = 1 ) before return of the hs - ffq in 1998 , were missing the date of diagnosis of diabetes ( n = 616 ) , or had a history of diabetes ( n = 137 ) , gestational diabetes ( n = 1,875 ) , cancer except nonmelanoma skin cancer , or cardiovascular disease ( n = 2,818 ) reported in 1999 . after exclusions , a total of 37,038 participants remained for the analysis . high school dietary intake was assessed using the hs - ffq , a 124-item ffq that asked participants about their diet during high school . subjects were asked how often they consumed a specified amount of each item using nine responses ranging from never or less than once per month to six or more per day . categories on the hs - ffq included main dishes , bread and cereals , fruits , vegetables , snack foods , and dairy . the hs - ffq was designed to include foods that were consumed when these women were in high school . nutrient intakes on the hs - ffq were computed by multiplying the frequency of consumption of each unit of food by the nutrient content of the specified portions and then summing the contributions . recall of adolescent diet among nhs ii participants and a similar cohort was reproducible ( 8,9 ) , and the hs - ffq has shown reasonable validity ( 9,10 ) . adult dietary patterns were assessed by 131-item ffqs administered in 1991 , 1995 , 1999 , and 2003 . these ffqs assessed usual dietary intake during the past year , and food items and response categories were similar to the hs - ffq . the validity of ffqs similar to those used in the nhs ii was reasonable ( 11 ) . to identify dietary patterns , food items on the ffqs were first aggregated into 37 food groups for the hs - ffq and 40 food groups for the nhs ii ffqs , on the basis of similarities in nutrient profile and food preferences . similar food groupings were used previously ( 5 ) . factor analysis with the orthogonal rotation procedure varimax briefly , factor analysis aggregates correlated variables , and each obtained factor ( dietary pattern ) is a linear combination of all food groups that are weighted by their factor loadings and explains as much variation in the food groups as possible ( 12 ) . food groups with positive loadings contribute to the dietary pattern , whereas those with negative loadings are inversely associated . food groups with absolute factor loadings 0.30 were considered as significantly contributing to the pattern ( 12 ) . prudent and western patterns , on the basis of eigenvalues ( > 1 ) , scree tests ( a graphical representation of eigenvalues > 1 ) , and interpretability of the factors ( 5 ) . scree tests allowed us to identify two major patterns with the largest eigenvalues ( > 2.75 ) . dietary pattern scores were calculated by summing the standardized intake of food groups , weighted by the factor loading of the food groups ( 12 ) . these scores were used to rank participants according to the degree to which they conformed to a given dietary pattern . the reproducibility and validity of dietary patterns derived by this method were reasonable ( 13 ) . because of potential overreporting or underreporting on the ffqs , dietary patterns were adjusted for total energy intake using the residual method ( 14 ) . women reporting a new diagnosis of diabetes on any of the biennial questionnaires were sent supplementary questionnaires to confirm the self - report . we used the american diabetes association criteria for diagnosis ( fasting glucose levels 7 mmol [ 126 mg / dl ] ) . in substudies of the nhs , 98% of self - reported diabetes cases documented by the same supplementary questionnaire dietary pattern scores were divided into quintiles , and cox proportional hazards regression was used to estimate incidence rate ratios comparing each quintile to the lowest . person - time of follow - up was calculated for each participant from 1997 until june 2005 , or date of t2 dm diagnosis , or death . test for trends across quintiles was conducted by modeling the median value for each category as a continuous variable . in the analysis of high school dietary patterns , multivariate models were adjusted for bmi at age 18 years ( < 18.5 , 18.5 to < 25 , 25 to < 30 , or 30 kg / m ) , total energy intake in high school ( quintiles , kcal / day ) , smoking between ages 15 and 19 years ( none or 14 , 514 , or 15 + cigarettes / day ) , and high school physical activity ( quintiles , metabolic equivalents [ mets]/week ) . we also adjusted for adult risk factors , including current smoking status ( never , past , current : 114 cigarettes / day , 15 cigarettes / day ) , current physical activity ( quintiles , mets / week ) , history of t2 dm in parents or siblings ( yes , no ) , oral contraceptive use ( never , past , current ) , postmenopausal hormone use ( never , ever ) , total energy ( quintiles , kcal / day ) , and alcohol ( 0 , 0.14.9 , 59.9 , 10 g / day ) . sensitivity tests were conducted by additionally adjusting for current prudent or western dietary pattern and weight change since age 18 years , which are potential mediators . effect modification of the association between high school dietary pattern and t2 dm by bmi at age 18 years , and physical activity during high school , was also assessed . interaction tests were performed by including a product term with the respective stratification variable and the median score of dietary pattern quintiles as a continuous variable and examining wald p values . the interaction between high school and adult dietary patterns was evaluated by modeling a cross - product term using median values of tertiles . to evaluate current dietary patterns in relation to t2 dm risk , the cumulative average of patterns obtained from the 1991 , 1995 , 1999 , and 2003 ffq cycles were used , and multivariate models were adjusted for adult risk factors described above . current bmi ( < 23 , 2324.9 , 2526.9 , 2728.9 , 2930.9 , 3132.9 , 3334.9 , 3536.9 , 3738.9 , 3940.9 , 4142.9 , 4344.9 , 45 kg / m ) and hypertension ( yes , no ) cumulative averages were used to reduce random within - person variation and best reflect long - term dietary intake ; however , a parallel analysis using baseline patterns was also evaluated . the correlation between high school and adult dietary pattern scores was evaluated by spearman correlation coefficients . to assess the joint effect of high school and adult western dietary pattern on t2 dm risk , dietary pattern scores were categorized into tertiles of low , medium , and high and cross - classified into a single variable , which was evaluated categorically using low - low as the reference in the multivariate model . all statistical tests were two - sided and performed using sas version 9 for unix ( sas institute , cary , nc ) . the nurses health study ii ( nhs ii ) is a prospective cohort of 116,671 female registered nurses aged 2444 years at baseline in 1989 . this cohort is followed using biennial mailed questionnaires on lifestyle , diet , and medical history . the study has maintained a response rate of 90% for every 2-year period ( 7 ) . in 1997 , participants were asked if they would be willing to complete a questionnaire about diet during high school ( hs - ffq [ high school food frequency questionnaire ] ) , at which time they were 3453 years of age . comparison of these women with the entire cohort suggests that they do not differ with regard to baseline dietary intake or t2 dm risk factors . this analysis is restricted to the 47,355 women who returned the hs - ffq in 1998 ( 73% of those sent the questionnaire ) . we excluded participants if they reported implausible daily caloric intakes of < 500 or 3,500 kcal / day for current intake levels ( n = 2,920 ) and < 500 or 5,000 kcal / day for high school intake levels ( n = 1,181 ) , or left 10 items blank ( n = 225 ) . in addition , we excluded participants if they had a confirmed diagnosis of diabetes ( n = 502 t2 dm , n = 42 type 1 diabetes ) or death ( n = 1 ) before return of the hs - ffq in 1998 , were missing the date of diagnosis of diabetes ( n = 616 ) , or had a history of diabetes ( n = 137 ) , gestational diabetes ( n = 1,875 ) , cancer except nonmelanoma skin cancer , or cardiovascular disease ( n = 2,818 ) reported in 1999 . after exclusions , a total of 37,038 participants remained for the analysis . high school dietary intake was assessed using the hs - ffq , a 124-item ffq that asked participants about their diet during high school . subjects were asked how often they consumed a specified amount of each item using nine responses ranging from never or less than once per month to six or more per day . categories on the hs - ffq included main dishes , bread and cereals , fruits , vegetables , snack foods , and dairy . the hs - ffq was designed to include foods that were consumed when these women were in high school . nutrient intakes on the hs - ffq were computed by multiplying the frequency of consumption of each unit of food by the nutrient content of the specified portions and then summing the contributions . recall of adolescent diet among nhs ii participants and a similar cohort was reproducible ( 8,9 ) , and the hs - ffq has shown reasonable validity ( 9,10 ) . adult dietary patterns were assessed by 131-item ffqs administered in 1991 , 1995 , 1999 , and 2003 . these ffqs assessed usual dietary intake during the past year , and food items and response categories were similar to the hs - ffq . the validity of ffqs similar to those used in the nhs ii was reasonable ( 11 ) . to identify dietary patterns , food items on the ffqs were first aggregated into 37 food groups for the hs - ffq and 40 food groups for the nhs ii ffqs , on the basis of similarities in nutrient profile and food preferences . factor analysis with the orthogonal rotation procedure varimax was applied to the food groups to generate dietary patterns ( 12 ) . briefly , factor analysis aggregates correlated variables , and each obtained factor ( dietary pattern ) is a linear combination of all food groups that are weighted by their factor loadings and explains as much variation in the food groups as possible ( 12 ) . food groups with positive loadings contribute to the dietary pattern , whereas those with negative loadings are inversely associated . food groups with absolute factor loadings 0.30 were considered as significantly contributing to the pattern ( 12 ) . prudent and western patterns , on the basis of eigenvalues ( > 1 ) , scree tests ( a graphical representation of eigenvalues > 1 ) , and interpretability of the factors ( 5 ) . scree tests allowed us to identify two major patterns with the largest eigenvalues ( > 2.75 ) . dietary pattern scores were calculated by summing the standardized intake of food groups , weighted by the factor loading of the food groups ( 12 ) . these scores were used to rank participants according to the degree to which they conformed to a given dietary pattern . the reproducibility and validity of dietary patterns derived by this method were reasonable ( 13 ) . because of potential overreporting or underreporting on the ffqs , dietary patterns were adjusted for total energy intake using the residual method ( 14 ) . women reporting a new diagnosis of diabetes on any of the biennial questionnaires were sent supplementary questionnaires to confirm the self - report . we used the american diabetes association criteria for diagnosis ( fasting glucose levels 7 mmol [ 126 mg / dl ] ) . in substudies of the nhs , 98% of self - reported diabetes cases documented by the same supplementary questionnaire dietary pattern scores were divided into quintiles , and cox proportional hazards regression was used to estimate incidence rate ratios comparing each quintile to the lowest . person - time of follow - up was calculated for each participant from 1997 until june 2005 , or date of t2 dm diagnosis , or death . test for trends across quintiles was conducted by modeling the median value for each category as a continuous variable . in the analysis of high school dietary patterns , multivariate models were adjusted for bmi at age 18 years ( < 18.5 , 18.5 to < 25 , 25 to < 30 , or 30 kg / m ) , total energy intake in high school ( quintiles , kcal / day ) , smoking between ages 15 and 19 years ( none or 14 , 514 , or 15 + cigarettes / day ) , and high school physical activity ( quintiles , metabolic equivalents [ mets]/week ) . we also adjusted for adult risk factors , including current smoking status ( never , past , current : 114 cigarettes / day , 15 cigarettes / day ) , current physical activity ( quintiles , mets / week ) , history of t2 dm in parents or siblings ( yes , no ) , oral contraceptive use ( never , past , current ) , postmenopausal hormone use ( never , ever ) , total energy ( quintiles , kcal / day ) , and alcohol ( 0 , 0.14.9 , 59.9 , 10 g / day ) . sensitivity tests were conducted by additionally adjusting for current prudent or western dietary pattern and weight change since age 18 years , which are potential mediators . effect modification of the association between high school dietary pattern and t2 dm by bmi at age 18 years , and physical activity during high school , was also assessed . interaction tests were performed by including a product term with the respective stratification variable and the median score of dietary pattern quintiles as a continuous variable and examining wald p values . the interaction between high school and adult dietary patterns was evaluated by modeling a cross - product term using median values of tertiles . to evaluate current dietary patterns in relation to t2 dm risk , the cumulative average of patterns obtained from the 1991 , 1995 , 1999 , and 2003 ffq cycles were used , and multivariate models were adjusted for adult risk factors described above . current bmi ( < 23 , 2324.9 , 2526.9 , 2728.9 , 2930.9 , 3132.9 , 3334.9 , 3536.9 , 3738.9 , 3940.9 , 4142.9 , 4344.9 , 45 kg / m ) and hypertension ( yes , no ) were additionally added to models . cumulative averages were used to reduce random within - person variation and best reflect long - term dietary intake ; however , a parallel analysis using baseline patterns was also evaluated . the correlation between high school and adult dietary pattern scores was evaluated by spearman correlation coefficients . to assess the joint effect of high school and adult western dietary pattern on t2 dm risk , dietary pattern scores were categorized into tertiles of low , medium , and high and cross - classified into a single variable , which was evaluated categorically using low - low as the reference in the multivariate model . all statistical tests were two - sided and performed using sas version 9 for unix ( sas institute , cary , nc ) . two major dietary patterns were identified from the hs - ffq ( supplementary table 1 ) . the pattern labeled prudent was characterized by a high consumption of vegetables , fruit , legumes , fish , and better - quality grains and low consumption of snacks and soda . in contrast , the pattern labeled western was characterized by a high consumption of desserts , snacks , processed meat , red meat , french fries , and refined grains and low consumption of vegetables , fruit , and fish . prudent and western , with some variation in the 2003 patterns ( supplementary table 2 ) . high school and current baseline characteristics of the study population according to quintile of high school dietary patterns are shown in table 1 . women with higher prudent pattern scores during high school were more physically active , less likely to smoke , had healthier dietary habits during high school and in adulthood , and gained less weight since age 18 years than women with a lower score for this pattern . women with higher western pattern scores during high school were less physically active , more likely to smoke , and tended to have less favorable dietary habits during high school and in adulthood , gained more weight since age 18 , and were more likely to have hypertension or a family history of diabetes . the correlation between high school and adult dietary pattern scores was 0.49 for the prudent pattern and 0.40 for the western pattern . age - adjusted high school and baseline characteristics by quintile of energy - adjusted dietary patterns among nhs ii participants during high school during 290,703 person - years of follow - up , we confirmed 550 cases of t2 dm . as shown in table 2 , the prudent pattern during high school was not associated with risk of t2 dm . however , higher western pattern scores during high school were associated with increased risk of t2 dm . after adjusting for age , bmi at age 18 years , total energy intake during high school , and physical activity and smoking during high school , participants in the highest quintile of western pattern score had a 62% greater risk of t2 dm than participants in the lowest quintile ( relative risk [ rr ] 1.62 ; 95% ci 1.272.07 ; p trend < 0.0001 ) . this association persisted after adjustment for adult risk factors ( physical activity , family history of diabetes , smoking , postmenopausal hormone use , oral contraceptive use , total energy intake , and alcohol use ) ( 1.39 ; 1.081.78 ; p trend : 0.006 ) . additional adjustment for adult western pattern score weakened the association ( 1.29 ; 1.001.66 ; p trend : 0.04 ) , and the association became nonsignificant after adjustment for weight change since age 18 years . rr of t2 dm among nhs ii participants ( 19972005 ) according to quintile of energy - adjusted high school dietary pattern the association between high school western dietary pattern and t2 dm was modified by bmi at age 18 years ( p for interaction : 0.04 ) , with the association being stronger among individuals with bmi at age 18 years of < 25 kg / m than individuals who were overweight at age 18 years ( supplementary table 3 ) . a higher score for the prudent pattern was associated with reduced risk of t2 dm after adjusting for age ( rr for highest vs. lowest quintile : 0.52 ; 95% ci 0.400.67 ; p trend : < 0.0001 ) ( supplementary table 4 ) . in contrast , a higher adult western pattern score was associated with a substantially higher risk for t2 dm . the association persisted after adjustment for potential confounders ( rr 2.14 ; 95% ci 1.582.88 ; p trend : < 0.0001 ) , but disappeared after adjustment for bmi ( 1.24 ; 0.911.69 ; p trend : 0.23 ) . additional adjustment for hypertension did not change the associations ( 0.85 , 0.641.12 , and 1.20 , 0.881.62 , for prudent and western patterns , respectively ) . similarly , results were obtained from the analysis using baseline dietary patterns ( data not shown ) . as shown in fig . 1 , individuals who had high western dietary pattern scores in adulthood and during high school had the greatest risk of t2 dm compared with individuals with low scores ( rr 1.82 ; 95% ci 1.352.45 ) . the strength of the association for the western pattern during high school appeared to decrease with lower levels of adult western pattern . adjustment for current bmi attenuated this association ( 1.15 ; 0.851.56 ) ( not shown ) . joint analysis between adult and high school western dietary pattern score in relation to t2 dm risk . tertiles ( low , medium , high ) of high school and adult western dietary pattern score were cross - classified into a single categorical variable and evaluated for risk of t2 dm using the low - low category as the reference . high levels of high school western dietary pattern are depicted by black bars , medium levels by gray bars , and low levels by white bars . data are adjusted for age , total calories , family history of diabetes , smoking status , physical activity , oral contraceptive use , hormone replacement therapy , and alcohol . the current pattern high school patterns [ rr ( 95% ci ) ] were as follows : low - low ( ref ) , 1.00 ; medium - low , 0.95 ( 0.661.37 ) ; high - low , 1.55 ( 1.102.19 ) ; low - medium , 0.94 ( 0.611.44 ) ; medium - medium , 1.12 ( 0.781.62 ) ; high - medium , 1.45 ( 1.022.05 ) ; low - high , 0.94 ( 0.581.53 ) ; medium - high , 1.28 ( 0.881.85 ) ; and high - high , 1.82 ( 1.352.45 ) . current median western pattern scores were as follows : low , 0.53 ; medium , 0.05 ; and high , 0.44 . high school median western pattern scores were as follows : low , 0.43 ; medium , 0.02 ; and high , 0.42 . in this large prospective cohort study of u.s . women , we identified two major dietary patterns during high school . greater adherence to the prudent pattern , characterized by a high intake of healthy foods , was not associated with risk of t2 dm , which is consistent with previous studies in our cohorts ( 3,5 ) . however , greater adherence to the western pattern , characterized by a high intake of desserts , processed meat , and refined grains , was associated with a higher risk of t2 dm independent of other high school and adult risk factors . to our knowledge , this is the first study that has examined adolescent dietary patterns in relation to risk of t2 dm decades later . the association between western patterns and t2 dm appeared to be partly mediated through weight gain . in this cohort , greater adherence to a western dietary pattern was previously associated with weight gain , whereas a prudent pattern was shown to facilitate weight maintenance ( 4 ) . our findings that adjustment for weight change since age 18 years and current bmi attenuated the associations between high school and current western dietary patterns with t2dm support a role for adiposity as a mediator . this result is supported by our finding that both high school dietary patterns predicted weight change since age 18 years ( p < 0.0001 ) ( not shown ) . the interaction between high school western dietary pattern and bmi at age 18 years suggests that this pattern may also affect future risk of t2 dm in previously normal - weight ( bmi < 25 kg / m ) girls , although the mechanism is not clear and should be interpreted cautiously . dietary components in the pattern that may affect t2 dm risk include higher saturated fat ( 16 ) , glycemic load ( 1 ) , heme iron ( 17 ) , and advanced glycation end products ( 18 ) from a higher consumption of processed meat , refined grains , and desserts . it is most likely a combination of dietary factors that was responsible for the robust associations . higher scores for the western pattern have been correlated with higher fasting insulin , and c - peptide levels ( 19 ) and inflammatory markers ( 20 ) . in our study , women who had high western pattern scores both in adolescence and in adulthood had the greatest risk of t2 dm , suggesting that adhering to a western dietary pattern during adolescence may affect risk of t2 dm beyond that imparted by high western pattern scores in adulthood , possibly by persistence of an unhealthy dietary pattern through the life course . little is known about how diet during early life may affect risk of t2 dm years later . greater adult height , which may represent adequate childhood nutritional status , has been associated with decreased risk of t2 dm , possibly by increasing childhood igf-1 levels ( 21,22 ) , although this relation has not been fully established . it is possible that greater adherence to a western pattern during adolescence increases risk of t2 dm in adulthood via a cumulative effect on physiological processes underlying disease development . findings from the bogalusa heart study indicate that high blood pressure , hyperinsulinemia , and dyslipidemia begin to cluster during childhood and can predict adult cardiovascular risk factors ( 23 ) . this clustering of risk factors has been linked to unhealthy dietary habits during childhood ( 24 ) . it may also be possible that greater adherence to the western dietary pattern is associated with an unhealthy lifestyle , which may track through life , affecting risk of t2 dm . despite adjusting for potential confounders , residual confounding the validity of adult recall of adolescent diet 1535 years earlier has not been established , since it is difficult to disentangle the effects of waning memory with age , influence of current diet on recall of past diet , and temporal changes in diet ( 8,9 ) . however , lack of an interaction with age at return of the hs - ffq suggests that fading memory of high school diet with age may not be a significant factor in these data , although this effect is difficult to isolate ( p for interaction : 0.89 for prudent pattern and 0.43 for western pattern ; supplementary table 5 ) . the validity of the hs - ffq was assessed by administering it to 80 young adults who had completed three 24-h diet recalls and two similar ffqs 10 years earlier while in high school ( 10 ) . the average nutrient correlation between the hs - ffq and 24-h recalls was 0.45 ( range 0.160.68 ) and between the hs - ffq and ffqs was 0.58 ( 0.400.88 ) ( 10 ) . validity of the hs - ffq was also assessed by comparing the nurses self - reports with those of their mothers ( 9 ) . the mean nutrient correlation between the mothers and nurses self - reports was 0.40 ( 0.130.59 ) and for foods 0.30 ( 0.100.61 ) ( 9 ) . other studies that have examined the validity of diet recalled 1124 years have reported moderate correlations for food ( range 0.290.4 ) and nutrients ( 0.230.59 ) ( 8) , which are similar to those reported for the hs - ffq . survey methodology shows that provision of a clear definition of the reference period ( i.e. , high school ) enhances recall ( 25 ) . these findings suggest that the hs - ffq provides a reasonable record of adolescent diet , although some attenuation of results can be expected because of nondifferential measurement error . there are also limitations in dietary pattern analysis such as subjectivity in grouping foods and determining the number of factors to retain . the reason why current patterns from the 2003 ffq were slightly different from previous years may be due to temporal changes in the diet . exclusion of 2003 ffq data from the cumulative average did not change our results . the prospective design of our study , in which cases were ascertained after return of the hs - ffq , limits the likelihood of recall bias caused by knowledge of disease status . an important strength of dietary pattern analysis is the ability to detect the combined effect of foods , since the physical properties of foods and interactions between nutrients may affect glucose homeostasis . our findings confirmed those from previous studies that have found positive associations between the western dietary pattern and risk of t2 dm . in addition , we showed that greater adherence to this pattern during adolescence was associated with increased risk of t2 dm independently and jointly with adult western patterns . women who had high western pattern scores during adolescence and in adulthood had the greatest risk of t2 dm compared with individuals with consistent low scores , suggesting that persistence of a western - type dietary pattern into adulthood may have particularly adverse effects on t2 dm risk , possibly through weight gain . for optimal prevention of t2 dm , a healthy diet should be adopted in early life and maintained throughout the life course .

objectivewhether dietary habits early in life can affect risk of type 2 diabetes ( t2 dm ) in adulthood is unknown . we evaluated the relationship between dietary patterns during adolescence and risk of t2 dm in midlife.research design and methodswe examined the 7-year incidence of t2 dm in relation to dietary patterns during high school among 37,038 participants in the nurses health study ii cohort , who completed a food - frequency questionnaire about their diet during high school . dietary patterns were derived by factor analysis . cox proportional hazards regression was used to estimate relative risk ( rr ) and 95% ci.resultsthe prudent pattern , characterized by healthy foods , was not associated with risk of t2 dm . the western pattern , characterized by desserts , processed meats , and refined grains , was associated with 29% greater risk of t2 dm ( rr 1.29 ; 95% ci 1.001.66 ; p trend 0.04 ) , after adjusting for high school and adult risk factors comparing extreme quintiles , but was attenuated after adjusting for adult weight change ( 1.19 ; 0.921.54 ) . women who had high western pattern scores in high school and adulthood had an elevated risk of t2 dm compared with women who had consistent low scores ( 1.82 ; 1.352.45 ) , and this association was partly mediated by adult bmi ( 1.15 ; 0.851.56).conclusionsa western dietary pattern during adolescence may increase risk of t2 dm in later life , partly through adult weight gain . preventive measures should be aimed at developing healthy dietary habits that begin in early life and continue through adulthood .

RESEARCH DESIGN AND METHODS Study population Assessment of high school and adult dietary patterns Outcome assessment Statistical analysis RESULTS CONCLUSIONS

the nurses health study ii ( nhs ii ) is a prospective cohort of 116,671 female registered nurses aged 2444 years at baseline in 1989 . in 1997 , participants were asked if they would be willing to complete a questionnaire about diet during high school ( hs - ffq [ high school food frequency questionnaire ] ) , at which time they were 3453 years of age . comparison of these women with the entire cohort suggests that they do not differ with regard to baseline dietary intake or t2 dm risk factors . we excluded participants if they reported implausible daily caloric intakes of < 500 or 3,500 kcal / day for current intake levels ( n = 2,920 ) and < 500 or 5,000 kcal / day for high school intake levels ( n = 1,181 ) , or left 10 items blank ( n = 225 ) . in addition , we excluded participants if they had a confirmed diagnosis of diabetes ( n = 502 t2 dm , n = 42 type 1 diabetes ) or death ( n = 1 ) before return of the hs - ffq in 1998 , were missing the date of diagnosis of diabetes ( n = 616 ) , or had a history of diabetes ( n = 137 ) , gestational diabetes ( n = 1,875 ) , cancer except nonmelanoma skin cancer , or cardiovascular disease ( n = 2,818 ) reported in 1999 . high school dietary intake was assessed using the hs - ffq , a 124-item ffq that asked participants about their diet during high school . categories on the hs - ffq included main dishes , bread and cereals , fruits , vegetables , snack foods , and dairy . the hs - ffq was designed to include foods that were consumed when these women were in high school . recall of adolescent diet among nhs ii participants and a similar cohort was reproducible ( 8,9 ) , and the hs - ffq has shown reasonable validity ( 9,10 ) . adult dietary patterns were assessed by 131-item ffqs administered in 1991 , 1995 , 1999 , and 2003 . factor analysis with the orthogonal rotation procedure varimax briefly , factor analysis aggregates correlated variables , and each obtained factor ( dietary pattern ) is a linear combination of all food groups that are weighted by their factor loadings and explains as much variation in the food groups as possible ( 12 ) . these scores were used to rank participants according to the degree to which they conformed to a given dietary pattern . the reproducibility and validity of dietary patterns derived by this method were reasonable ( 13 ) . in substudies of the nhs , 98% of self - reported diabetes cases documented by the same supplementary questionnaire dietary pattern scores were divided into quintiles , and cox proportional hazards regression was used to estimate incidence rate ratios comparing each quintile to the lowest . in the analysis of high school dietary patterns , multivariate models were adjusted for bmi at age 18 years ( < 18.5 , 18.5 to < 25 , 25 to < 30 , or 30 kg / m ) , total energy intake in high school ( quintiles , kcal / day ) , smoking between ages 15 and 19 years ( none or 14 , 514 , or 15 + cigarettes / day ) , and high school physical activity ( quintiles , metabolic equivalents [ mets]/week ) . we also adjusted for adult risk factors , including current smoking status ( never , past , current : 114 cigarettes / day , 15 cigarettes / day ) , current physical activity ( quintiles , mets / week ) , history of t2 dm in parents or siblings ( yes , no ) , oral contraceptive use ( never , past , current ) , postmenopausal hormone use ( never , ever ) , total energy ( quintiles , kcal / day ) , and alcohol ( 0 , 0.14.9 , 59.9 , 10 g / day ) . sensitivity tests were conducted by additionally adjusting for current prudent or western dietary pattern and weight change since age 18 years , which are potential mediators . effect modification of the association between high school dietary pattern and t2 dm by bmi at age 18 years , and physical activity during high school , was also assessed . the interaction between high school and adult dietary patterns was evaluated by modeling a cross - product term using median values of tertiles . to evaluate current dietary patterns in relation to t2 dm risk , the cumulative average of patterns obtained from the 1991 , 1995 , 1999 , and 2003 ffq cycles were used , and multivariate models were adjusted for adult risk factors described above . current bmi ( < 23 , 2324.9 , 2526.9 , 2728.9 , 2930.9 , 3132.9 , 3334.9 , 3536.9 , 3738.9 , 3940.9 , 4142.9 , 4344.9 , 45 kg / m ) and hypertension ( yes , no ) cumulative averages were used to reduce random within - person variation and best reflect long - term dietary intake ; however , a parallel analysis using baseline patterns was also evaluated . the correlation between high school and adult dietary pattern scores was evaluated by spearman correlation coefficients . to assess the joint effect of high school and adult western dietary pattern on t2 dm risk , dietary pattern scores were categorized into tertiles of low , medium , and high and cross - classified into a single variable , which was evaluated categorically using low - low as the reference in the multivariate model . the nurses health study ii ( nhs ii ) is a prospective cohort of 116,671 female registered nurses aged 2444 years at baseline in 1989 . in 1997 , participants were asked if they would be willing to complete a questionnaire about diet during high school ( hs - ffq [ high school food frequency questionnaire ] ) , at which time they were 3453 years of age . we excluded participants if they reported implausible daily caloric intakes of < 500 or 3,500 kcal / day for current intake levels ( n = 2,920 ) and < 500 or 5,000 kcal / day for high school intake levels ( n = 1,181 ) , or left 10 items blank ( n = 225 ) . in addition , we excluded participants if they had a confirmed diagnosis of diabetes ( n = 502 t2 dm , n = 42 type 1 diabetes ) or death ( n = 1 ) before return of the hs - ffq in 1998 , were missing the date of diagnosis of diabetes ( n = 616 ) , or had a history of diabetes ( n = 137 ) , gestational diabetes ( n = 1,875 ) , cancer except nonmelanoma skin cancer , or cardiovascular disease ( n = 2,818 ) reported in 1999 . high school dietary intake was assessed using the hs - ffq , a 124-item ffq that asked participants about their diet during high school . adult dietary patterns were assessed by 131-item ffqs administered in 1991 , 1995 , 1999 , and 2003 . briefly , factor analysis aggregates correlated variables , and each obtained factor ( dietary pattern ) is a linear combination of all food groups that are weighted by their factor loadings and explains as much variation in the food groups as possible ( 12 ) . because of potential overreporting or underreporting on the ffqs , dietary patterns were adjusted for total energy intake using the residual method ( 14 ) . in substudies of the nhs , 98% of self - reported diabetes cases documented by the same supplementary questionnaire dietary pattern scores were divided into quintiles , and cox proportional hazards regression was used to estimate incidence rate ratios comparing each quintile to the lowest . person - time of follow - up was calculated for each participant from 1997 until june 2005 , or date of t2 dm diagnosis , or death . in the analysis of high school dietary patterns , multivariate models were adjusted for bmi at age 18 years ( < 18.5 , 18.5 to < 25 , 25 to < 30 , or 30 kg / m ) , total energy intake in high school ( quintiles , kcal / day ) , smoking between ages 15 and 19 years ( none or 14 , 514 , or 15 + cigarettes / day ) , and high school physical activity ( quintiles , metabolic equivalents [ mets]/week ) . we also adjusted for adult risk factors , including current smoking status ( never , past , current : 114 cigarettes / day , 15 cigarettes / day ) , current physical activity ( quintiles , mets / week ) , history of t2 dm in parents or siblings ( yes , no ) , oral contraceptive use ( never , past , current ) , postmenopausal hormone use ( never , ever ) , total energy ( quintiles , kcal / day ) , and alcohol ( 0 , 0.14.9 , 59.9 , 10 g / day ) . sensitivity tests were conducted by additionally adjusting for current prudent or western dietary pattern and weight change since age 18 years , which are potential mediators . effect modification of the association between high school dietary pattern and t2 dm by bmi at age 18 years , and physical activity during high school , was also assessed . the interaction between high school and adult dietary patterns was evaluated by modeling a cross - product term using median values of tertiles . to evaluate current dietary patterns in relation to t2 dm risk , the cumulative average of patterns obtained from the 1991 , 1995 , 1999 , and 2003 ffq cycles were used , and multivariate models were adjusted for adult risk factors described above . current bmi ( < 23 , 2324.9 , 2526.9 , 2728.9 , 2930.9 , 3132.9 , 3334.9 , 3536.9 , 3738.9 , 3940.9 , 4142.9 , 4344.9 , 45 kg / m ) and hypertension ( yes , no ) were additionally added to models . the correlation between high school and adult dietary pattern scores was evaluated by spearman correlation coefficients . to assess the joint effect of high school and adult western dietary pattern on t2 dm risk , dietary pattern scores were categorized into tertiles of low , medium , and high and cross - classified into a single variable , which was evaluated categorically using low - low as the reference in the multivariate model . two major dietary patterns were identified from the hs - ffq ( supplementary table 1 ) . in contrast , the pattern labeled western was characterized by a high consumption of desserts , snacks , processed meat , red meat , french fries , and refined grains and low consumption of vegetables , fruit , and fish . high school and current baseline characteristics of the study population according to quintile of high school dietary patterns are shown in table 1 . women with higher prudent pattern scores during high school were more physically active , less likely to smoke , had healthier dietary habits during high school and in adulthood , and gained less weight since age 18 years than women with a lower score for this pattern . women with higher western pattern scores during high school were less physically active , more likely to smoke , and tended to have less favorable dietary habits during high school and in adulthood , gained more weight since age 18 , and were more likely to have hypertension or a family history of diabetes . the correlation between high school and adult dietary pattern scores was 0.49 for the prudent pattern and 0.40 for the western pattern . age - adjusted high school and baseline characteristics by quintile of energy - adjusted dietary patterns among nhs ii participants during high school during 290,703 person - years of follow - up , we confirmed 550 cases of t2 dm . as shown in table 2 , the prudent pattern during high school was not associated with risk of t2 dm . however , higher western pattern scores during high school were associated with increased risk of t2 dm . after adjusting for age , bmi at age 18 years , total energy intake during high school , and physical activity and smoking during high school , participants in the highest quintile of western pattern score had a 62% greater risk of t2 dm than participants in the lowest quintile ( relative risk [ rr ] 1.62 ; 95% ci 1.272.07 ; p trend < 0.0001 ) . this association persisted after adjustment for adult risk factors ( physical activity , family history of diabetes , smoking , postmenopausal hormone use , oral contraceptive use , total energy intake , and alcohol use ) ( 1.39 ; 1.081.78 ; p trend : 0.006 ) . additional adjustment for adult western pattern score weakened the association ( 1.29 ; 1.001.66 ; p trend : 0.04 ) , and the association became nonsignificant after adjustment for weight change since age 18 years . rr of t2 dm among nhs ii participants ( 19972005 ) according to quintile of energy - adjusted high school dietary pattern the association between high school western dietary pattern and t2 dm was modified by bmi at age 18 years ( p for interaction : 0.04 ) , with the association being stronger among individuals with bmi at age 18 years of < 25 kg / m than individuals who were overweight at age 18 years ( supplementary table 3 ) . a higher score for the prudent pattern was associated with reduced risk of t2 dm after adjusting for age ( rr for highest vs. lowest quintile : 0.52 ; 95% ci 0.400.67 ; p trend : < 0.0001 ) ( supplementary table 4 ) . in contrast , a higher adult western pattern score was associated with a substantially higher risk for t2 dm . the association persisted after adjustment for potential confounders ( rr 2.14 ; 95% ci 1.582.88 ; p trend : < 0.0001 ) , but disappeared after adjustment for bmi ( 1.24 ; 0.911.69 ; p trend : 0.23 ) . 1 , individuals who had high western dietary pattern scores in adulthood and during high school had the greatest risk of t2 dm compared with individuals with low scores ( rr 1.82 ; 95% ci 1.352.45 ) . the strength of the association for the western pattern during high school appeared to decrease with lower levels of adult western pattern . adjustment for current bmi attenuated this association ( 1.15 ; 0.851.56 ) ( not shown ) . joint analysis between adult and high school western dietary pattern score in relation to t2 dm risk . tertiles ( low , medium , high ) of high school and adult western dietary pattern score were cross - classified into a single categorical variable and evaluated for risk of t2 dm using the low - low category as the reference . high levels of high school western dietary pattern are depicted by black bars , medium levels by gray bars , and low levels by white bars . the current pattern high school patterns [ rr ( 95% ci ) ] were as follows : low - low ( ref ) , 1.00 ; medium - low , 0.95 ( 0.661.37 ) ; high - low , 1.55 ( 1.102.19 ) ; low - medium , 0.94 ( 0.611.44 ) ; medium - medium , 1.12 ( 0.781.62 ) ; high - medium , 1.45 ( 1.022.05 ) ; low - high , 0.94 ( 0.581.53 ) ; medium - high , 1.28 ( 0.881.85 ) ; and high - high , 1.82 ( 1.352.45 ) . high school median western pattern scores were as follows : low , 0.43 ; medium , 0.02 ; and high , 0.42 . women , we identified two major dietary patterns during high school . greater adherence to the prudent pattern , characterized by a high intake of healthy foods , was not associated with risk of t2 dm , which is consistent with previous studies in our cohorts ( 3,5 ) . however , greater adherence to the western pattern , characterized by a high intake of desserts , processed meat , and refined grains , was associated with a higher risk of t2 dm independent of other high school and adult risk factors . to our knowledge , this is the first study that has examined adolescent dietary patterns in relation to risk of t2 dm decades later . the association between western patterns and t2 dm appeared to be partly mediated through weight gain . in this cohort , greater adherence to a western dietary pattern was previously associated with weight gain , whereas a prudent pattern was shown to facilitate weight maintenance ( 4 ) . our findings that adjustment for weight change since age 18 years and current bmi attenuated the associations between high school and current western dietary patterns with t2dm support a role for adiposity as a mediator . this result is supported by our finding that both high school dietary patterns predicted weight change since age 18 years ( p < 0.0001 ) ( not shown ) . the interaction between high school western dietary pattern and bmi at age 18 years suggests that this pattern may also affect future risk of t2 dm in previously normal - weight ( bmi < 25 kg / m ) girls , although the mechanism is not clear and should be interpreted cautiously . dietary components in the pattern that may affect t2 dm risk include higher saturated fat ( 16 ) , glycemic load ( 1 ) , heme iron ( 17 ) , and advanced glycation end products ( 18 ) from a higher consumption of processed meat , refined grains , and desserts . higher scores for the western pattern have been correlated with higher fasting insulin , and c - peptide levels ( 19 ) and inflammatory markers ( 20 ) . in our study , women who had high western pattern scores both in adolescence and in adulthood had the greatest risk of t2 dm , suggesting that adhering to a western dietary pattern during adolescence may affect risk of t2 dm beyond that imparted by high western pattern scores in adulthood , possibly by persistence of an unhealthy dietary pattern through the life course . little is known about how diet during early life may affect risk of t2 dm years later . greater adult height , which may represent adequate childhood nutritional status , has been associated with decreased risk of t2 dm , possibly by increasing childhood igf-1 levels ( 21,22 ) , although this relation has not been fully established . it is possible that greater adherence to a western pattern during adolescence increases risk of t2 dm in adulthood via a cumulative effect on physiological processes underlying disease development . it may also be possible that greater adherence to the western dietary pattern is associated with an unhealthy lifestyle , which may track through life , affecting risk of t2 dm . however , lack of an interaction with age at return of the hs - ffq suggests that fading memory of high school diet with age may not be a significant factor in these data , although this effect is difficult to isolate ( p for interaction : 0.89 for prudent pattern and 0.43 for western pattern ; supplementary table 5 ) . the validity of the hs - ffq was assessed by administering it to 80 young adults who had completed three 24-h diet recalls and two similar ffqs 10 years earlier while in high school ( 10 ) . other studies that have examined the validity of diet recalled 1124 years have reported moderate correlations for food ( range 0.290.4 ) and nutrients ( 0.230.59 ) ( 8) , which are similar to those reported for the hs - ffq . our findings confirmed those from previous studies that have found positive associations between the western dietary pattern and risk of t2 dm . in addition , we showed that greater adherence to this pattern during adolescence was associated with increased risk of t2 dm independently and jointly with adult western patterns . women who had high western pattern scores during adolescence and in adulthood had the greatest risk of t2 dm compared with individuals with consistent low scores , suggesting that persistence of a western - type dietary pattern into adulthood may have particularly adverse effects on t2 dm risk , possibly through weight gain . for optimal prevention of t2 dm , a healthy diet should be adopted in early life and maintained throughout the life course .

helicobacter pylori is a gram - negative gastrointestinal bacterium that has coevolved with its human host for at least 58.000 years , and worldwide still half of the human population is infected . h. pylori infection leads to chronic inflammation of the gastric mucosa , which remains often without clinical symptoms . however , serious diseases as peptic ulcer disease , mucosa - associated lymphoid tissue ( malt ) lymphoma , and gastric adenocarcinoma develop in 15 percent of the infected population . whereas h. pylori infection triggers a vigorous immune response , resulting in high titres of h. pylori - specific antibodies , this response is not sufficient for eradication of the pathogen and when remaining untreated with antibiotics , infection will remain for life . therefore , h. pylori is often regarded a model organism for persistent bacterial infection in man . h. pylori is heterogeneous and outcome of infection depends on both bacterial virulence factors and host genetics , as reviewed in . whereas appropriate host immune responses lead to asymptomatic persistent colonization , h. pylori - associated pathology is the result of aberrant inflammation . h. pylori induces a strong innate immune response that involves various components of the innate immune system , including nucleotide - binding oligomerization domain protein i ( nod1 ) and secretion of antimicrobial peptides . in addition , studies report that h. pylori induces pro - inflammatory gene expression in host cells via toll - like receptor ( tlr)2 , tlr4 , tlr5 , and tlr9 [ 710 ] . however , several of these reports present conflicting data and this area requires further research , which should take into account tlr expression dynamics and the actual repertoire of expressed tlr in the infected gastric mucosa in vivo . the innate immune response to h. pylori results in inflammation of the gastric epithelium ( gastritis ) and induces an influx of neutrophils and other immune cell though the release of chemokines and cytokines , as reviewed in , thereby being essential for the initiation of an acquired immune response to h. pylori . the murine h. felis infection model provides a useful model resembling gastric pathological changes in human h. pylori infection . h. felis infection of b and t cell - deficient ( rag-1 ) mice and t cell - deficient ( tcr ) mice does not result in gastric pathology despite high levels of colonization , whereas infection of b cell - deficient mice results in severe gastric alteration , identical to those seen in immunocompetent mice . these results indicate that t cells are required for protection against helicobacter , but also that gastric immune mediated damage is dependent on t cells , and not by b cells or antibody secretion . cd4 t cells can be categorized according to their cytokine - secretion profile and cytotoxic potential . th1 cells secrete tumour necrosis factor ( tnf ) and interferon- ( ifn- ) , lyse antigen - loaded target cells through mechanisms that are mediated by perforin or fas ( also known as cd95 ) , and elicit macrophage activation . th2 cells secrete interleukin-4 ( il-4 ) , il-5 , and il-10 , are involved in down regulation of th1cell - mediated inflammatory events , and facilitate production of antibodies by b cells . the host cytokine response is regarded an important determinant in onset of gastritis and the development of disease . vaccination studies in different mouse strains have shown that , dependent on mouse strain or helicobacter species , both th2 [ 14 , 15 ] and th1 [ 16 , 17 ] cytokine responses are involved in the adaptive immune response against h. pylori and that protection from disease may require the ability of the host to mount a balanced th1/th2 response upon infection . whereas the severity of gastritis due to innate immunity influences the risk of disease , it seems that the pattern of inflammation in the stomach determines which disease will develop : chronic antral - predominant inflammation is associated with increased acid production and predisposes tot duodenal ulceration , whereas corpus - predominant or pan - gastritis is associated with reduced acid production and predisposes to gastric ulceration and gastric adenocarcinoma . whether h. pylori - associated gastroduodenal disease develops , or asymptomatic chronic gastritis , is related to the predominant t - helper phenotype of the acquired immune response that eventually arises upon infection . human gastric t cells in h. pylori infection have a predominant th1 phenotype [ 18 , 19 ] , which is associated with pathology . dendritic cells ( dc ) in the gastric mucosa take up antigens and migrate to nearby lymph nodes where they activate nave t cells and orchestrate the subsequent immune response . in vitro , dc cultured in the presence of h. pylori are activated and secrete cytokines , including interleukin ( il)-6 , il-8 , il-10 , il-12 , il-1 , and tnf [ 22 , 23 ] . h. pylori - pulsed dc induce increased il-2 , ifn- , and tnf in cd4cd45ra nave t cells , which is in agreement with the th1 cytokine profile produced by t cells from gastric mucosal biopsies [ 18 , 19 , 24 ] . virulence factors that contribute to the predominance of th1 responses in h. pylori infection include the h. pylori neutrophil activating protein ( hp - nap ) , which induces il-12 secretion by neutrophils and monocytes and strongly promotes th1-skewing of antigen - specific cd4 t cells , and a plasticity region locus jhp0947-jhp0949 that is associated with duodenal ulcer disease . in addition , the th1/th2 balance is influenced by h. pylori genomic dna recombination and phase - variable expression of lps lewis blood - group antigens which bind to the dc receptor dc - sign , further enhancing h. pylori capacity to adapt to the host immune system in order to achieve persistent infection . in h. pylori- infected individuals , peptic ulcer disease ( pud ) is associated with th1 polarization of gastric cd4 t cell responses , whereas uncomplicated chronic gastritis ( ucg ) is associated with a mixed th1/th2 response . t cell clones from the gastric antral mucosa of patients with or without peptic ulcer display a different preferential antigen - specificity . approximately half of the h. pylori - reactive th cell clones derived from peptic ulcer patients were specific for the cytotoxin - associated protein ( caga ) , whereas approximately one forth of h. pylori - reactive th clones from nonulcer gastritis patients were specific for h. pylori urease [ 18 , 20 ] . upon antigen - specific stimulation , over eighty percent of the h. pylori - reactive th cells from peptic ulcer disease patients showed a polarized th1 profile , with high production of ifn- and no il-4 . in contrast , in non - ulcer gastritis patients about two - third of the h. pylori - specific antral clones displayed a mixed th1/th2 cytokine profile , whereas only one - third was polarized to th1 . these results are in agreement with several studies that indicate that th1 polarization of the h. pylori - specific t cell response is associated with more severe disease [ 18 , 19 , 24 , 26 , 29 , 30 ] . in the h. pylori - infected gastric mucosa , il-12 drives the th1 signalling pathway . the pathogenic capacity of polarized th1 responses in h. pylori infection is indirectly supported by the phenomenon often referred to as the african enigma , that is , despite a high prevalence of h. pylori infection in africa , peptic ulcer disease and gastric cancer are uncommon . the notion that concurrent th2 responses could reduce th1-mediated gastro - duodenal pathology and development of gastric cancer is supported by the observation that coinfection of mice with h. felis and a natural murine helminth ( heligmosomoides polygyrus ) reduced th1-mediated gastric atrophy , which is regarded as a premalignant lesion . therefore , one possible explanation for the low prevalence of h. pylori - associated diseases in africa is that the endemic high parasitic burden causes predominant th2 responses that protect h. pylori - individuals from developing peptic ulcers and gastric cancer . an accumulating body of evidence indicates that cd4cd25 regulatory t cells ( treg ) do not only prevent development of autoimmune diseases , but also regulate pathogen- induced inflammation and disease , including in h. pylori infection . in mice , cd4cd25 treg reduce gastric immunopathology , despite increased h. pylori colonization of the gastric mucosa , and in vitro suppress antigen - specific proliferation of h. pylori - specific cd4 t effector cells . in vivo , depletion of cd4cd25foxp treg in h. pylori infected mice leads to severe gastritis , influx of b cells , t cells , and macrophages into the gastric mucosa , increased titers of h. pylori - specific igg1 , and igg2c and , subsequently , reduced bacterial load . the mechanisms of effector t cell suppression by cd4cd25 treg remain to be elucidated , but they include ctla-4-mediated anergy and tgf- secretion . once cd4 t cells are rendered anergic by cd4cd25 treg , the presence of these treg is not required to maintain hyporesponsiveness . also in humans , natural cd4cd25foxp treg are recruited during h. pylori infection and correlate with increased bacterial colonization and mucosal tgf- gene expression . when compared to adults , h. pylori - infected children have increased numbers of treg and increased expression of the treg - cytokines tgf- and il-10 and lower level of gastritis , despite similar levels of h. pylori colonization . in addition , cd4cd25foxp treg cell numbers are increased in areas of duodenal gastric metaplasia in duodenal ulcer patients where they are localized in cd4 t cell aggregates . together , these results suggest that h. pylori infection induces cd4cd25foxp treg that contribute to the asymptomatic persistent infection , seen in the majority of people , and that individuals with inadequate regulatory t cell responses will develop gastro - duodenal pathology . this profound role of regulatory t cells in outcome of h. pylori infection may provide another explanation for the african enigma : in endemic areas long - term helminth infections induce a robust anti - inflammatory , regulatory t cell network that protects h. pylori - infected individuals from peptic ulcer disease and gastric cancer . several clinical and epidemiological studies indicate that most patients with autoimmune gastritis ( aig ) have or have had h. pylori infection . aig is characterized by autoimmune- mediated destruction of the secretory glands in the corpus of the stomach , leading to loss of gastric acid producing parietal cells and zymogenic cells , referred to as corpus atrophy . there are striking similarities between classical aig and h. pylori - induced corpus atrophic gastritis , and histologically defined preclinical stages of aig can be successfully treated by h. pylori eradication [ 4850 ] . h. pylori induces autoantibodies reactive with gastric mucosal antigens in approximately half of the infected individuals [ 5155 ] . gastric hk - atpase , the proton pump located in the secretory canaliculi of the parietal cells , is the autoantigen that drives immunopathology in aig . hk - atpase has also been identified as the single major autoantigen in chronic h. pylori gastritis with corpus atrophy . in mice , h. pylori infection induces gastric autoantibodies through mimicry between lps lewis blood - group antigens and lewis antigens on the glycosylated -subunit of hk - atpase . however , in humans , h. pylori - associated anti - hk - atpase autoantibodies are directed against protein epitopes , and their formation does not involve lewis mimicry . in humans , h. pylori infection leads to the activation of gastric t cells , of which a proportion is cross - reactive against both h. pylori peptide antigens and hk - atpase , and circulating antigastric autoantibodies are not pathogenic themselves , but mark ongoing t cell - mediated gastric autoimmunity . our knowledge of the pathogenesis of human aig is based largely on mouse models of experimental autoimmune gastritis ( eaig ) . eaig spontaneously develops in t cell receptor transgenic ( tcr - tg ) mice in which the cd4 t cells express an tcr that recognize an epitope of the hk - atpase -subunit ( 630641 ) or in neonatally thymectomized mice . in eaig and aig , the gastric inflammatory infiltrate comprises cd4 and cd8 t cells , macrophages and b cells , and the histopathological lesions in mouse models are similar to those observed in humans with chronic aig [ 62 , 63 ] . eaig is mediated by hk - atpase - specific cd4 t cells and gastritogenic t cell epitopes on both and subunits on hk - atpase have been identified [ 6567 ] . in human aig , a proportion of autoreactive gastric t cells recognize hk - atpase epitopes identical or partly overlapping with those in eaig [ 68 , 69 ] . the relevance of such partly conserved epitopes in ( e)aig remains unclear , but availability of an epitope for tcr recognition , rather than affinity for the tcr , enhances the gastritogenic potential of autoreactive t cells . eaig can be induced by either th1 , th2 or th17 ( il-17 secreting ) cells specific for hk - atpase . however , a major role has been attributed to ifn- , and gastric mucosa infiltrating th1 cells in onset and pathology of eaig . this is in concordance with our observation that in aig , the majority ( 87% ) of hk - atpase - specific t cell clones produced ifn- upon stimulation with the appropriate antigenic peptide epitope . these striking similarities between eaig and aig indicate that eaig is an excellent model for human aig . since eaig is one of the best studied animal models for human autoimmunity , advances in this model may contribute to our knowledge of organ - specific autoimmune disease in general . recently , the role of naturally occurring cd4cd25 treg in th1- , th2- , and th17-mediated gastritis has been described and tgf-induced autoantigen - specific , foxp regulatory t cells ( itreg ) have been shown to prevent autoimmunity by suppressing activation of autoantigen - presenting dendritic cells . even if data in h. pylori model is still relatively sparce , the balance between il-12 , il-23 , and treg responses seems to play an important role in the type of inflammatory responses in mouse models of inflammatory bowel disease . the gastrointestinal tract of mammals is inhabited by many different species of commensal micro - organisms that exist in a mutualistic relationship with the host . the colonization of the small intestine of mice with a single commensal microbe , such as segmented filamentous bacterium , was sufficient to induce the appearance of th cells able to produce il-17 and il-22 in the lamina propria with related activation of inflammatory genes . using indeed mouse models of h. hepaticus - induced t - cell dependent colitis , it was demonstrated that il-23 and not il-12 is essential for the development of a severe disease . although il-23 has been implicated in the genesis of other inflammatory disorders via the activation of il-17-secreting th cells , in this h. hepaticus model of colitis , il-17 in the absence of ifn- did not appear sufficient to induce disease , the maximum intestinal inflammation depending on both ifn- and il-17 . in aig patients without evidence of previous or actual h. pylori infection , large numbers of t cells are present in the gastric mucosa . around 25% of the corpus derived t cells recognized gastric hk - atpase , whereas only a few ( 3% ) of the antral t cells proliferated upon stimulation with this autoantigen . as in eaig , in which administration of depleting anti - cd4 antibodies but not anti - cd8 antibodies reduces the incidence of gastritis , all human hk - atpase - specific t cell clones were cd4 [ 68 , 69 ] . t cell proliferation to purified hk - atpase is dose - dependent and hla class ii restricted , and involves different tcr v rearrangements and submolecular epitopes , indicating that human aig is driven by antigen - specific polyclonal activation of autoreactive t cells , and not due to expansion of a single- autoreactive t cell clone . from aig patients with a concurrent h. pylori infection , gastric cd4 t cells reactive to hk - atpase and cd4 t cells reactive to h. pylori can be cloned , but also cd4 t cells that recognize both hk - atpase and h. pylori antigens . identification of these h. pylori / hk - atpase - specific t cells has provided a functional mechanism that can explain the long known association between h. pylori infection and corpus atrophic gastritis . a library of overlapping 15-mer synthetic peptides , together comprising the entire and subunits of human gastric hk - atpase , was used to identify the submolecular epitopes recognized by hk - atpase - specific and h. pylori / hk - atpase - specific cd4 t cells from h. pylori- infected aig patients [ 59 , 68 , 69 ] . subsequently , candidate h. pylori epitopes were predicted by alignment of identified hk - atpase epitopes with the genomes of the h. pylori j99 and 26695 strains , taking into account the mhc class ii peptide binding motifs of each individual t cell donor . the repertoire of hk - atpase and subunit epitopes recognized by hk - atpase - specific cd4 t cell clones is similar in h. pylori- infected and noninfected aig patients [ 59 , 69 ] . all h. pylori / hk - atpase - cross - reactive t cells find their autoantigenic epitope in the subunit of hk - atpase , which is much longer ( 1034 amino acids ) than the subunit ( 270 amino acids long ) . surprisingly , none of the identified h. pylori peptides recognized by the h. pylori / hk - atpase cross - reactive t cells belong to the known immunodominant h. pylori proteins , that is , vaca , caga , and urease , which have been identified as major targets of gastric t cells in h. pylori- infected patients with chronic antral gastritis and peptic ulcer . instead , the h. pylori peptide epitopes recognized by cross - reactive t cells are predominantly derived from products of h. pylori house - hold genes . antigen - specific stimulation of h. pylori / hk - atpase - cross - reactive t cells is hla - dr restricted . h. pylori / hk - atpase - reactive t cell clones , derived from two different donors that share the expression of hla - drb1 * 04 and recognize the same hk - atpase peptide , also find their bacterial epitope in the same h. pylori . and vice versa , two t cell clones recognizing the same hk - atpase epitope , but derived from different donors , one expressing hla - drb1 * 0301 and the other expressing hla - drb1 * 1303 , find their cross - reactive bacterial peptide in different h. pylori proteins . upon activation with the appropriate antigenic peptide , all hk - atpase - reactive gastric t cell or hk - atpase / h . pylori - cross - reactive t cell clones from aig patients have a th1 phenotype , characterized by production of ifn- and neither il-4 nor il-5 [ 59 , 68 , 69 ] . after activation , all the autoreactive and cross - reactive hk - atpase - specific th clones were able to induce cell death via either fas - fas ligand(fasl)-mediated apoptosis or perforin - mediated cytotoxicity against target cells . the expression of fas on gastric epithelial cells is increased by the th1 cytokines ifn- and tnf and fas / fasl interactions contribute to death of gastric epithelial cells . together , these observations are in agreement with at least two possible scenario 's of the role of h. pylori infection in the onset or acceleration of gastric autoimmunity . in most infected individuals , as yet not completely understood , interactions between h. pylori and the host immune system lead to asymptomatic chronic inflammation of the gastric mucosa , kept under control by cd4cd25 treg . however , also apparently healthy individuals may harbour hk - atpase - autoreactive cd4 t cells that have escaped from negative selection in the thymus , as reflected by the occasional presence of hk - atpase - specific autoantibodies . in such individuals , h. pylori - driven chronic th1-mediated inflammation may be sufficient to abrogate cd4cd25 treg suppression , leading to activation of hk - atpase - specific cd4 t cells and subsequent gastric autoimmunity . a second scenario also involves thymic escape by hk - atpase - specific autoreactive t cells , but in addition , genetics predisposes a host to develop gastric autoimmunity if this host expresses particular hla alleles that can facilitate the presentation of both hk - atpase epitopes and h. pylori peptides [ 45 , 79 ] . this second scenario is supported by the significant association of increased risk for development of pa with expression of hla - dr2 /hla - dr4 or hla - dr4/hla - dr5 , and observations that a substantial proportion of patients with pa are or were infected with h. pylori [ 81 , 82 ] . extranodal marginal zone b cell lymphoma of mucosa - associated lymphoid tissue ( malt lymphoma ) represents the third commonest form of non - hodgkin lymphoma [ 83 , 84 ] . the most frequent site of malt lymphomas is the stomach , where they were first recognized as a distinct entity . a link of h. pylori infection with gastric malt lymphoma was provided by the identification of h. pylori in the majority of the lymphoma specimens . pylori - related low - grade gastric malt lymphoma represents a model for studying the interplay between chronic infection , immune response , and lymphomagenesis . this lymphoma represents the first described neoplasia susceptible of regression following antibiotic therapy resulting in h. pylori eradication . a prerequisite for lymphomagenesis is the development of secondary inflammatory malt induced by h. pylori . tumor cells of low - grade gastric malt lymphoma ( maltoma ) are memory b cells still responsive to differentiation signals , such as cd40 costimulation and cytokines produced by antigen - stimulated th cells , and dependent for their growth on the stimulation by h. pylori - specific t cells [ 8891 ] . in early phases , this tumor is sensitive to withdrawal of h. pylori - induced t - cell help , providing an explanation for both the tumor 's tendency to remain localized to its primary site and its regression after h. pylori eradication . the analysis of the antigen - induced b - cell help exerted by h. pylori - reactive gastric t - cell clones provided detailed information on the molecular and cellular mechanisms associated with the onset of low - grade gastric maltoma . in the stomach of maltoma patients , a high percentage of th cells were specific for h. pylori ( between 3% and 20% in each case ) . in particular , 25% were specific for urease , 4% for vaca , and none for caga or hsp ; 71% of th clones proliferated in response to h. pylori antigens , different from urease , caga , vaca , or hsp . each h. pylori - specific th clone derived from gastric maltoma produced il-2 and a variety of b - cell - stimulating cytokines , such as il-4 and il-13 . in vitro stimulation with the appropriate h. pylori antigens induced h. pylori - specific th clones derived from gastric maltoma to express powerful help for b - cell activation and proliferation . b cells from maltoma patients proliferate in response to h. pylori , but the b - cell proliferation induced by h. pylori antigens was strictly t - cell - dependent because it could not take place with h. pylori and without t helper cells . in chronic gastritis patients , either with or without ulcer , the helper function towards b cells exerted by h. pylori antigen - stimulated gastric t - cell clones was negatively regulated by the concomitant cytolytic killing of b cells [ 18 , 20 ] . in contrast , gastric t - cell clones from maltoma were unable to downmodulate their antigen - induced help for b - cell proliferation . indeed , none of these clones was able to express perforin - mediated cytotoxicity against autologous b cells . moreover , the majority of th clones from uncomplicated chronic gastritis induced fas - fas ligand - mediated apoptosis in target cells , whereas only a small fraction of h. pylori - specific gastric clones from maltoma were able to induce apoptosis in target cells , including autologous b cells . both defective perforin - mediated cytotoxicity and poor ability to induce fas - fas ligand - mediated apoptosis were restricted to maltoma - infiltrating t cells , since h. pylori - specific th cells derived from the peripheral blood of the same patients expressed the same cytolytic potential and proapoptotic activity as that shown by th cells from chronic gastritis patients . accordingly , mice lacking t cell and nk cell cytotoxic effector pathways have also been shown to develop spontaneous tumors ( table 1 ) [ 92101 ] . for example , mice that lack perforin , a cytotoxic molecule used by cytotoxic cells such as cd8 t cells and nk cells to form membrane pores in target cells , develop lymphomas with age . these spontaneous lymphomas are of b cell origin , develop in older mice ( > 1 year of age ) regardless of the mouse strain [ 94 , 95 ] , and , when transplanted into wt mice , are rejected by cd8 t cells . b cell lymphomas also arise in mice lacking both perforin and 2 m , and tumor onset is earlier and occurs with increased prevalence compared with mice lacking only perforin . in addition , b cell lymphomas derived from mice lacking both perforin and 2 m are rejected by either nk cells or t cells following transplantation to wt mice , rather than by cd8 t cells ( as in tumors derived from mice lacking only perforin ) , demonstrating that cell surface expression of mhc class i molecules by tumor cells can be an important factor in determining which effector cells mediate immune protective effects . intriguingly , mutations in the gene encoding perforin have also been identified in subsets of lymphoma patients , although it is not clear whether this contributes to disease . mice lacking the death - inducing molecule tnf - related apoptosis - inducing ligand ( trail ) or expressing a defective mutant form of the death - inducing molecule fasl have also been shown to be susceptible to spontaneous lymphomas that develop with late onset [ 97 , 98 ] . these aging studies have clearly demonstrated a critical role for cytotoxic pathways in immunoregulation and/or immunosuppression of spontaneous tumor development in mice . the reason why gastric t cells of maltoma , while delivering powerful help to b cells , are deficient in mechanisms involved in the control of b - cell growth still remains unclear . it has been shown that vaca toxin inhibits antigen processing in apcs and t cells , but not the exocytosis of perforin - containing granules of nk cells [ 103 , 104 ] . it is possible that , in some h. pylori - infected individuals , some bacterial components affect the development or the expression in gastric t cells of regulatory cytotoxic mechanisms on b - cell proliferation , allowing exhaustive and unbalanced b - cell help and lymphomagenesis to occur [ 91 , 105 , 106 ] . helicobacter pylori is able to induce a huge variety of antigen - specific t - cell responses in the stomach , due to host genetics , age , sex , different bacterial and environmental factors , or other concomitant infections . the cytolytic effector functions of gastric h. pylori - specific t cells are extremely different between patients with autoimmune gastritis or malt lymphoma ( figure 1 ) . in some patients , due to genetic and environmental factors not yet fully elucidated , h. pylori infection triggers an abnormal activation at gastric level of cytotoxic , and proapoptotic cross - reactive t cells leading to gastric autoimmunity via molecular mimicry . conversely in a minority of infected patients , h. pylori is able to induce the development of specific t cells defective of both perforin- and fas ligand - mediated cytotoxicity , which consequently promotes both b - cell overgrowth and exhaustive b - cell proliferation , finally leading to the onset of low - grade gastric malt lymphoma .

helicobacter pylori infection is the major cause of gastroduodenal pathologies , but only a minority of infected patients develop gastric b - cell lymphoma , gastric autoimmunity , or other life threatening diseases , as gastric cancer or peptic ulcer . the type of host immune response against h. pylori , particularly the cytolytic effector functions of t cells , is crucial for the outcome of the infection . t cells are potentially able to kill a target via different mechanisms , such as perforins or fas - fas ligand interaction . in h. pylori - infected patients with gastric autoimmunity cytolytic t cells , that cross - recognize different epitopes of h. pylori proteins and h+k+-atpase autoantigen , infiltrate the gastric mucosa and lead to gastric atrophy via long - lasting activation of fas ligand - mediated appotosis and perforin - induced cytotoxicity . on the other hand , gastric t cells from malt lymphoma exhibit defective perforin- and fas - fas ligand - mediated killing of b cells , with consequent abnormal help for b - cell proliferation , suggesting that deregulated and exhaustive h. pylori - induced t cell - dependent b - cell activation can support both the onset and the promotion of low - grade b - cell lymphoma .

1. Introduction 2. 3. 4. Concluding Remarks

h. pylori infection leads to chronic inflammation of the gastric mucosa , which remains often without clinical symptoms . however , serious diseases as peptic ulcer disease , mucosa - associated lymphoid tissue ( malt ) lymphoma , and gastric adenocarcinoma develop in 15 percent of the infected population . whereas h. pylori infection triggers a vigorous immune response , resulting in high titres of h. pylori - specific antibodies , this response is not sufficient for eradication of the pathogen and when remaining untreated with antibiotics , infection will remain for life . whereas appropriate host immune responses lead to asymptomatic persistent colonization , h. pylori - associated pathology is the result of aberrant inflammation . h. pylori induces a strong innate immune response that involves various components of the innate immune system , including nucleotide - binding oligomerization domain protein i ( nod1 ) and secretion of antimicrobial peptides . the innate immune response to h. pylori results in inflammation of the gastric epithelium ( gastritis ) and induces an influx of neutrophils and other immune cell though the release of chemokines and cytokines , as reviewed in , thereby being essential for the initiation of an acquired immune response to h. pylori . h. felis infection of b and t cell - deficient ( rag-1 ) mice and t cell - deficient ( tcr ) mice does not result in gastric pathology despite high levels of colonization , whereas infection of b cell - deficient mice results in severe gastric alteration , identical to those seen in immunocompetent mice . these results indicate that t cells are required for protection against helicobacter , but also that gastric immune mediated damage is dependent on t cells , and not by b cells or antibody secretion . vaccination studies in different mouse strains have shown that , dependent on mouse strain or helicobacter species , both th2 [ 14 , 15 ] and th1 [ 16 , 17 ] cytokine responses are involved in the adaptive immune response against h. pylori and that protection from disease may require the ability of the host to mount a balanced th1/th2 response upon infection . whether h. pylori - associated gastroduodenal disease develops , or asymptomatic chronic gastritis , is related to the predominant t - helper phenotype of the acquired immune response that eventually arises upon infection . human gastric t cells in h. pylori infection have a predominant th1 phenotype [ 18 , 19 ] , which is associated with pathology . dendritic cells ( dc ) in the gastric mucosa take up antigens and migrate to nearby lymph nodes where they activate nave t cells and orchestrate the subsequent immune response . h. pylori - pulsed dc induce increased il-2 , ifn- , and tnf in cd4cd45ra nave t cells , which is in agreement with the th1 cytokine profile produced by t cells from gastric mucosal biopsies [ 18 , 19 , 24 ] . virulence factors that contribute to the predominance of th1 responses in h. pylori infection include the h. pylori neutrophil activating protein ( hp - nap ) , which induces il-12 secretion by neutrophils and monocytes and strongly promotes th1-skewing of antigen - specific cd4 t cells , and a plasticity region locus jhp0947-jhp0949 that is associated with duodenal ulcer disease . t cell clones from the gastric antral mucosa of patients with or without peptic ulcer display a different preferential antigen - specificity . approximately half of the h. pylori - reactive th cell clones derived from peptic ulcer patients were specific for the cytotoxin - associated protein ( caga ) , whereas approximately one forth of h. pylori - reactive th clones from nonulcer gastritis patients were specific for h. pylori urease [ 18 , 20 ] . upon antigen - specific stimulation , over eighty percent of the h. pylori - reactive th cells from peptic ulcer disease patients showed a polarized th1 profile , with high production of ifn- and no il-4 . in contrast , in non - ulcer gastritis patients about two - third of the h. pylori - specific antral clones displayed a mixed th1/th2 cytokine profile , whereas only one - third was polarized to th1 . these results are in agreement with several studies that indicate that th1 polarization of the h. pylori - specific t cell response is associated with more severe disease [ 18 , 19 , 24 , 26 , 29 , 30 ] . in the h. pylori - infected gastric mucosa , il-12 drives the th1 signalling pathway . the pathogenic capacity of polarized th1 responses in h. pylori infection is indirectly supported by the phenomenon often referred to as the african enigma , that is , despite a high prevalence of h. pylori infection in africa , peptic ulcer disease and gastric cancer are uncommon . therefore , one possible explanation for the low prevalence of h. pylori - associated diseases in africa is that the endemic high parasitic burden causes predominant th2 responses that protect h. pylori - individuals from developing peptic ulcers and gastric cancer . an accumulating body of evidence indicates that cd4cd25 regulatory t cells ( treg ) do not only prevent development of autoimmune diseases , but also regulate pathogen- induced inflammation and disease , including in h. pylori infection . in mice , cd4cd25 treg reduce gastric immunopathology , despite increased h. pylori colonization of the gastric mucosa , and in vitro suppress antigen - specific proliferation of h. pylori - specific cd4 t effector cells . in vivo , depletion of cd4cd25foxp treg in h. pylori infected mice leads to severe gastritis , influx of b cells , t cells , and macrophages into the gastric mucosa , increased titers of h. pylori - specific igg1 , and igg2c and , subsequently , reduced bacterial load . when compared to adults , h. pylori - infected children have increased numbers of treg and increased expression of the treg - cytokines tgf- and il-10 and lower level of gastritis , despite similar levels of h. pylori colonization . together , these results suggest that h. pylori infection induces cd4cd25foxp treg that contribute to the asymptomatic persistent infection , seen in the majority of people , and that individuals with inadequate regulatory t cell responses will develop gastro - duodenal pathology . this profound role of regulatory t cells in outcome of h. pylori infection may provide another explanation for the african enigma : in endemic areas long - term helminth infections induce a robust anti - inflammatory , regulatory t cell network that protects h. pylori - infected individuals from peptic ulcer disease and gastric cancer . several clinical and epidemiological studies indicate that most patients with autoimmune gastritis ( aig ) have or have had h. pylori infection . there are striking similarities between classical aig and h. pylori - induced corpus atrophic gastritis , and histologically defined preclinical stages of aig can be successfully treated by h. pylori eradication [ 4850 ] . h. pylori induces autoantibodies reactive with gastric mucosal antigens in approximately half of the infected individuals [ 5155 ] . gastric hk - atpase , the proton pump located in the secretory canaliculi of the parietal cells , is the autoantigen that drives immunopathology in aig . in humans , h. pylori infection leads to the activation of gastric t cells , of which a proportion is cross - reactive against both h. pylori peptide antigens and hk - atpase , and circulating antigastric autoantibodies are not pathogenic themselves , but mark ongoing t cell - mediated gastric autoimmunity . eaig spontaneously develops in t cell receptor transgenic ( tcr - tg ) mice in which the cd4 t cells express an tcr that recognize an epitope of the hk - atpase -subunit ( 630641 ) or in neonatally thymectomized mice . in eaig and aig , the gastric inflammatory infiltrate comprises cd4 and cd8 t cells , macrophages and b cells , and the histopathological lesions in mouse models are similar to those observed in humans with chronic aig [ 62 , 63 ] . the relevance of such partly conserved epitopes in ( e)aig remains unclear , but availability of an epitope for tcr recognition , rather than affinity for the tcr , enhances the gastritogenic potential of autoreactive t cells . even if data in h. pylori model is still relatively sparce , the balance between il-12 , il-23 , and treg responses seems to play an important role in the type of inflammatory responses in mouse models of inflammatory bowel disease . the colonization of the small intestine of mice with a single commensal microbe , such as segmented filamentous bacterium , was sufficient to induce the appearance of th cells able to produce il-17 and il-22 in the lamina propria with related activation of inflammatory genes . using indeed mouse models of h. hepaticus - induced t - cell dependent colitis , it was demonstrated that il-23 and not il-12 is essential for the development of a severe disease . in aig patients without evidence of previous or actual h. pylori infection , large numbers of t cells are present in the gastric mucosa . around 25% of the corpus derived t cells recognized gastric hk - atpase , whereas only a few ( 3% ) of the antral t cells proliferated upon stimulation with this autoantigen . t cell proliferation to purified hk - atpase is dose - dependent and hla class ii restricted , and involves different tcr v rearrangements and submolecular epitopes , indicating that human aig is driven by antigen - specific polyclonal activation of autoreactive t cells , and not due to expansion of a single- autoreactive t cell clone . from aig patients with a concurrent h. pylori infection , gastric cd4 t cells reactive to hk - atpase and cd4 t cells reactive to h. pylori can be cloned , but also cd4 t cells that recognize both hk - atpase and h. pylori antigens . identification of these h. pylori / hk - atpase - specific t cells has provided a functional mechanism that can explain the long known association between h. pylori infection and corpus atrophic gastritis . a library of overlapping 15-mer synthetic peptides , together comprising the entire and subunits of human gastric hk - atpase , was used to identify the submolecular epitopes recognized by hk - atpase - specific and h. pylori / hk - atpase - specific cd4 t cells from h. pylori- infected aig patients [ 59 , 68 , 69 ] . subsequently , candidate h. pylori epitopes were predicted by alignment of identified hk - atpase epitopes with the genomes of the h. pylori j99 and 26695 strains , taking into account the mhc class ii peptide binding motifs of each individual t cell donor . surprisingly , none of the identified h. pylori peptides recognized by the h. pylori / hk - atpase cross - reactive t cells belong to the known immunodominant h. pylori proteins , that is , vaca , caga , and urease , which have been identified as major targets of gastric t cells in h. pylori- infected patients with chronic antral gastritis and peptic ulcer . instead , the h. pylori peptide epitopes recognized by cross - reactive t cells are predominantly derived from products of h. pylori house - hold genes . antigen - specific stimulation of h. pylori / hk - atpase - cross - reactive t cells is hla - dr restricted . and vice versa , two t cell clones recognizing the same hk - atpase epitope , but derived from different donors , one expressing hla - drb1 * 0301 and the other expressing hla - drb1 * 1303 , find their cross - reactive bacterial peptide in different h. pylori proteins . pylori - cross - reactive t cell clones from aig patients have a th1 phenotype , characterized by production of ifn- and neither il-4 nor il-5 [ 59 , 68 , 69 ] . after activation , all the autoreactive and cross - reactive hk - atpase - specific th clones were able to induce cell death via either fas - fas ligand(fasl)-mediated apoptosis or perforin - mediated cytotoxicity against target cells . together , these observations are in agreement with at least two possible scenario 's of the role of h. pylori infection in the onset or acceleration of gastric autoimmunity . in most infected individuals , as yet not completely understood , interactions between h. pylori and the host immune system lead to asymptomatic chronic inflammation of the gastric mucosa , kept under control by cd4cd25 treg . in such individuals , h. pylori - driven chronic th1-mediated inflammation may be sufficient to abrogate cd4cd25 treg suppression , leading to activation of hk - atpase - specific cd4 t cells and subsequent gastric autoimmunity . a second scenario also involves thymic escape by hk - atpase - specific autoreactive t cells , but in addition , genetics predisposes a host to develop gastric autoimmunity if this host expresses particular hla alleles that can facilitate the presentation of both hk - atpase epitopes and h. pylori peptides [ 45 , 79 ] . a link of h. pylori infection with gastric malt lymphoma was provided by the identification of h. pylori in the majority of the lymphoma specimens . pylori - related low - grade gastric malt lymphoma represents a model for studying the interplay between chronic infection , immune response , and lymphomagenesis . tumor cells of low - grade gastric malt lymphoma ( maltoma ) are memory b cells still responsive to differentiation signals , such as cd40 costimulation and cytokines produced by antigen - stimulated th cells , and dependent for their growth on the stimulation by h. pylori - specific t cells [ 8891 ] . in early phases , this tumor is sensitive to withdrawal of h. pylori - induced t - cell help , providing an explanation for both the tumor 's tendency to remain localized to its primary site and its regression after h. pylori eradication . the analysis of the antigen - induced b - cell help exerted by h. pylori - reactive gastric t - cell clones provided detailed information on the molecular and cellular mechanisms associated with the onset of low - grade gastric maltoma . each h. pylori - specific th clone derived from gastric maltoma produced il-2 and a variety of b - cell - stimulating cytokines , such as il-4 and il-13 . in vitro stimulation with the appropriate h. pylori antigens induced h. pylori - specific th clones derived from gastric maltoma to express powerful help for b - cell activation and proliferation . b cells from maltoma patients proliferate in response to h. pylori , but the b - cell proliferation induced by h. pylori antigens was strictly t - cell - dependent because it could not take place with h. pylori and without t helper cells . in chronic gastritis patients , either with or without ulcer , the helper function towards b cells exerted by h. pylori antigen - stimulated gastric t - cell clones was negatively regulated by the concomitant cytolytic killing of b cells [ 18 , 20 ] . in contrast , gastric t - cell clones from maltoma were unable to downmodulate their antigen - induced help for b - cell proliferation . indeed , none of these clones was able to express perforin - mediated cytotoxicity against autologous b cells . moreover , the majority of th clones from uncomplicated chronic gastritis induced fas - fas ligand - mediated apoptosis in target cells , whereas only a small fraction of h. pylori - specific gastric clones from maltoma were able to induce apoptosis in target cells , including autologous b cells . both defective perforin - mediated cytotoxicity and poor ability to induce fas - fas ligand - mediated apoptosis were restricted to maltoma - infiltrating t cells , since h. pylori - specific th cells derived from the peripheral blood of the same patients expressed the same cytolytic potential and proapoptotic activity as that shown by th cells from chronic gastritis patients . for example , mice that lack perforin , a cytotoxic molecule used by cytotoxic cells such as cd8 t cells and nk cells to form membrane pores in target cells , develop lymphomas with age . these spontaneous lymphomas are of b cell origin , develop in older mice ( > 1 year of age ) regardless of the mouse strain [ 94 , 95 ] , and , when transplanted into wt mice , are rejected by cd8 t cells . the reason why gastric t cells of maltoma , while delivering powerful help to b cells , are deficient in mechanisms involved in the control of b - cell growth still remains unclear . it has been shown that vaca toxin inhibits antigen processing in apcs and t cells , but not the exocytosis of perforin - containing granules of nk cells [ 103 , 104 ] . it is possible that , in some h. pylori - infected individuals , some bacterial components affect the development or the expression in gastric t cells of regulatory cytotoxic mechanisms on b - cell proliferation , allowing exhaustive and unbalanced b - cell help and lymphomagenesis to occur [ 91 , 105 , 106 ] . helicobacter pylori is able to induce a huge variety of antigen - specific t - cell responses in the stomach , due to host genetics , age , sex , different bacterial and environmental factors , or other concomitant infections . the cytolytic effector functions of gastric h. pylori - specific t cells are extremely different between patients with autoimmune gastritis or malt lymphoma ( figure 1 ) . in some patients , due to genetic and environmental factors not yet fully elucidated , h. pylori infection triggers an abnormal activation at gastric level of cytotoxic , and proapoptotic cross - reactive t cells leading to gastric autoimmunity via molecular mimicry . conversely in a minority of infected patients , h. pylori is able to induce the development of specific t cells defective of both perforin- and fas ligand - mediated cytotoxicity , which consequently promotes both b - cell overgrowth and exhaustive b - cell proliferation , finally leading to the onset of low - grade gastric malt lymphoma .

traditional remedies based on natural products could be traced back over five millennia to written documents of the early civilizations . particularly , traditional korean and chinese oriental medicines herbs ( omhs ) have attracted interest and acceptance in many countries with the merits of a few side effects , affordability , and local availability . moreover , their long historical clinical practice and reliable therapeutic efficacy make them excellent sources for discovering natural bioactive compounds . among them , acer ( a. ) tegmentosum ( aceraceae , sancheong - mok in korean ) is a type of deciduous tree distributed in the northeast asia including korea , russia , and china . in korea , the leaves and stem of a. tegmentosum have been traditionally used for the treatment of hepatic disorders such as hepatitis , hepatic cancer , hepatic cirrhosis , and liver detoxification . previous studies have shown that extracts of the stem of a. tegmentosum possess various pharmacological properties such as antioxidative , anti - inflammatory , antigastrophatic , antiadipogenic , anticancer , and cytotoxic activities [ 57 ] . however , the phytochemical constituents and efficient method for investigating bioactivity of a. tegmentosum have not been reported . also , a variety of approaches have been developed for the extraction of useful components from a. tegmentosum , for instance , soxhlet extraction ( se ) , heating reflux extraction ( hee ) , supercritical fluid extraction ( sfe ) , ultrasonic assisted extraction ( uae ) , and microwave - assisted extraction ( mae ) [ 810 ] . water , methanol , ethanol , and ethyl acetate ( ea ) were commonly used solvents for the extraction of bioactive compounds from plant materials and omhs . the identification and relative amounts of five types of compounds in a. tegmentosum were determined by lc - ms , nmr , and on - line screening hplc - abts assay . these techniques included on - line screening with hplc post - column assay involving the abts radical technique , allowing spectrophotometric monitoring of bioactive compounds . generally , dpph ( abts ) radical is another simple , rapid on - line method for the detection of antioxidants from crude plant extracts . it combines hplc with an assay involving a stable radical species [ 1,1-diphenyl-2-picrylhydrazyl radical ( dpph ) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid ) radical cation ( abts ) ] in the hplc - dpph ( abts ) method [ 13 , 14 ] . moreover , this method was successfully applied for the screening and identification of natural bioactive compounds from complex mixtures , particularly for the extracts of omhs [ 1517 ] . the chemical structures of the five types of compounds were confirmed by spectroscopic methods such as h - nmr , c - nmr , and lc / ms . in this study , five phenolic compounds were isolated from hot water extract of a. tegmentosum bychromatographic separation . using spectroscopic methods , the structures of these compounds were determined as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) . moreover , we investigated their anti - inflammatory effects on lps - stimulated raw 264.7 cells . we also investigated the applications of on - line screening hplc - abts assays for bioactivity screening to find a more practical approach toward the use of on - line screening hplc - abts assays for the rapid pinpointing of peaks in chromatograms corresponding to bioactive compounds . the stem of a. tegmentosum was purchased from yeongcheon traditional herbal market ( gyeongsangbuk - do , yeongcheon , korea ) . all voucher specimens were deposited in the herbal bank of km - based herbal drug development group , korea institute of oriental medicine . the following reagents were used for radical scavenging assays : abts ( 2,2-azino - bis-3-ethylbenzothiazoline-6-sulfonic acid ) , potassium persulfate , and trifluoroacetic acid ( tfa ) were purchased from sigma co. ( usa ) . the hplc grade methanol ( meoh ) and acetonitrile ( acn ) were purchased from j. t. baker ( philipsburg , nj , usa ) . the hexane , dichloromethane ( dcm ) , ethyl acetate ( ea ) , and normal butyl alcohol ( n - buoh ) were purchased from daejung chemical ( gyeonggi - do , shiheung , korea ) . the triple distilled water was filtered with a 0.2 m membrane filter ( advantec , tokyo , japan ) before analysis . lps , bovine serum albumin ( bsa ) , and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) were purchased from sigma ( st . louis , mo , usa ) . antibodies for elisa were obtained from ebioscience ( san diego , ca , usa ) . the high purity isolated compounds ( higher than > 95% ) were prepared by dissolving 2 mg of the standard chemicals feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) in 10 ml of methanol and adjusting the concentration to 200 ppm . a 2 mm abts stock solution containing 3.5 mm potassium persulfate was prepared and was kept in the dark at room temperature for 16 h to allow the completion of radical generation and was then diluted with water ( 1 : 29 , v / v ) . dry samples ( 3 kg ) from the powders of the a. tegmentosum were loaded ( 10-times volume ) in hot water extraction system . the extraction was performed by heating for 3 h at 100c ( gyeongseo extractor cosmos-600 , inchon , korea ) . then , the solution was filtered using standard testing sieves ( 150 m , retsch , haan , germany ) , freeze - dried , and maintained in desiccators at 4c prior to use . for large amount of extractions , 20 g freeze - dry samples were loaded ( 1 : 1 , extracted thrice ) and extracted successively using dcm , ea , and n - buoh . the contents of feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) in a. tegmentosum were remarkably higher in the ea extract ( 1.24 g ) . then , the samples were filtered through a 0.2 m membrane filter prior to on - line screening hplc - abts analysis . nmr spectra were obtained using a varian inova 400 mhz and 600 mhz nmr ( varian , usa ) . the isolated compounds feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) were confirmed vialc - ms analysis using agilent 1100 + g1958 ( agilent , usa ) lc systems ( table 1 ) . open column chromatography was performed using silica gel ( kieselgel 60 , merck , germany ) and octadecylsilane ( ods ) li - chropre rp-18 ( merck , germany ) . thin layer chromatography ( tlc ) analysis was performed using silica gel glass plates ( kieselgel 60 f254 and rp-18 f254s , merck , germany ) and developed using a mobile phase composed of chloroform - methanol - water and stained using 10% h2so4 to detect the ea extract and pure compounds . a. tegmentosum extract was injected into a dionex ultimate 3000 hplc system ( thermo scientific ) . the chromatographic columns used in this experiment were commercially available and were purchased from rs - tech ( 0.46 25 cm , 5 m , c18 , daejeon , korea ) . the injection volume was 10 l , and the flow rate of the mobile phase was 1.0 ml / min . the wavelength of the uv detector was fixed at 210 , 254 , 280 , and 320 nm . the composition of the mobile phases was a : 99.9% water / trifluoroacetic acid ( 99.9/0.1 vol% ) and b : 100% acetonitrile . the run time was 60 min , and the solvent program was the linear gradient method ( 90 : 1060 : 40 a : b vol% , 70 min : initial condition ) ( table 2 ) . figure 3 shows a schematic diagram of the on - line coupling of a hplc to a dad ( diode array detector ) and the continuous flow abts assay . the abts at a flow rate of 0.5 ml / min was delivered using a dionex ultimate 3000 pump . after mixing through a 1 ml loop , maintained at 40c , the absorbance was measured using a multiple wavelength detector ( mwd ) at 734 nm . raw 264.7 cells were purchased from korea cell line bank ( seoul , korea ) and grown in rpmi 1640 medium containing 10% fbs and 100 u / ml of antibiotics sulfate . the cells were incubated in humidified 5% co2 atmosphere at 37c . to stimulate the cells , the medium was changed with fresh rpmi 1640 medium , and lps ( 200 ng / ml ) was added in the presence or absence of five compounds ( 10 , 30 , 50 , and 100 m ) for 24 h. cytotoxicity was analyzed using the mtt assay . five compounds were added to the cells and incubated for 24 h at 37c with 5% co2 . mtt solutions were added to each well and the cells were incubated for another 4 h. the formazan melted in dimethyl sulfoxide ( dmso ) , and then the optical density was read at 570 nm using an elisa reader ( infinite m200 , tecan , mnnedorf , switzerland ) . no production was analyzed by measuring the nitrite in the supernatants of cultured macrophage cells . the cells were pretreated with five compounds and stimulated with lps for 24 h. the supernatant was mixed with the same volume of griess reagent ( 1% sulfanilamide , 0.1% naphthylethylenediamine dihydrochloride , and 2.5% phosphoric acid ) and incubated at room temperature ( rt ) for 5 min . cells were seeded at a density of 5 10 cells / ml in 24-well culture plates and pretreated with various concentrations of five compounds for 30 min before the lps stimulation . elisa plates ( nunc , roskilde , denmark ) were coated overnight at 4c with capture antibody diluted in a coating buffer ( 0.1 m carbonate , ph 9.5 ) and then washed five times with phosphate - buffered saline ( pbs ) containing 0.05% tween 20 . the nonspecific protein - binding sites were blocked with assay diluent buffer ( pbs containing 10% fbs , ph 7.0 ) for > 1 h. the samples and standards were added to the wells promptly . after 2 hours of incubation at rt or overnight at 4c , the working detector solution ( biotinylated detection antibody and streptavidin - hrp reagent ) was added and incubated for 1 h. subsequently , the substrate solution ( tetramethylbenzidine ) was added to the wells and incubated for 30 min in the dark before the reaction was quenched with stop solution ( nh3po4 ) . statistical significance of each treated group was compared to the control and determined by student 's t - tests . the stems of a. tegmentosum were extracted using boiling water and then partitioned successively using dichloromethane ( dcm ) , ethyl acetate ( ea ) , normal butyl alcohol ( n - buoh ) , and water ( h2o ) . the ea extract ( 1.24 g ) ( 4 10 cm ) and eluted using chloroform- ( chcl3 ) meoh h2o [ 15 : 3 : 1 ( 1.2 l ) 13 : 3 : 1 ( 1.3 l ) 10 : 3 : 1 ( 1.2 l ) 7 : 3 : 1 ( 1 l ) 4 : 3 : 1 ( 1 l ) meoh ] . [ 1.5 45 cm , 50% meoh ( 180 ml ) 70% meoh ( 300 ml ) 100% meoh ] yielding seven fractions ( os1e-6 - 17 ) and ultimately produced compounds 1 ( os1e-6 - 2 , 6.0 mg ) and 2 ( os1e-6 - 4 , 5.9 mg ) . [ 1.5 44 cm , 80% meoh ( 400 ml ) 100% meoh ] yielding 10 fractions ( os1e-7 - 110 ) . ( 1.5 8 cm ) and eluted with meoh h2o [ 1 : 6 ( 900 ml ) meoh ] to afford compounds 3 ( os1e-7 - 6 - 2 , 30.7 mg ) and 4 ( os1e-7 - 6 - 4 , 14.2 mg ) . ( 2.5 8 cm ) and eluted with meoh h2o [ 1 : 3 ( 450 ml ) 1 : 2 ( 300 ml ) 1 : 1 ( 300 ml ) meoh ] yielding 13 fractions ( os1e-9 - 113 ) . fraction os1e-9 - 3 ( 77.8 mg ) was subjected to silica gel c.c . ( 1.5 10 cm ) and eluted with chcl3meoh h2o [ 10 : 3 : 1 ( 2.2 l ) 7 : 3 : 1 ( 1.6 l ) 4 : 3 : 1 ( 1.4 l ) meoh ] to afford compound 5 ( os1e-9 - 3 - 2 , 60.3 mg ) . based on the spectroscopic methods , such as h - nmr , c - nmr , and lc / ms , the chemical structures of the compounds were confirmed as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) by comparison of spectral data with those of references . moreover , the on - line screening hplc - abts assay method was rapid and efficient for the investigation of bioactivity of the isolated compounds from acer tegmentosum . compounds 1 , 2 , 3 , 4 , and 5 were developed on ods tlc with 10% h2so4 and the expected yellow or brown color was observed for the phenolic compounds . yellow amorphous powder ( meoh ) ; esi - ms m / z 435.1 [ m+h ] ; h - nmr ( 400 mhz , cd3od , h ) 7.73 ( 1h , d , j = 1.6 hz , h-2 ) , 7.55 ( 1h , dd , j = 8.4 , 1.6 hz , h-6 ) , 6.88 ( 1h , d , j = 8.4 hz , h-5 ) , 6.39 ( 1h , d , j = 1.6 hz , h-8 ) , 6.19 ( 1h , d , j = 1.6 hz , h-6 ) , 5.16 ( 1h , d , j = 6.0 hz , h-1 ) , 3.89 ( 1h , dd , j = 8.0 , 6.0 hz , h-2 ) , 3.82 ( 1h , h-5a ) , 3.80 ( 1h , m , h-4 ) , 3.64 ( 1h , dd , j = 8.0 , 2.8 hz , h-3 ) , 3.44 ( 1h , dd , j = 13.2 , 3.2 hz , h-5b ) ; c - nmr ( 100 mhz , cd3od , c ) ; see table 3 . the characterization data of compound 1 were compared to the literature value and combined with quercetin and arabinopyranoside to confirm the structure as feniculin . compound 2 ( avicularin ) . yellow amorphous powder ( meoh ) ; esi - ms m / z 435.1 [ m+h ] ; h - nmr ( 600 mhz , cd3od , h ) 7.52 ( 1h , d , j = 1.8 hz , h-2 ) , 7.49 ( 1h , dd , j = 8.4 , 1.8 hz , h-6 ) , 6.90 ( 1h , d , j = 8.4 hz , h-5 ) , 6.38 ( 1h , d , j = 1.8 hz , h-8 ) , 6.20 ( 1h , d , j = 1.8 hz , h-6 ) , 5.46 ( 1h , s , h-1 ) , 4.32 ( 1h , d , j = 2.4 hz , h-2 ) , 3.89 ( 1h , dd , j = 4.8 , 2.4 hz , h-3 ) , 3.861 ( 1h , m , h-4 ) , 3.494 ( 2h , t , j = 4.2 , h-5 ) ; c - nmr ( 150 mhz , cd3od , c ) ; see table 3 . the structure was confirmed by esi - ms m / z 435.1 [ m+h ] . compounds 1 and 2 were isolated from the plant for the first time . compound 3 [ ( + ) -catechin ] . brown powder ( meoh ) ; esi - ms m / z 291.0 [ m+h ] ; h - nmr ( 600 mhz , cd3od , h ) 6.83 ( 1h , d , j = 2.4 hz , h-2 ) , 6.76 ( 1h , d , j = 8.4 hz , h-5 ) , 6.71 ( 1h , dd , j = 8.4 , 2.4 hz , h-6 ) , 5.92 ( 1h , d , j = 2.4 hz , h-6 ) , 5.85 ( 1h , d , j = 2.4 hz , h-8 ) , 4.56 ( 1h , d , j = 7.2 hz , h-2 ) , 3.97 ( 1h , m , h-3 ) , 2.50 ( 1h , dd , j = 16.2 , 8.4 hz , h-4a ) , 2.84 ( 1h , dd , j = 16.2 , 6.0 hz , h-4b ) ; c - nmr ( 150 mhz , cd3od , c ) ; see table 3 brown powder ( meoh ) ; esi - ms m / z 291.0 [ m+h ] ; h - nmr ( 600 mhz , cd3od , h ) 6.96 ( 1h , d , j = 1.8 hz , h-2 ) , 6.79 ( 1h , dd , j = 8.4 , 1.8 hz , h-6 ) , 6.75 ( 1h , d , j = 8.4 hz , h-5 ) , 5.93 ( 1h , d , j = 1.8 hz , h-6 ) , 5.91 ( 1h , d , j = 1.8 hz , h-8 ) , 4.80 ( 1h , brs , h-2 ) , 4.16 ( 1h , m , h-3 ) , 2.86 ( 1h , dd , j = 16.8 , 4.8 hz , h-4a ) , 2.73 ( 1h , dd , j = 16.8 , 3.0 hz , h-4b ) ; c - nmr ( 150 mhz , cd3od , c ) ; see table 3 . compounds 3 and 4 were compared to the literature data for ( + ) -catechin and ( )-epicatechin for the structure identification . the structure was confirmed by the presence of m / z at 291.0 [ m+h ] in the esi - ms positive mode . compound 5 ( 6 -o - galloyl salidroside ) . pale yellow oil ( meoh ) ; esi - ms m / z 453.1 [ m+h ] ; h - nmr ( 600 mhz , cd3od , h ) 7.10 ( 2h , br s , h-2 , h-6 ) , 6.96 ( 2h , d , j = 8.4 hz , h-2 , 6 ) , 6.65 ( 2h , d , j = 8.4 hz , h-3 , h-5 ) , 6.65 ( 1h , d , j = 8.4 hz , h-5 ) , 4.52 ( 1h , dd , j = 11.4 , 2.4 hz , h-6a ) , 4.45 ( 1h , dd , j = 11.4 , 6.0 hz , h-6b ) , 4.32 ( 1h , d , j = 7.8 hz , h-1 ) , 3.95 ( 1h , m , h-8a ) , 3.70 ( 1h , m , h-8b ) , 3.55 ( 1h , m , h-5 ) , 3.42 ( 2h , t , j = 7.8 hz , h-3 , 4 ) , 3.22 ( 1h , t , j = 8.4 hz , h-2 ) , 2.77 ( 2h , m , h-7 ) ; c - nmr ( 150 mhz , cd3od , c ) ; see table 3 . this study investigated the bioactivity ( using abts assay ; radical scavenging activity ) and anti - inflammatory activity of the five isolated phenolic compounds that were measured . all the compounds 15 in the ea fraction ( each yield ; mg ) exhibited antioxidant activities ( table 4 ) . moreover , this on - line screening hplc - abts assay method was rapid and efficient for the investigation of bioactivity from a. tegmentosum and was obtained from rs - tech ( 0.46 25 cm , 5 m , c18 , daejeon , korea ) . the injection volume was 10 l , and the flow rate of the mobile phase was 1.0 ml / min . the wavelength of the uv detector was fixed at 210 , 254 , 280 , and 320 nm . the five phenolic compounds were characterized by comparing the hplc uv - dad maximum absorption peaks of the samples with those of the pure isolation standards ( figure 4 ) . the determination of antioxidant activity from the on - line hplc dpph ( abts ) assay was based on the decrease in absorbance at 517 and 734 nm after the postcolumn reaction of antioxidants separated from hplc with dpph ( abts ) , and the antioxidants present in a sample would be easily indicated by negative peaks . the composition of the mobile phases was a : 99.9 vol% water / trifluoroacetic acid ( 99.9/0.1 , vol% ) and b : 100% acetonitrile . the run time was 60 min , and the solvent program used the linear gradient method ( 90 : 1060 : 40 a : b vol% , 70 min : initial condition ) . the hplc separated analytes showed a postcolumn reaction with the abts and the reduction was detected as a negative peak using a mwd at 734 nm . the combined uv ( positive signals ) and abts quenching ( negative signals ) chromatograms of the different a. tegmentosum extracts ( 200 ppm ) are presented in figures 5 and 6 . several eluted fractions of the five phenolic compounds in the ea extract were detected as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) , giving a positive signal on the uv detector at 210 nm . the other compounds showed hydrogen - donating capacity ( negative peak ) toward the abts radical at the applied concentration . these results revealed that the method could be applied for quick bioactivity screening , or more precisely , to detect the radical - scavenging activity of compounds , indicating that ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) exhibited bioactivities . 6-o - galloyl salidroside ( 5 ) exhibited higher bioactivity , whereas feniculin ( 1 ) and avicularin ( 2 ) showed low bioactivity ( figure 5 ) . the retention time ( rt ) of feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) was 10.700 , 13.884 , 15.928 , 24.217 , and 25.062 min , respectively . the bioactivity appeared to be approximately proportional to the concentration of the five phenolic compounds in the extracts . the hplc analysis conditions for the best separation of the five compounds were successfully established by varying the open column treatment and solvent purification step ( figure 6 ) . this study confirms the feasibility of assessing the bioactivity of specific phytochemicals using an on - line screening hplc - abts assay method ( table 5 ) . this proposed method was successfully applied for the screening and identification of natural bioactive compounds from a. tegmentosum . figure 7 shows the hplc profile and lc - ms spectra of five isolated phenolic compounds from a. tegmentosum . lc - ms analysis indicated that the compounds from a. tegmentosum were isolated in highly pure form . each compound was dissolved in meoh at a concentration of 200 ppm , and the lc - ms analysis conditions are listed in table 1 . lc - ms analysis is a powerful tool in metabolic profiling and metabolomics research and it can accurately determine the content of specific metabolites even at low levels in plant samples . the comparison of uv spectra , mass spectra , and retention times of the five types of compounds with the data of standard compounds led to their unambiguous assignments . simultaneous analysis based on rt of feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) of 32.6 , 34.0 , 12.1 , 17.7 , and 21.3 min , respectively , was correlated with the molecular mass data and relative response . esi - ms analysis of compounds feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) showed intense [ m+h ] signals at 435.1 , 435.1 , 291.0 , 291.0 , and 453.1 m / z values , respectively . we evaluated the cytotoxicity of five compounds using the mtt assay to determine the optimal concentration effective for anti - inflammation with minimum toxicity . as shown in figure 8(a ) , all the five compounds did not affect cell viability up to 100 m , indicating that the compounds were not toxic to cells . we evaluated the effects of five compounds on no secretion in lps - stimulated raw 264.7 cells . the cells were pretreated with five compounds at various concentrations prior to the lps stimulation , and no production was measured . as a positive control , we employed 10 m dexamethasone , which is widely employed as an anti - inflammatory agent . all the compounds , except 6-o - galloyl salidroside , did not show any inhibitory effect on lps - induced no production ( figure 8(b ) ) . the inhibitory effect of the five compounds on the production of inflammatory cytokines , another parameter of inflammation , was investigated . in this study , we examined the effect of the five compounds on tnf- , il-6 , and il-1 expressions . figure 9(a ) shows that feniculin slightly repressed tnf- production at a concentration of 10 m ( figure 9(a ) ) . however , avicularin , ( + ) -catechin , ( )-epicatechin , and 6-o - galloyl salidroside did not show any inhibitory effect on lps - induced tnf- production . as shown in figure 9(b ) , consistent with tnf- results , feniculin slightly repressed il-6 production at a concentration of 10 m . moreover , avicularin inhibited il-6 production at concentrations of 30 and 50 m . ( + ) -catechin inhibited il-6 production at 30 m or more . ( )-epicatechin and 6-o - galloyl salidroside did not show any inhibitory effect on lps - induced il-6 secretion . however , all the compounds except 6-o - galloyl salidroside did not inhibit il-1 ( figure 9(c ) ) . this study showed that among the soluble fractions from the hot water extract of a. tegmentosum , the ea - soluble fraction possessed the highest bioactivity and free radical - scavenging activities . compounds in the dried twigs of a. tegmentosum were extracted with hot water and partitioned successively using dcm , ea , n - buoh , and water . the content of useful compounds in a. tegmentosum was remarkably higher in ea the extract ( 1.24 g ) . five phenolic compounds were isolated by the silica gel , octadecyl silica gel , and sephadex lh-20 column chromatography . the chemical structures of the isolated compounds were determined by spectroscopic methods , such as h - nmr , c - nmr , and lc / ms and were confirmed as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) by comparison of spectral data with those of references . the ea extract of a. tegmentosum containing five phenolic compounds exhibiting the best bioactivity was further monitored by an on - line screening hplc - abtsassay method . compounds 1 and 2 were isolated for the first time , and their anti - inflammatory activities were evaluated . moreover , the on - line screening hplc - abts assay method was rapid and efficient to search for bioactive compound from a. tegmentosum . furthermore , ( + ) -catechin and 6-o - galloyl salidroside exhibited the inhibitory activities on inflammatory mediator production such as tnf- , il-6 , and il-1 cytokines . in conclusion , a. tegmentosum can be used as a basic material for the development of new drugs in omhs .

the acer tegmentosum ( 3 kg ) was extracted using hot water , and the freeze - dried extract powder was partitioned successively using dichloromethane ( dcm ) , ethyl acetate ( ea ) , butyl alcohol ( n - buoh ) , and water . from the ea extract fraction ( 1.24 g ) , five phenolic compounds were isolated by the silica gel , octadecyl silica gel , and sephadex lh-20 column chromatography . based on spectroscopic methods such as 1h - nmr , 13c - nmr , and lc / ms the chemical structures of the compounds were confirmed as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) . moreover , a rapid on - line screening hplc - abts+ system for individual bioactivity of the ea - soluble fraction ( five phenolic compounds ) was developed . the results indicated that compounds 1 and 2 were first isolated from the a. tegmentosum . the anti - inflammatory activities and on - line screening hplc - abts+ assay method of these compounds in lps - stimulated murine macrophages were rapid and efficient for the investigation of bioactivity of a. tegmentosum .

1. Introduction 2. Experimental 3. Results and Discussion 4. Conclusions

particularly , traditional korean and chinese oriental medicines herbs ( omhs ) have attracted interest and acceptance in many countries with the merits of a few side effects , affordability , and local availability . in korea , the leaves and stem of a. tegmentosum have been traditionally used for the treatment of hepatic disorders such as hepatitis , hepatic cancer , hepatic cirrhosis , and liver detoxification . previous studies have shown that extracts of the stem of a. tegmentosum possess various pharmacological properties such as antioxidative , anti - inflammatory , antigastrophatic , antiadipogenic , anticancer , and cytotoxic activities [ 57 ] . however , the phytochemical constituents and efficient method for investigating bioactivity of a. tegmentosum have not been reported . also , a variety of approaches have been developed for the extraction of useful components from a. tegmentosum , for instance , soxhlet extraction ( se ) , heating reflux extraction ( hee ) , supercritical fluid extraction ( sfe ) , ultrasonic assisted extraction ( uae ) , and microwave - assisted extraction ( mae ) [ 810 ] . water , methanol , ethanol , and ethyl acetate ( ea ) were commonly used solvents for the extraction of bioactive compounds from plant materials and omhs . the identification and relative amounts of five types of compounds in a. tegmentosum were determined by lc - ms , nmr , and on - line screening hplc - abts assay . these techniques included on - line screening with hplc post - column assay involving the abts radical technique , allowing spectrophotometric monitoring of bioactive compounds . generally , dpph ( abts ) radical is another simple , rapid on - line method for the detection of antioxidants from crude plant extracts . the chemical structures of the five types of compounds were confirmed by spectroscopic methods such as h - nmr , c - nmr , and lc / ms . in this study , five phenolic compounds were isolated from hot water extract of a. tegmentosum bychromatographic separation . using spectroscopic methods , the structures of these compounds were determined as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) . moreover , we investigated their anti - inflammatory effects on lps - stimulated raw 264.7 cells . we also investigated the applications of on - line screening hplc - abts assays for bioactivity screening to find a more practical approach toward the use of on - line screening hplc - abts assays for the rapid pinpointing of peaks in chromatograms corresponding to bioactive compounds . the hexane , dichloromethane ( dcm ) , ethyl acetate ( ea ) , and normal butyl alcohol ( n - buoh ) were purchased from daejung chemical ( gyeonggi - do , shiheung , korea ) . lps , bovine serum albumin ( bsa ) , and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) were purchased from sigma ( st . the high purity isolated compounds ( higher than > 95% ) were prepared by dissolving 2 mg of the standard chemicals feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) in 10 ml of methanol and adjusting the concentration to 200 ppm . dry samples ( 3 kg ) from the powders of the a. tegmentosum were loaded ( 10-times volume ) in hot water extraction system . then , the solution was filtered using standard testing sieves ( 150 m , retsch , haan , germany ) , freeze - dried , and maintained in desiccators at 4c prior to use . for large amount of extractions , 20 g freeze - dry samples were loaded ( 1 : 1 , extracted thrice ) and extracted successively using dcm , ea , and n - buoh . the contents of feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) in a. tegmentosum were remarkably higher in the ea extract ( 1.24 g ) . then , the samples were filtered through a 0.2 m membrane filter prior to on - line screening hplc - abts analysis . the isolated compounds feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) were confirmed vialc - ms analysis using agilent 1100 + g1958 ( agilent , usa ) lc systems ( table 1 ) . open column chromatography was performed using silica gel ( kieselgel 60 , merck , germany ) and octadecylsilane ( ods ) li - chropre rp-18 ( merck , germany ) . thin layer chromatography ( tlc ) analysis was performed using silica gel glass plates ( kieselgel 60 f254 and rp-18 f254s , merck , germany ) and developed using a mobile phase composed of chloroform - methanol - water and stained using 10% h2so4 to detect the ea extract and pure compounds . the injection volume was 10 l , and the flow rate of the mobile phase was 1.0 ml / min . the wavelength of the uv detector was fixed at 210 , 254 , 280 , and 320 nm . the run time was 60 min , and the solvent program was the linear gradient method ( 90 : 1060 : 40 a : b vol% , 70 min : initial condition ) ( table 2 ) . figure 3 shows a schematic diagram of the on - line coupling of a hplc to a dad ( diode array detector ) and the continuous flow abts assay . mtt solutions were added to each well and the cells were incubated for another 4 h. the formazan melted in dimethyl sulfoxide ( dmso ) , and then the optical density was read at 570 nm using an elisa reader ( infinite m200 , tecan , mnnedorf , switzerland ) . the stems of a. tegmentosum were extracted using boiling water and then partitioned successively using dichloromethane ( dcm ) , ethyl acetate ( ea ) , normal butyl alcohol ( n - buoh ) , and water ( h2o ) . the ea extract ( 1.24 g ) ( 4 10 cm ) and eluted using chloroform- ( chcl3 ) meoh h2o [ 15 : 3 : 1 ( 1.2 l ) 13 : 3 : 1 ( 1.3 l ) 10 : 3 : 1 ( 1.2 l ) 7 : 3 : 1 ( 1 l ) 4 : 3 : 1 ( 1 l ) meoh ] . fraction os1e-9 - 3 ( 77.8 mg ) was subjected to silica gel c.c . based on the spectroscopic methods , such as h - nmr , c - nmr , and lc / ms , the chemical structures of the compounds were confirmed as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) by comparison of spectral data with those of references . moreover , the on - line screening hplc - abts assay method was rapid and efficient for the investigation of bioactivity of the isolated compounds from acer tegmentosum . compounds 1 , 2 , 3 , 4 , and 5 were developed on ods tlc with 10% h2so4 and the expected yellow or brown color was observed for the phenolic compounds . yellow amorphous powder ( meoh ) ; esi - ms m / z 435.1 [ m+h ] ; h - nmr ( 600 mhz , cd3od , h ) 7.52 ( 1h , d , j = 1.8 hz , h-2 ) , 7.49 ( 1h , dd , j = 8.4 , 1.8 hz , h-6 ) , 6.90 ( 1h , d , j = 8.4 hz , h-5 ) , 6.38 ( 1h , d , j = 1.8 hz , h-8 ) , 6.20 ( 1h , d , j = 1.8 hz , h-6 ) , 5.46 ( 1h , s , h-1 ) , 4.32 ( 1h , d , j = 2.4 hz , h-2 ) , 3.89 ( 1h , dd , j = 4.8 , 2.4 hz , h-3 ) , 3.861 ( 1h , m , h-4 ) , 3.494 ( 2h , t , j = 4.2 , h-5 ) ; c - nmr ( 150 mhz , cd3od , c ) ; see table 3 . compounds 1 and 2 were isolated from the plant for the first time . compound 3 [ ( + ) -catechin ] . compounds 3 and 4 were compared to the literature data for ( + ) -catechin and ( )-epicatechin for the structure identification . compound 5 ( 6 -o - galloyl salidroside ) . pale yellow oil ( meoh ) ; esi - ms m / z 453.1 [ m+h ] ; h - nmr ( 600 mhz , cd3od , h ) 7.10 ( 2h , br s , h-2 , h-6 ) , 6.96 ( 2h , d , j = 8.4 hz , h-2 , 6 ) , 6.65 ( 2h , d , j = 8.4 hz , h-3 , h-5 ) , 6.65 ( 1h , d , j = 8.4 hz , h-5 ) , 4.52 ( 1h , dd , j = 11.4 , 2.4 hz , h-6a ) , 4.45 ( 1h , dd , j = 11.4 , 6.0 hz , h-6b ) , 4.32 ( 1h , d , j = 7.8 hz , h-1 ) , 3.95 ( 1h , m , h-8a ) , 3.70 ( 1h , m , h-8b ) , 3.55 ( 1h , m , h-5 ) , 3.42 ( 2h , t , j = 7.8 hz , h-3 , 4 ) , 3.22 ( 1h , t , j = 8.4 hz , h-2 ) , 2.77 ( 2h , m , h-7 ) ; c - nmr ( 150 mhz , cd3od , c ) ; see table 3 . this study investigated the bioactivity ( using abts assay ; radical scavenging activity ) and anti - inflammatory activity of the five isolated phenolic compounds that were measured . all the compounds 15 in the ea fraction ( each yield ; mg ) exhibited antioxidant activities ( table 4 ) . moreover , this on - line screening hplc - abts assay method was rapid and efficient for the investigation of bioactivity from a. tegmentosum and was obtained from rs - tech ( 0.46 25 cm , 5 m , c18 , daejeon , korea ) . the injection volume was 10 l , and the flow rate of the mobile phase was 1.0 ml / min . the five phenolic compounds were characterized by comparing the hplc uv - dad maximum absorption peaks of the samples with those of the pure isolation standards ( figure 4 ) . the determination of antioxidant activity from the on - line hplc dpph ( abts ) assay was based on the decrease in absorbance at 517 and 734 nm after the postcolumn reaction of antioxidants separated from hplc with dpph ( abts ) , and the antioxidants present in a sample would be easily indicated by negative peaks . the run time was 60 min , and the solvent program used the linear gradient method ( 90 : 1060 : 40 a : b vol% , 70 min : initial condition ) . several eluted fractions of the five phenolic compounds in the ea extract were detected as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) , giving a positive signal on the uv detector at 210 nm . these results revealed that the method could be applied for quick bioactivity screening , or more precisely , to detect the radical - scavenging activity of compounds , indicating that ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) exhibited bioactivities . 6-o - galloyl salidroside ( 5 ) exhibited higher bioactivity , whereas feniculin ( 1 ) and avicularin ( 2 ) showed low bioactivity ( figure 5 ) . the retention time ( rt ) of feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) was 10.700 , 13.884 , 15.928 , 24.217 , and 25.062 min , respectively . the bioactivity appeared to be approximately proportional to the concentration of the five phenolic compounds in the extracts . the hplc analysis conditions for the best separation of the five compounds were successfully established by varying the open column treatment and solvent purification step ( figure 6 ) . this study confirms the feasibility of assessing the bioactivity of specific phytochemicals using an on - line screening hplc - abts assay method ( table 5 ) . figure 7 shows the hplc profile and lc - ms spectra of five isolated phenolic compounds from a. tegmentosum . lc - ms analysis indicated that the compounds from a. tegmentosum were isolated in highly pure form . the comparison of uv spectra , mass spectra , and retention times of the five types of compounds with the data of standard compounds led to their unambiguous assignments . simultaneous analysis based on rt of feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) of 32.6 , 34.0 , 12.1 , 17.7 , and 21.3 min , respectively , was correlated with the molecular mass data and relative response . esi - ms analysis of compounds feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) showed intense [ m+h ] signals at 435.1 , 435.1 , 291.0 , 291.0 , and 453.1 m / z values , respectively . as shown in figure 8(a ) , all the five compounds did not affect cell viability up to 100 m , indicating that the compounds were not toxic to cells . we evaluated the effects of five compounds on no secretion in lps - stimulated raw 264.7 cells . all the compounds , except 6-o - galloyl salidroside , did not show any inhibitory effect on lps - induced no production ( figure 8(b ) ) . however , avicularin , ( + ) -catechin , ( )-epicatechin , and 6-o - galloyl salidroside did not show any inhibitory effect on lps - induced tnf- production . ( + ) -catechin inhibited il-6 production at 30 m or more . ( )-epicatechin and 6-o - galloyl salidroside did not show any inhibitory effect on lps - induced il-6 secretion . however , all the compounds except 6-o - galloyl salidroside did not inhibit il-1 ( figure 9(c ) ) . this study showed that among the soluble fractions from the hot water extract of a. tegmentosum , the ea - soluble fraction possessed the highest bioactivity and free radical - scavenging activities . compounds in the dried twigs of a. tegmentosum were extracted with hot water and partitioned successively using dcm , ea , n - buoh , and water . the content of useful compounds in a. tegmentosum was remarkably higher in ea the extract ( 1.24 g ) . five phenolic compounds were isolated by the silica gel , octadecyl silica gel , and sephadex lh-20 column chromatography . the chemical structures of the isolated compounds were determined by spectroscopic methods , such as h - nmr , c - nmr , and lc / ms and were confirmed as feniculin ( 1 ) , avicularin ( 2 ) , ( + ) -catechin ( 3 ) , ( )-epicatechin ( 4 ) , and 6-o - galloyl salidroside ( 5 ) by comparison of spectral data with those of references . the ea extract of a. tegmentosum containing five phenolic compounds exhibiting the best bioactivity was further monitored by an on - line screening hplc - abtsassay method . compounds 1 and 2 were isolated for the first time , and their anti - inflammatory activities were evaluated . moreover , the on - line screening hplc - abts assay method was rapid and efficient to search for bioactive compound from a. tegmentosum . furthermore , ( + ) -catechin and 6-o - galloyl salidroside exhibited the inhibitory activities on inflammatory mediator production such as tnf- , il-6 , and il-1 cytokines . in conclusion , a. tegmentosum can be used as a basic material for the development of new drugs in omhs .

determining the relative effects of single- and double - row suture anchor repairs of the rotator cuff will allow physicians to be better equipped to treat patients with rotator cuff disease . the prevalence of full and partial thickness defects increases linearly with age after 50 years , and by the age of 80 years , they exist in 80% of the population . the influence of rotator cuff pathology on glenohumeral kinematics is not well documented in the literature , although several authors have speculated on the wide disparity of functional abilities of patients with grossly similar pathology . a number of cadaveric studies have examined the effects of rotator cuff pathology on shoulder kinematics . collectively , these studies suggest that increasing the size of the rotator cuff tear will result in an increased alteration of kinematics . currently , there is little information available on the effect of rotator cuff tears on active kinematics in - vitro . examined the effects of sequential one , two , and three cm rotator cuff tears that were created in the supraspinatus tendon and extended into the subscapularis tendon . the humeral head was displaced posteriorly with progressively larger tears of the rotator cuff during active shoulder elevation . the authors concluded that increasing the size of the rotator cuff tear resulted in greater alterations in glenohumeral kinematics . the goals of surgical repair of rotator cuff tears are to restore the anatomic relationship between the tendons and their osseous footprints on the humerus . common open and arthroscopic techniques include transosseous suture repair or the use of suture anchors in either a single- or double - row repair configuration . several recent studies have examined the biomechanical properties and clinical outcomes of the different repair techniques . biomechanical studies have found double - row constructs to be stronger than single - row constructs in both cadaveric and animal models . in addition , double - row repairs can restore the tendon footprint more accurately and are associated with higher rates of tendon healing than single - row repairs . it has also been suggested that double - row repairs are preferable for cases with retracted tendons . despite the biomechanical advantage , clinical studies have failed to show a distinct advantage favoring the double - row repair technique . in addition to ensuring mechanical integrity of these reconstructions , the resulting glenohumeral joint motion is also important . however , we are unaware of any studies that have examined the effect of different suture anchor repairs on shoulder translations . hence , the purpose of this study was to determine active glenohumeral kinematics with simulated 2 cm - sized rotator cuff tears . in addition , the ability of single- and double - row suture anchor repair to restore intact glenohumeral kinematics was also compared . we hypothesized that ( 1 ) two cm defects in the rotator cuff would have a minor effect on simulated active glenohumeral kinematics and that ( 2 ) both single- and double - row suture anchor fixation would restore normal active intact motion . eight pairs of fresh - frozen cadaveric upper forequarters ( mean age : 66 9 years ; range : 46 - 74 years ; 5 male ) were used . all specimens had no evidence of previous surgery , trauma , glenohumeral arthritis , or rotator cuff tendonopathy . specimens were thawed to room temperature ( mean 23c ( 2c ) ) and were kept moist with 0.9% saline throughout testing . a shoulder testing simulator that creates active unconstrained glenohumeral elevation was employed . specimens were prepared by osteotomy of the distal humerus at mid - shaft , exposure of the anterior deltoid , and exposure of the inferior border of the scapula . a 316 stainless - steel rod , with weights added to be equivalent to the intact arm , was cemented into the distal humeral canal . equivalence between the intact arm and the weighted rod was verified with a spring scale . each specimen was mounted to the simulator by securing the inferior pole of the scapula into a custom pot with polymethylmethacrylate . muscle simulation was achieved by suturing pneumatically actuated cables to the tendons of the rotator cuff muscles supraspinatus , subscapularis , and infraspinatus / teres minor , and attaching three cables to the deltoid tuberosity to simulate the three heads of the deltoid muscle . forces were applied to the individual cables using a previously described set of loading ratios . these were based upon electromyographic ( emg ) data and the relative physiological cross - sectional areas of the muscles . the forces varied according to the angle of elevation of the humerus and were different for each specimen . the relative motion of the humerus with respect to the scapula was collected with an electromagnetic tracking device ( flock of birds , ascension technologies , burlington , usa ) . the receivers for the tracking system were fixed to the superior border of the scapula and the rod that replaced the distal humerus . passive glenohumeral elevation in the plane of the scapula was conducted to ensure no impingement occurred as a result of fixation . small tone loads of between 20n and 30n were applied to the rotator cuff muscles at the start of each cycle of motion . these loads were selected to ensure that the humeral head was centered in the glenoid socket when the motion began . five trials of both passive and active motions were conducted to allow quantification of repeatability . elevation was defined as movement in the scapular plane from approximately 0 to 90 of glenohumeral elevation . following intact testing , a deltoid splitting technique was used to expose the rotator cuff for creation of a 1 cm ( mediolateral ) by 2 cm ( anteroposterior ) defect in the anterior supraspinatus , which began at the rotator interval and propagated posteriorly . one centimeter of the supraspinatus tendon was resected to simulate the clinical scenario of tendon retraction . the deltoid muscle and skin incisions were closed with a running suture prior to recording the simulated active motion of the new torn state . following testing , one shoulder from each of the eight pairs was randomized to undergo rotator cuff repair using a single - row suture anchor technique , while the contralateral shoulder was repaired using a double - row suture anchor technique . all repairs were performed with 5.5 mm threaded metal anchors double - loaded with # 2 high strength suture ( arthrex , naples , usa ) . the single row repair was performed by placing one anchor into the midportion of the greater tuberosity footprint . the rotator cuff was repaired with simple sutures placed into the lateral edge of the tendon . the double - row repair was performed by placing one anchor adjacent to the articular margin , and a second anchor was placed at the lateral edge of the greater tuberosity . the medial row was repaired using two mattress sutures , and the lateral row was repaired with two simple sutures . an overview of the experimental procedure repeatability of the trials was quantified by using the standard deviation of the five trials at each position . a segment - fixed co - ordinate system was created on each of the scapula and humerus to allow calculation of the glenohumeral joint kinematics . the plane of abduction and the position of the humeral head relative to the fixed scapula were analyzed at every 10 of glenohumeral elevation . the relative motion of the humeral head with respect to the scapula was also quantified . statistical analyses were performed using a friedman repeated measures analysis of variance ( anova ) on ranks . the dependent variables considered were the angle of abduction and the torn or repaired state . specimens were prepared by osteotomy of the distal humerus at mid - shaft , exposure of the anterior deltoid , and exposure of the inferior border of the scapula . a 316 stainless - steel rod , with weights added to be equivalent to the intact arm , was cemented into the distal humeral canal . equivalence between the intact arm and the weighted rod was verified with a spring scale . each specimen was mounted to the simulator by securing the inferior pole of the scapula into a custom pot with polymethylmethacrylate . muscle simulation was achieved by suturing pneumatically actuated cables to the tendons of the rotator cuff muscles supraspinatus , subscapularis , and infraspinatus / teres minor , and attaching three cables to the deltoid tuberosity to simulate the three heads of the deltoid muscle . forces were applied to the individual cables using a previously described set of loading ratios . these were based upon electromyographic ( emg ) data and the relative physiological cross - sectional areas of the muscles . the forces varied according to the angle of elevation of the humerus and were different for each specimen . the relative motion of the humerus with respect to the scapula was collected with an electromagnetic tracking device ( flock of birds , ascension technologies , burlington , usa ) . the receivers for the tracking system were fixed to the superior border of the scapula and the rod that replaced the distal humerus . passive glenohumeral elevation in the plane of the scapula was conducted to ensure no impingement occurred as a result of fixation . small tone loads of between 20n and 30n were applied to the rotator cuff muscles at the start of each cycle of motion . these loads were selected to ensure that the humeral head was centered in the glenoid socket when the motion began . five trials of both passive and active motions were conducted to allow quantification of repeatability . elevation was defined as movement in the scapular plane from approximately 0 to 90 of glenohumeral elevation . following intact testing , a deltoid splitting technique was used to expose the rotator cuff for creation of a 1 cm ( mediolateral ) by 2 cm ( anteroposterior ) defect in the anterior supraspinatus , which began at the rotator interval and propagated posteriorly . one centimeter of the supraspinatus tendon was resected to simulate the clinical scenario of tendon retraction . the deltoid muscle and skin incisions were closed with a running suture prior to recording the simulated active motion of the new torn state . following testing , one shoulder from each of the eight pairs was randomized to undergo rotator cuff repair using a single - row suture anchor technique , while the contralateral shoulder was repaired using a double - row suture anchor technique . all repairs were performed with 5.5 mm threaded metal anchors double - loaded with # 2 high strength suture ( arthrex , naples , usa ) . the single row repair was performed by placing one anchor into the midportion of the greater tuberosity footprint . the rotator cuff was repaired with simple sutures placed into the lateral edge of the tendon . the double - row repair was performed by placing one anchor adjacent to the articular margin , and a second anchor was placed at the lateral edge of the greater tuberosity . the medial row was repaired using two mattress sutures , and the lateral row was repaired with two simple sutures . repeatability of the trials was quantified by using the standard deviation of the five trials at each position . a segment - fixed co - ordinate system was created on each of the scapula and humerus to allow calculation of the glenohumeral joint kinematics . the plane of abduction and the position of the humeral head relative to the fixed scapula were analyzed at every 10 of glenohumeral elevation . the relative motion of the humeral head with respect to the scapula was also quantified . statistical analyses were performed using a friedman repeated measures analysis of variance ( anova ) on ranks . the dependent variables considered were the angle of abduction and the torn or repaired state . within all pairs of intact specimens , no statistically significant side - to - side differences were observed . therefore , the measurements for the left and right cases were averaged and included in the friedman repeated measures anova as one entity . in addition , no significant differences were found between passive and active intact glenohumeral elevation of the specimens through the range of motion . statistical analyses were performed at every 10 of elevation , through an arc of 20 to 80 of glenohumeral elevation . the plane of elevation exhibited was altered between the intact and the simulated tear states ( p = 0.01 ) [ figure 2 ] . the 2 cm defect resulted in posterior angulation of the plane of elevation through the arc of abduction . this was greatest at the mid - portion of abduction ( 40-60 ) with an overall average of 8 posterior shift when compared to the intact kinematic state . the angle of internal - external rotation of the humerus was not affected by the simulated tear [ figure 3 ] . similarly , no differences in the position of the humeral head were observed as a result of the simulated defect [ figure 4 ] . change in plane of glenohumeral elevation from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in internal - external rotation angle from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in ( a ) superior - inferior and ( b ) anterior - posterior position of the humeral head from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation once repaired , a significantly different plane of elevation was achieved [ figure 2 ] . this was true of both the single row suture anchor repair ( p < 0.001 ) and the double - row suture anchor repair ( p = 0.001 ) . the internal - external rotation angle and position of the humeral head were not altered by the repair [ figures 3 and 4 ] . single- and double - row suture anchor repairs were able to restore the intact kinematic state of the glenohumeral joint for 2 cm rotator cuff defects ( p = 0.10 ) [ figure 2 ] . single row suture anchor repair resulted in a slightly anterior angulation of the plane of elevation on average when compared to the intact kinematic state . double - row suture anchor repair resulted in a similar plane of elevation as that observed in the intact state ( p = 1.00 ) . no differences in the plane of elevation were observed between the two repairs ( p = 0.10 ) . both single- and double - row repairs resulted in increased variability in the change of the internal - external rotation angle of the humerus [ figure 3 ] , but no statistically significant differences were observed . likewise , the position of the humeral head was not altered as a result of the repairs . differences in the plane of glenohumeral elevation summarized for all specimens in all angles of elevation . no differences were observed in the internal - external rotation angle , superior - inferior position , and anterior - posterior position the plane of elevation exhibited was altered between the intact and the simulated tear states ( p = 0.01 ) [ figure 2 ] . the 2 cm defect resulted in posterior angulation of the plane of elevation through the arc of abduction . this was greatest at the mid - portion of abduction ( 40-60 ) with an overall average of 8 posterior shift when compared to the intact kinematic state . the angle of internal - external rotation of the humerus was not affected by the simulated tear [ figure 3 ] . similarly , no differences in the position of the humeral head were observed as a result of the simulated defect [ figure 4 ] . change in plane of glenohumeral elevation from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in internal - external rotation angle from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in ( a ) superior - inferior and ( b ) anterior - posterior position of the humeral head from the intact state as a function of elevation angle for all states . once repaired , a significantly different plane of elevation was achieved [ figure 2 ] . this was true of both the single row suture anchor repair ( p < 0.001 ) and the double - row suture anchor repair ( p = 0.001 ) . the internal - external rotation angle and position of the humeral head were not altered by the repair [ figures 3 and 4 ] . single- and double - row suture anchor repairs were able to restore the intact kinematic state of the glenohumeral joint for 2 cm rotator cuff defects ( p = 0.10 ) [ figure 2 ] . single row suture anchor repair resulted in a slightly anterior angulation of the plane of elevation on average when compared to the intact kinematic state . double - row suture anchor repair resulted in a similar plane of elevation as that observed in the intact state ( p = 1.00 ) . no differences in the plane of elevation were observed between the two repairs ( p = 0.10 ) . both single- and double - row repairs resulted in increased variability in the change of the internal - external rotation angle of the humerus [ figure 3 ] , but no statistically significant differences were observed . likewise , the position of the humeral head was not altered as a result of the repairs . differences in the plane of glenohumeral elevation summarized for all specimens in all angles of elevation . no differences were observed in the internal - external rotation angle , superior - inferior position , and anterior - posterior position over the last decade , arthroscopic shoulder techniques and equipment have improved the ability to perform minimally invasive repairs to the rotator cuff tendons . the literature has demonstrated that single- and double - row repairs are safe and effective . the purpose of this study , unlike prior biomechanical studies that examined the relative strength double - row cuff repair , was to illustrate the kinematic effects of single- and double - row suture anchor repairs of 2 cm supraspinatus tears on simulated active glenohumeral joint kinematics . the effects of a simulated 2 cm defect in the supraspinatus tendon in this study were similar to the results found previously by kedgley et al . as theorized , no changes were found in the internal - external rotation of the humerus or the position of the humeral head with elevation , though the 2 cm defect did result in an alteration in the plane of elevation with a mean change of 8 posteriorly . these results would perhaps be expected ; however , compensation likely occurs in - vivo with a medium - sized tear such as this . our observations did not demonstrate any significant differences in the plane of elevation between single- and double - row repairs . sugaya et al . , in a retrospective cohort study , compared the functional and structural outcomes of single - row versus double - row rotator cuff repair . at a follow - up 35 months , no statistically significant differences in clinical outcome could be appreciated between the two different repair techniques . franceschi et al . , in a randomized controlled clinical trial , compared 30 patients who received a single - row repair and 30 patients who received a double - row repair . at two years follow - up , there was no clinically significant difference between the two different repair techniques . in this study , there are a number of possible reasons that may account for the results . first , all rotator cuff repairs were performed with suture anchors that create focal sites of tendon reattachment to the greater tuberosity . in an in - vitro study , integration of the tendon to the tuberosity does not occur , and restoration of normal force distribution from a pennate muscle across the tendon - bone interface is unlikely . in both repair conditions , in - vivo repair studies may be capable of identifying kinematic differences among repair techniques that result in proportionately different rates of healing at the bone - tendon interface . . a 2 cm defect may behave differently in a small specimen than in a large specimen and may have affected our ability to detect a change following the simulated tears . simulating tears that are proportional to the size of the specimen rather than based on an absolute dimension may have resulted in more physiologically relevant results , although they should not have influenced the comparison of the repair types . this supports the generalized trend toward more accurate restoration of the intact glenohumeral kinematics was seen in double - row suture anchor repair for a 2 cm defect of the supraspinatus . third , rotator cuff pathology is often a chronic and progressive disorder that may occur in conjunction with adaptive changes in adjacent muscles . in contrast , this study examined the kinematics of an acute lesion , and repair of the rotator cuff and may not accurately reflect the more common clinical scenario seen in patients with chronic rotator cuff disease . the primary factors that contribute to shoulder stability remained functional , including the glenohumeral articular structures and capsulolabral complex . in a clinical situation , this would likely not be the case , as some degree of degradation would have occurred throughout the cuff tissue . however , had the specimens already exhibited pathology of the rotator cuff , we would have been unable to compare the repaired and intact states . fourth , this study simulated a chronic clinical scenario by creating an acute defect in the rotator cuff to mimic tendon retraction . shortening the tendon may have inadvertently created the anterior angulation seen in the plane of elevation with our suture anchor repairs . however , there are also theories that a biochemical mediator may cause a damaged rotator cuff to retract . similarly , the re - attachment of the tendon most likely imposed the observed increased variability in the internal - external rotation angle of the humerus . unfortunately , the use of a deltoid split to produce the rotator cuff tear may not represent the same biomechanics , as that which would be observed had an arthroscopic approach been taken . however , no deltoid failures occurred during testing , and a previous study showed very little alteration of glenohumeral kinematics with a 1 cm rotator cuff tear created in the same way . the paired , randomized , and controlled nature of this study did generate results of significant merit . furthermore , a unique , validated shoulder simulator capable of simulating active glenohumeral joint motion using a physiologically - based muscle loading protocol was employed . in summary , a 2 cm defect in the supraspinatus alters the kinematics of the glenohumeral joint by moving the humeral plane of elevation posteriorly . both single- and double - row repair techniques resulted in restoration of normal kinematics when compared to the intact state . we did not find a significant difference in glenohumeral kinematics between the two repair techniques . further studies are warranted to ascertain which repairs afford patients optimal functional and clinical results . with an improved understanding of the kinematics of the glenohumeral joint

purpose : double - row suture anchor fixation of the rotator cuff was developed to reduce repair failure rates . the purpose of this study was to determine the effects of simulated rotator cuff tears and subsequent repairs using single- and double - row suture anchor fixation on three - dimensional shoulder kinematics . it was hypothesized that both single- and double - row repairs would be effective in restoring active intact kinematics of the shoulder.materials and methods : sixteen fresh - frozen cadaveric shoulder specimens ( eight matched pairs ) were tested using a custom loading apparatus designed to simulate unconstrained motion of the shoulder . in each specimen , the rotator cuff was sectioned to create a medium - sized ( 2 cm ) tear . within each pair , one specimen was randomized to a single - row suture anchor repair , while the contralateral side underwent a double - row suture anchor repair . joint kinematics were recorded for intact , torn , and repaired scenarios using an electromagnetic tracking device.results:active kinematics confirmed that a medium - sized rotator cuff tear affected glenohumeral kinematics when compared to the intact state . single- and double - row suture anchor repairs restored the kinematics of the intact specimen.conclusions:this study illustrates the effects of medium - sized rotator cuff tears and their repairs on active glenohumeral kinematics . no significant difference ( p 0.10 ) was found between the kinematics of single- and double - row techniques in medium - sized rotator cuff repairs.clinical relevance : determining the relative effects of single- and double - row suture anchor repairs of the rotator cuff will allow physicians to be better equipped to treat patients with rotator cuff disease .

Clinical Relevance: INTRODUCTION MATERIALS AND METHODS Testing procedure Outcome variables and statistical analysis RESULTS Intact versus defect condition Defect versus repair condition Intact versus repair condition DISCUSSION CONCLUSIONS

determining the relative effects of single- and double - row suture anchor repairs of the rotator cuff will allow physicians to be better equipped to treat patients with rotator cuff disease . the prevalence of full and partial thickness defects increases linearly with age after 50 years , and by the age of 80 years , they exist in 80% of the population . the influence of rotator cuff pathology on glenohumeral kinematics is not well documented in the literature , although several authors have speculated on the wide disparity of functional abilities of patients with grossly similar pathology . a number of cadaveric studies have examined the effects of rotator cuff pathology on shoulder kinematics . collectively , these studies suggest that increasing the size of the rotator cuff tear will result in an increased alteration of kinematics . currently , there is little information available on the effect of rotator cuff tears on active kinematics in - vitro . examined the effects of sequential one , two , and three cm rotator cuff tears that were created in the supraspinatus tendon and extended into the subscapularis tendon . the humeral head was displaced posteriorly with progressively larger tears of the rotator cuff during active shoulder elevation . the authors concluded that increasing the size of the rotator cuff tear resulted in greater alterations in glenohumeral kinematics . the goals of surgical repair of rotator cuff tears are to restore the anatomic relationship between the tendons and their osseous footprints on the humerus . common open and arthroscopic techniques include transosseous suture repair or the use of suture anchors in either a single- or double - row repair configuration . several recent studies have examined the biomechanical properties and clinical outcomes of the different repair techniques . biomechanical studies have found double - row constructs to be stronger than single - row constructs in both cadaveric and animal models . in addition , double - row repairs can restore the tendon footprint more accurately and are associated with higher rates of tendon healing than single - row repairs . it has also been suggested that double - row repairs are preferable for cases with retracted tendons . despite the biomechanical advantage , clinical studies have failed to show a distinct advantage favoring the double - row repair technique . in addition to ensuring mechanical integrity of these reconstructions , the resulting glenohumeral joint motion is also important . however , we are unaware of any studies that have examined the effect of different suture anchor repairs on shoulder translations . hence , the purpose of this study was to determine active glenohumeral kinematics with simulated 2 cm - sized rotator cuff tears . in addition , the ability of single- and double - row suture anchor repair to restore intact glenohumeral kinematics was also compared . we hypothesized that ( 1 ) two cm defects in the rotator cuff would have a minor effect on simulated active glenohumeral kinematics and that ( 2 ) both single- and double - row suture anchor fixation would restore normal active intact motion . eight pairs of fresh - frozen cadaveric upper forequarters ( mean age : 66 9 years ; range : 46 - 74 years ; 5 male ) were used . all specimens had no evidence of previous surgery , trauma , glenohumeral arthritis , or rotator cuff tendonopathy . specimens were prepared by osteotomy of the distal humerus at mid - shaft , exposure of the anterior deltoid , and exposure of the inferior border of the scapula . a 316 stainless - steel rod , with weights added to be equivalent to the intact arm , was cemented into the distal humeral canal . equivalence between the intact arm and the weighted rod was verified with a spring scale . each specimen was mounted to the simulator by securing the inferior pole of the scapula into a custom pot with polymethylmethacrylate . muscle simulation was achieved by suturing pneumatically actuated cables to the tendons of the rotator cuff muscles supraspinatus , subscapularis , and infraspinatus / teres minor , and attaching three cables to the deltoid tuberosity to simulate the three heads of the deltoid muscle . forces were applied to the individual cables using a previously described set of loading ratios . these were based upon electromyographic ( emg ) data and the relative physiological cross - sectional areas of the muscles . the forces varied according to the angle of elevation of the humerus and were different for each specimen . the relative motion of the humerus with respect to the scapula was collected with an electromagnetic tracking device ( flock of birds , ascension technologies , burlington , usa ) . the receivers for the tracking system were fixed to the superior border of the scapula and the rod that replaced the distal humerus . small tone loads of between 20n and 30n were applied to the rotator cuff muscles at the start of each cycle of motion . following intact testing , a deltoid splitting technique was used to expose the rotator cuff for creation of a 1 cm ( mediolateral ) by 2 cm ( anteroposterior ) defect in the anterior supraspinatus , which began at the rotator interval and propagated posteriorly . one centimeter of the supraspinatus tendon was resected to simulate the clinical scenario of tendon retraction . the deltoid muscle and skin incisions were closed with a running suture prior to recording the simulated active motion of the new torn state . following testing , one shoulder from each of the eight pairs was randomized to undergo rotator cuff repair using a single - row suture anchor technique , while the contralateral shoulder was repaired using a double - row suture anchor technique . all repairs were performed with 5.5 mm threaded metal anchors double - loaded with # 2 high strength suture ( arthrex , naples , usa ) . the single row repair was performed by placing one anchor into the midportion of the greater tuberosity footprint . the rotator cuff was repaired with simple sutures placed into the lateral edge of the tendon . the double - row repair was performed by placing one anchor adjacent to the articular margin , and a second anchor was placed at the lateral edge of the greater tuberosity . an overview of the experimental procedure repeatability of the trials was quantified by using the standard deviation of the five trials at each position . a segment - fixed co - ordinate system was created on each of the scapula and humerus to allow calculation of the glenohumeral joint kinematics . the plane of abduction and the position of the humeral head relative to the fixed scapula were analyzed at every 10 of glenohumeral elevation . the relative motion of the humeral head with respect to the scapula was also quantified . statistical analyses were performed using a friedman repeated measures analysis of variance ( anova ) on ranks . specimens were prepared by osteotomy of the distal humerus at mid - shaft , exposure of the anterior deltoid , and exposure of the inferior border of the scapula . a 316 stainless - steel rod , with weights added to be equivalent to the intact arm , was cemented into the distal humeral canal . equivalence between the intact arm and the weighted rod was verified with a spring scale . each specimen was mounted to the simulator by securing the inferior pole of the scapula into a custom pot with polymethylmethacrylate . muscle simulation was achieved by suturing pneumatically actuated cables to the tendons of the rotator cuff muscles supraspinatus , subscapularis , and infraspinatus / teres minor , and attaching three cables to the deltoid tuberosity to simulate the three heads of the deltoid muscle . forces were applied to the individual cables using a previously described set of loading ratios . these were based upon electromyographic ( emg ) data and the relative physiological cross - sectional areas of the muscles . the forces varied according to the angle of elevation of the humerus and were different for each specimen . the relative motion of the humerus with respect to the scapula was collected with an electromagnetic tracking device ( flock of birds , ascension technologies , burlington , usa ) . the receivers for the tracking system were fixed to the superior border of the scapula and the rod that replaced the distal humerus . passive glenohumeral elevation in the plane of the scapula was conducted to ensure no impingement occurred as a result of fixation . small tone loads of between 20n and 30n were applied to the rotator cuff muscles at the start of each cycle of motion . following intact testing , a deltoid splitting technique was used to expose the rotator cuff for creation of a 1 cm ( mediolateral ) by 2 cm ( anteroposterior ) defect in the anterior supraspinatus , which began at the rotator interval and propagated posteriorly . one centimeter of the supraspinatus tendon was resected to simulate the clinical scenario of tendon retraction . the deltoid muscle and skin incisions were closed with a running suture prior to recording the simulated active motion of the new torn state . following testing , one shoulder from each of the eight pairs was randomized to undergo rotator cuff repair using a single - row suture anchor technique , while the contralateral shoulder was repaired using a double - row suture anchor technique . all repairs were performed with 5.5 mm threaded metal anchors double - loaded with # 2 high strength suture ( arthrex , naples , usa ) . the single row repair was performed by placing one anchor into the midportion of the greater tuberosity footprint . the rotator cuff was repaired with simple sutures placed into the lateral edge of the tendon . the double - row repair was performed by placing one anchor adjacent to the articular margin , and a second anchor was placed at the lateral edge of the greater tuberosity . the medial row was repaired using two mattress sutures , and the lateral row was repaired with two simple sutures . a segment - fixed co - ordinate system was created on each of the scapula and humerus to allow calculation of the glenohumeral joint kinematics . the plane of abduction and the position of the humeral head relative to the fixed scapula were analyzed at every 10 of glenohumeral elevation . the relative motion of the humeral head with respect to the scapula was also quantified . in addition , no significant differences were found between passive and active intact glenohumeral elevation of the specimens through the range of motion . the plane of elevation exhibited was altered between the intact and the simulated tear states ( p = 0.01 ) [ figure 2 ] . the 2 cm defect resulted in posterior angulation of the plane of elevation through the arc of abduction . this was greatest at the mid - portion of abduction ( 40-60 ) with an overall average of 8 posterior shift when compared to the intact kinematic state . similarly , no differences in the position of the humeral head were observed as a result of the simulated defect [ figure 4 ] . change in plane of glenohumeral elevation from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in internal - external rotation angle from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in ( a ) superior - inferior and ( b ) anterior - posterior position of the humeral head from the intact state as a function of elevation angle for all states . this was true of both the single row suture anchor repair ( p < 0.001 ) and the double - row suture anchor repair ( p = 0.001 ) . single- and double - row suture anchor repairs were able to restore the intact kinematic state of the glenohumeral joint for 2 cm rotator cuff defects ( p = 0.10 ) [ figure 2 ] . single row suture anchor repair resulted in a slightly anterior angulation of the plane of elevation on average when compared to the intact kinematic state . double - row suture anchor repair resulted in a similar plane of elevation as that observed in the intact state ( p = 1.00 ) . no differences in the plane of elevation were observed between the two repairs ( p = 0.10 ) . both single- and double - row repairs resulted in increased variability in the change of the internal - external rotation angle of the humerus [ figure 3 ] , but no statistically significant differences were observed . likewise , the position of the humeral head was not altered as a result of the repairs . no differences were observed in the internal - external rotation angle , superior - inferior position , and anterior - posterior position the plane of elevation exhibited was altered between the intact and the simulated tear states ( p = 0.01 ) [ figure 2 ] . the 2 cm defect resulted in posterior angulation of the plane of elevation through the arc of abduction . this was greatest at the mid - portion of abduction ( 40-60 ) with an overall average of 8 posterior shift when compared to the intact kinematic state . the angle of internal - external rotation of the humerus was not affected by the simulated tear [ figure 3 ] . change in plane of glenohumeral elevation from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in internal - external rotation angle from the intact state as a function of elevation angle for all states . mean ( 1 sd ) for all specimens presented for 30 to 80 of elevation change in ( a ) superior - inferior and ( b ) anterior - posterior position of the humeral head from the intact state as a function of elevation angle for all states . this was true of both the single row suture anchor repair ( p < 0.001 ) and the double - row suture anchor repair ( p = 0.001 ) . single- and double - row suture anchor repairs were able to restore the intact kinematic state of the glenohumeral joint for 2 cm rotator cuff defects ( p = 0.10 ) [ figure 2 ] . single row suture anchor repair resulted in a slightly anterior angulation of the plane of elevation on average when compared to the intact kinematic state . double - row suture anchor repair resulted in a similar plane of elevation as that observed in the intact state ( p = 1.00 ) . no differences in the plane of elevation were observed between the two repairs ( p = 0.10 ) . both single- and double - row repairs resulted in increased variability in the change of the internal - external rotation angle of the humerus [ figure 3 ] , but no statistically significant differences were observed . likewise , the position of the humeral head was not altered as a result of the repairs . no differences were observed in the internal - external rotation angle , superior - inferior position , and anterior - posterior position over the last decade , arthroscopic shoulder techniques and equipment have improved the ability to perform minimally invasive repairs to the rotator cuff tendons . the literature has demonstrated that single- and double - row repairs are safe and effective . the purpose of this study , unlike prior biomechanical studies that examined the relative strength double - row cuff repair , was to illustrate the kinematic effects of single- and double - row suture anchor repairs of 2 cm supraspinatus tears on simulated active glenohumeral joint kinematics . the effects of a simulated 2 cm defect in the supraspinatus tendon in this study were similar to the results found previously by kedgley et al . as theorized , no changes were found in the internal - external rotation of the humerus or the position of the humeral head with elevation , though the 2 cm defect did result in an alteration in the plane of elevation with a mean change of 8 posteriorly . these results would perhaps be expected ; however , compensation likely occurs in - vivo with a medium - sized tear such as this . our observations did not demonstrate any significant differences in the plane of elevation between single- and double - row repairs . , in a retrospective cohort study , compared the functional and structural outcomes of single - row versus double - row rotator cuff repair . at a follow - up 35 months , no statistically significant differences in clinical outcome could be appreciated between the two different repair techniques . , in a randomized controlled clinical trial , compared 30 patients who received a single - row repair and 30 patients who received a double - row repair . at two years follow - up , there was no clinically significant difference between the two different repair techniques . first , all rotator cuff repairs were performed with suture anchors that create focal sites of tendon reattachment to the greater tuberosity . in an in - vitro study , integration of the tendon to the tuberosity does not occur , and restoration of normal force distribution from a pennate muscle across the tendon - bone interface is unlikely . a 2 cm defect may behave differently in a small specimen than in a large specimen and may have affected our ability to detect a change following the simulated tears . simulating tears that are proportional to the size of the specimen rather than based on an absolute dimension may have resulted in more physiologically relevant results , although they should not have influenced the comparison of the repair types . this supports the generalized trend toward more accurate restoration of the intact glenohumeral kinematics was seen in double - row suture anchor repair for a 2 cm defect of the supraspinatus . in contrast , this study examined the kinematics of an acute lesion , and repair of the rotator cuff and may not accurately reflect the more common clinical scenario seen in patients with chronic rotator cuff disease . however , had the specimens already exhibited pathology of the rotator cuff , we would have been unable to compare the repaired and intact states . fourth , this study simulated a chronic clinical scenario by creating an acute defect in the rotator cuff to mimic tendon retraction . shortening the tendon may have inadvertently created the anterior angulation seen in the plane of elevation with our suture anchor repairs . however , there are also theories that a biochemical mediator may cause a damaged rotator cuff to retract . similarly , the re - attachment of the tendon most likely imposed the observed increased variability in the internal - external rotation angle of the humerus . unfortunately , the use of a deltoid split to produce the rotator cuff tear may not represent the same biomechanics , as that which would be observed had an arthroscopic approach been taken . however , no deltoid failures occurred during testing , and a previous study showed very little alteration of glenohumeral kinematics with a 1 cm rotator cuff tear created in the same way . the paired , randomized , and controlled nature of this study did generate results of significant merit . furthermore , a unique , validated shoulder simulator capable of simulating active glenohumeral joint motion using a physiologically - based muscle loading protocol was employed . in summary , a 2 cm defect in the supraspinatus alters the kinematics of the glenohumeral joint by moving the humeral plane of elevation posteriorly . both single- and double - row repair techniques resulted in restoration of normal kinematics when compared to the intact state . we did not find a significant difference in glenohumeral kinematics between the two repair techniques . with an improved understanding of the kinematics of the glenohumeral joint

amines and their derivatives are frequently added to engine oils for anticorrosive or lubricating purposes . however , the nature of their interactions with the surfaces that they protect remains unclear , particularly when used in conjunction with other additive species , such as fatty acids or zinc dialkylthiophosphates ( zddps ) . for example , it remains open to debate whether they directly bind to the surface or are used rather to improve the binding of other additives . such questions remain unresolved because of the inability of traditional methods to study these additives in situ and to construct a molecular - level model of the additive interaction with the surface . in this work , we combine a number of surface - specific and surface - sensitive techniques to address the detailed adsorption behavior and surface molecular structure of a model additive , 1-hexadecylamine adsorbed from hexadecane onto an iron surface . zhu et al . demonstrated that 1-hexadecylamine in a tetradecane system between two mica surfaces shows similar performance to a commercial automatic transmission fluid in terms of the hard - wall thickness and rheological effects under shear . the amine molecules were reported to form monolayers at concentrations as low as 0.1 wt % in the oil , with film thicknesses of around 18 , separated by a small layer of oil and excess amine , as measured using a surface force balance . neutron reflectometry ( nr ) is a well - established technique for the study of the air / liquid and solid / liquid interfaces . in this approach , the generated reflectivity profile , obtained by reflecting a neutron beam at grazing angles close to the critical angle , has features that depend upon structural parameters of the adsorbed species , such as layer composition , thickness , and roughness . here , in a modification of the standard approach , polarized neutron reflectometry ( pnr ) has been used ; neutrons polarized either parallel ( + ) or antiparallel ( ) ( up- or down-spin ) to an external magnetic field interact differently with magnetic materials , such as iron ( magnetic moment , = 2.2 b ) . for magnetic materials that are magnetized in the plane of the surface using an external field , the neutron refractive index , n the measured reflectivity , r , is spin - dependent , and the refractive index must be written as a combination of the nuclear ( nnuclear ) and magnetic ( nmagnetic ) contributionswhere n is the number density , c is a constant ( 0.265 10 b cm ) , b is the scattering length , and is the moment per atom . when two separate reflectivity profiles are acquired for the same sample using up- and down - spin neutrons successively , two independent data sets are obtained from the same sample . this additional information is important when extracting the structural model of the interface by data fitting , because the number of possible models that can be fitted to both sets of data is significantly reduced . the scattering power of each layer at the interface is quantified in the scattering length density ( sld ) . the slds for the materials used in this work are given in table 1 . the model - fitting technique is a recognized method used to analyze reflectivity data . the interfacial region is divided into a finite number of layers , each characterized by a thickness , d , scattering length density , , and roughness , from which the reflectivity is calculated using the optical matrix method . a flat background is generally added to each point , and instrumental resolution may also be taken into consideration in the calculations . the calculated reflectivity is then compared to the experimental data , and the goodness of the fit is evaluated in terms of :where n is the number of data points , rf and rm are the fitted and measured reflectivities , respectively , and is the error bar associated with the measured reflectivity . the value of is minimized by the least - squares routine . each parameter ( number of layers , thickness , etc . ) can be kept fixed or varied , and the reflectivity is calculated from the model until the value for has reached a minimum . the optimum fitted parameters combine to give the sld profile of the sample , which represents the variation in composition perpendicular to the interface . from the fitted reflectivity data , the adsorbed amount in the layer , , may be calculated using the following formula : where ibi is the sum of the coherent scattering lengths for each species and na is avogadro s number . the reflectivity data can not generally be directly inverted to give a unique structural solution because of the usual phase problem between reciprocal and real space . when fitting the model , we aim to find structural solutions consistent with the experimental data ( within the experimental error ) , but often more than one structural model may be consistent with a given set of data . this results in an effective error bar for the experimentally determined structural parameters . we have previously characterized the adsorption behavior of palmitic acid , the carboxylic acid analogue to the amine studied here , using pnr . a monolayer of the acid , with a layer thickness of 16 ( 5 ) was identified , with an additional diffuse layer in the surrounding oil . the amount of adsorbed acid was estimated from the scattering length density of the fitted layer , which indicated that full monolayer coverage was attained by a concentration of 1000 ppm ( 3.9 10 mol dm ) . in sum - frequency generation spectroscopy ( sfg ) , another surface - specific technique , a vibrational spectrum of the interfacial molecules , is obtained by spatially and temporally overlapping a fixed visible and tunable infrared ( ir ) laser beam to generate an output laser beam with frequency equal to the sum of the two incident beams . the high energy of the laser beams gives rise to nonlinear optical effects , in particular the second - order susceptibility , , which will only have a non - zero value in a non - centrosymmetric environment . hence , importantly for surface studies , there is no signal from the bulk material , and only the interface , where the symmetry is broken and has a non - zero value , is specifically investigated . the sfg technique can be used to extract several interfacial parameters , including the tilt angle of various moieties within the molecule ( most frequently methyl or methylene groups ) to the surface normal , molecular conformation , and polar orientation . these may be determined by variation of the polarization combinations of the incident and reflected beams . the non - resonant contribution present for metal surfaces can be used to unambiguously identify the absolute orientation of a molecule on a surface . depletion isotherms are used to quantitatively determine the adsorbed amount of a species as a function of the solution concentration . in this technique , solutions of different initial adsorbate concentrations are added to a high - surface - area powdered substrate and allowed to equilibrate . adsorption leads to a reduction of the adsorbate concentration in the solution , which may then be measured , in this case , using fourier transform infrared ( ftir ) spectroscopy . in many cases , the data can be best interpreted in terms of the langmuir isotherm , where adsorption increases rapidly until monolayer coverage is attained . x - ray photoelectron spectroscopy ( xps ) was used to characterize the nature of the iron surface to determine the metal surface composition and oxidation state of the iron . the iron oxide substrates used for pnr and sfg experiments were sputtered to a thickness of 200 onto a polished silicon substrate , ( 111 ) orientation , ( n ) type , with a diameter of 55 mm and thickness of 5 mm , at the helmholtz zentrum , berlin , germany , using reactive magnetron sputtering . the iron oxide powder was purchased from sigma - aldrich ( > 99% purity , 69.7% fe , determined by titration ) , and brunauer emmett teller ( bet ) surface area ( determined by n2 adsorption fitted to the bet isotherm equation ) was 4.15 m g. hexadecylamine [ 98.1% , determined by gas chromatography ( gc ) ] and dodecane ( 97.1% , determined by gc ) were also purchased from sigma - aldrich . deuterated hexadecane [ 99.2% , determined by proton nuclear magnetic resonance ( h nmr ) ] was purchased from cambridge isotopes laboratories , inc . , and deuterated hexadecylamine ( 99.2% , determined by gc ) was purchased from qmx laboratories . they were used without further purification . a solution depletion isotherm was measured by tumbling samples of 10 ml of hexadecyl - d33-amine in dodecane ( over the concentration range of 0.03.5 10 mol dm ) with iron oxide powder ( 0.5 g ) . samples were allowed to equilibrate over 24 h , before centrifuging to separate the solid from the supernatant , before analysis of the final concentration by integration of peaks in the c d stretching region of the ir spectra compared to a set of calibration standards . the pnr experiments were conducted on the polref instrument at isis , rutherford appleton laboratory , u.k . briefly , a pulsed neutron beam from the spallation target is directed toward the sample at grazing angles and collimated by slits . polarized neutrons were used to provide extra contrasts ; polarization was achieved by the use of a transmission polarizer , current sheet , and mirror polarizer . the neutron polarization was maintained by a series of permanent magnets ( approximately 0.03 t ) along the flight path . the reflection data were collected at three incident angles of 0.25 , 1.5 , and 2.5 by tilting the sample in the beam . experimental setup for the pnr : ( a ) side view schematic of the neutron reflectometry cell and ( b ) top view of the polarized neutron reflection . the deuterated oil was pipetted directly onto a roughened silicon surface , and the sputtered iron surface [ cleaned using ultraviolet ( uv)/ozone ] was placed over this , separated from the base by a polytetrafluoroethylene ( ptfe ) o - ring , to create a thin oil layer and held in place by means of a heavy weight . the lower silicon surface was roughened to minimize unwanted reflection from this surface and additional background which might arise . the iron substrate was characterized by pnr under air and in hexadecane - d34 . solutions of hexadecylamine in the deuterated hexadecane were then added to the experimental cell in increasing concentrations , and reflectivity profiles were measured with both polarization states . the sfg measurements were taken using an ekspla picosecond spectrometer ( 30 ps pulses at 20 hz ) at the department of chemistry at the university of cambridge . the same iron - sputtered silicon substrates were used as for the pnr experiments and also cleaned using uv / ozone , after which no hydrocarbon residue remained . ppp and ssp ( where the order is sum - frequency , visible , and ir ) polarization combinations over the c although the n h stretching region ( 35003300 cm ) was examined , no peak of significant intensity was observed and reference to the ftir and raman spectra of the amine showed overlap of only one peak at 3400 cm , which had significantly lower intensity in the raman compared to the c h stretches . ppp spectra were also recorded for the oppositely deuterated system , i.e. , hexadecyl - d33-amine in protonated hexadecane , over both the c h and c d regions ( 20002300 cm ) . a co - propagating geometry was used , with angles of 53 and 60 to the surface normal for the ir and visible laser beams , respectively . the spectra were normalized to the product of the ir and visible beam intensities using the ekspla normalization facility . xps measurements were taken at the nexus laboratory in newcastle using the axis nova xps spectrometer . small silicon wafers , 1 1 cm , were sputtered with iron using magnetron sputtering and cleaned for 30 min using uv / ozone . both a bare substrate , i.e. , under air , and a substrate treated with a sample of 4.25 10 mol dm hexadecylamine in dodecane before being allowed to dry were analyzed , with three measurements taken from each sample to ensure representative data . fe 2p and c 1s spectra were recorded from the bare substrate to characterize the oxide layer and cleanliness of the surface , respectively , and n 1s spectra were additionally recorded for the sample treated with the amine sample , to assess the n surface interactions , if any , resulting from the presence of the nitrogen - containing additive . an isotherm of hexadecyl - d33-amine measured using the depletion method is given in figure 4 . the data exhibit a rapid rise in adsorption at low concentrations before reaching a plateau of approximately 3 10 mol m at higher concentrations . bet measurements of the powder give a specific surface area of 4.15 m g. hence , the adsorption at the plateau in the isotherm corresponds to each amine molecule occupying 55 on the substrate surface . this is reasonable given that the amine headgroup surface area is expected to be about 20 , allowing for space to accommodate slightly bulkier alkyl chains . the most common cleavage plane of the iron oxide ( -fe2o3 ) surface is the hexagonal ( 0001 ) plane , with a unit cell size of 5 ( as reported by shaikhutdinov et al . ) between the uppermost iron atoms on the surface . considering the size of the amine molecule , an adsorption model in which each of these fe sites is occupied seems implausible given the extent of crowding this would entail ; if each site were occupied , the area per molecule would be 21.7 , assuming a perfect homogeneous surface across the entire powder sample . a more disperse model ( e.g. , with alternate sites occupied ) therefore seems more probable . adsorption at the solid / solution interface is an exchange process and can be described by a number of models , particularly those of everett . however , where the adsorbate adsorbs strongly this adsorption may be approximated by the langmuir model . figure 2a presents the neutron reflection data from the substrate under deuterated hexadecane compared to the data from the surface when exposed to 1.25 10 mol dm hexadecylamine in deuterated hexadecane . the figure shows data obtained with both up- and down - spin neutron polarizations , clearly demonstrating the pronounced difference in reflectivity obtained for each , most markedly in the down - spin profiles . the iron - sputtered substrate was initially measured under air and then under the deuterated solvent to identify and characterize the metal and oxide layers present . the up - spin data exhibit a series of fringes , the spacing of which are inversely related to the thicknesses of the surface layers . the gradients of the reflectivity curves are determined by roughnesses at each interface . from a careful fitting of these data using the polly software , the iron layer thickness of 202 corresponded to that estimated by xrr data provided by the helmholtz zentrum , berlin , germany , where the substrates were sputtered . ( a ) reflectivity data obtained with both up- and down - spin neutrons for the bare substrate under dodecane ( ) and with the addition of 1.25 10 mol dm amine in deuterated solvent ( ) . ( b ) down - spin reflectivity curves for solvent only ( ) , with the addition of 1.25 10 mol dm amine ( ) , and with the addition of 4.25 10 mol dm amine ( ) in the deuterated solvent . for clarity , each of these profiles is shifted by a factor of 10 . the magnetic behavior of the iron and iron oxide layers is also contained in the reflectivity data and can be estimated by fitting . here , the magnetic moment , , of the oxide layer ( = 2.0 b ) was intermediate between fe2o3 ( non - magnetic , = 0.0 b ) and fe3o4 ( magnetic , = 4.1 b ) , suggesting that the layer consists of a mixture of the two oxides . examples of mixtures of such oxides have been reported for the sputtered iron layers in the literature . the addition of increasing concentrations of hexadecylamine in hexadecane - d34 led to changes in the reflectivity profiles generated , indicating that the amine molecules were interacting with the iron surface and adsorbing . the greatest changes were seen in the down - spin data , as expected . in figure 3 , the simulated sld profile is depicted schematically along the vertical z axis passing through the interface . for the down - spin simulation , there is only one major contrast , related to the adsorbed layer , whereas the presence of a larger contrast ( between the iron and silicon ) for the up - spin simulation causes the up - spin neutrons to be less sensitive to changes in the adsorbed layer . ( a ) for the up - spin neutrons , the contrast relating to the thin adsorbed layer is swamped by that between the iron and underlying silicon , ( b ) whereas there is only one major contrast for the down - spin neutrons ( shown by the bold arrows ) . examples of the down - spin data are shown in figure 2b where reflectivity profiles obtained with down - spin neutrons for the two highest concentrations , 1.25 10 and 4.25 10 mol dm amine , are compared to that for the substrate under the deuterated solvent only . models fitted to the experimental data for each concentration gave a layer thickness for the adsorbed amine of 16 ( 3 ) at the two lower concentrations ( 1.03 10 and 4.18 10 mol dm ) and 20 ( 3 ) as the concentration was increased to 1.25 10 and 4.25 10 mol dm ( the error in the layer thickness was estimated from the minimum and maximum thicknesses over a range of slds that could be fitted to the data ) . the variation in layer thickness with concentration ( from 16 to 20 ) is of the order of the experimental uncertainty , and hence any changes on layer thickness are rather modest . the extended hexadecylamine chain length is expected to be 21.5 , suggesting that the measured layer thickness is reasonable . the difference is proposed to arise from one or more of several possibilities ; the amine molecules may sit tilted on the surface or may extend into the porous oxide surface , or conformation disorder in the chains may exist , producing a slightly thinner layer than the fully extended chain . at higher concentrations , the increased adsorption appears to have led to some reorientation of the alkyl chains . the scattering length densities of the amine layer were also fitted and found to decrease with an increasing amine concentration . it is expected that the ratio of amine to solvent in the adsorbed layer will increase with more amine adsorption , and the sld should therefore tend from that of hexadecane - d34 , 6.717 10 , toward that of hexadecylamine , 0.260 10 , as observed . the fitted values are shown in table 2 , also expressed as percent coverage of the two components ( deduced from the amine / solvent ratio needed to attain the fitted scattering length density ) , along with the corresponding adsorbed amounts ( calculated assuming the layer thickness of 16 and molecular volumes of 5.127 10 and 4.867 10 m for the amine and solvent , respectively ; these were calculated from molecular weights of 241.46 and 259.97 g mol and bulk densities of 0.782 and 0.887 g ml for the amine and solvent , respectively ) . the concentration dependence of the reflection indicates that the amine shows a relatively high affinity for the iron surface even at low concentrations , reaching a coverage of around 50% ( in terms of the amine / solvent ratio calculated from the fitted sld ) at saturation . the adsorbed amounts determined by pnr have been plotted alongside the depletion isotherm data in figure 4 . while the curve shapes are very similar , with the half - maximum concentrations being the same for both sets of data , the pnr data show a plateau value significantly higher than that of the depletion isotherm , giving an area per amine molecule of around 30 , a value somewhat less than that given by the solution isotherms outlined above . these include the surface roughness of the iron oxide layer in the neutron reflection experiments , because the adsorbed amounts are calculated assuming a completely flat surface . however , as reported above , the surface roughness is significant ( approximately 10 ) , and hence the actual surface area is larger than the geometric area of the wafer . simple calculations based on the experimentally determined roughness indicate that this effect can account for the difference in adsorption between the isotherm and neutron reflection approaches . another potential contributing factor may be differences in the nature and composition of the oxide layer for the powder and sputtered thin film . comparison of the pnr ( ) and depletion isotherm ( ) data . h stretching region ( 28003000 cm ) for hexadecylamine adsorbed onto iron from hexadecane - d34 with the two polarization combinations ppp and ssp . there are five resolvable bands in the ppp spectrum , which are assigned to the methylene symmetric stretch ( d ) at 2847 cm and its associated fermi resonance at ca . 2900 cm , the methyl symmetric stretch ( r ) at 2872 cm with its fermi resonance at 2936 cm , and the methyl antisymmetric stretch ( r ) at 2964 cm . it is immediately apparent that the methyl fermi resonance is disproportionately strong in the spectra for both polarization combinations . because the fermi resonance draws intensity from the fundamental , its intensity should be no greater than equivalent to the fundamental band with which it is associated . in this case , the same effect has previously been reported by zhang et al . for a mild steel surface on which octadecane thiol has been self - assembled . in their paper , the increased intensity of the fermi resonance is attributed to the presence of iron oxide . because our surface is predominately composed of an oxide layer , as will be demonstrated by the xps data discussed subsequently , this strengthening of the fermi resonance band may also be attributed to the iron oxide film . because interpretation of the sfg spectra relies heavily on the relative intensities of the methyl and methylene bands , the ppp spectrum of deuterated hexadecylamine in the cd stretching region was also recorded ( figure 5b ) . this spectrum is markedly different from that observed in the ch region , being free from any distortion in band intensities arising from the fermi resonance . this region is dominated by the methyl stretching bands at 2070 , 2123 , and 2217 cm , corresponding to r , rfr+ , and r , respectively , with only two small shoulders apparent at 2098 and 2197 cm , corresponding to the d and d bands , respectively . h stretching region for ppp ( top ) and ssp ( bottom ) combinations . the amine concentration in both cases is 4.25 10 mol dm . from both qualitative inspection of the spectra and comparison of the modeled spectral intensities , it is clear that the methylene symmetric stretching peaks ( d , at 2846/2098 cm ) in the ppp spectra are of almost negligible intensity compared to the peaks that correspond to the terminal methyl group stretches ( at 2871/2070 , 2935/2123 , and 2964/2117 cm for the r , rfr+ , and r peaks , respectively , in the ch and cd regions ) . this occurs when the alkyl chain conformational order is predominately all trans and the methylene modes become locally centrosymmetric and , hence , sfg - inactive , and it is therefore concluded that , although a small number of gauche defects are present , the adsorbed layer shows a high degree of conformational order . this conclusion concerning the conformational order in the alkyl chains is supported by the work of miranda et al . , who report the sfg spectra of dioctadecyl dimethyl ammonium chloride ( doac ) from solvents of varying polarity onto quartz . for polar solvents , a high level of disorder was observed , but when a nonpolar solvent of comparable molecular length to doac was used , hexadecane , the monolayer formed was well - ordered . the authors attribute this increased ordering to interdigitation of the solvent molecule with the adsorbate to form a tightly packed layer . the fitted sld values observed in our neutron data also indicate that a significant proportion of solvent is present in the monolayer ; it seems probable therefore that a similar process of interdigitation occurs here . sfg spectra recorded in the ch region for the deuterated amine in hexadecane failed to produce any significant spectra for hexadecane . this is most likely due to a combination of the reduced ir intensity on the metal surface because of absorption by the bulk oil and the presumed near all - trans conformational order of the interdigitated hexadecane , which would result in a centrosymmetric and , therefore , sfg - inactive hexadecane layer . the polar orientation of the molecules on the surface has been inferred from a comparison of the spectra presented here to spectra reported in the literature of odt on mild steel , in which the adsorbed monolayer has a known orientation ; the signals attributed to methyl group stretches all appear as peaks with a modeled phase of 43 in close agreement with the 45 previously reported , suggesting that the terminal methyl groups are pointing away from the surface , and hence , the adsorbed layer sits with the amine headgroup oriented toward the iron surface , a result that is supported by the xps data that show evidence of a nitrogen adsorption peak . the tilt angle of the terminal methyl group to the surface normal was calculated by comparison of the ratio of symmetric and antisymmetric peak intensities in the ppp spectra to the theoretically simulated value derived from the method proposed by zhang et al . , found according towhere l and k are the nonlinear and linear fresnel factors , respectively , is the second - order susceptibility , and ep / s is the surface electric field for the p- or s - polarized light . because can be directly related to the tilt angle , , this permits the plotting of a curve of the ratio of intensities for the two peaks as a function of , from which the experimentally derived ratio may be used to determine the methyl tilt angle relative to the surface normal . this approach gave a value for the methyl group inclination of 32. however , this tilt angle should be treated as an estimate only given that the monolayer contains at least some gauche defects . from simple geometric calculations using the layer thickness value of 20 ( 3 ) at saturation determined by pnr and a chain length of 21.5 , a tilt angle of 22 to the surface normal is predicted . the fe 2p spectrum from the iron - sputtered silicon wafer is shown in figure 6a ; the broad peak at around 710 ev may be resolved into two peaks at 711.8 and 709.9 ev , which is consistent with the presence of the two types of iron oxide , fe2o3 and fe3o4 . many examples of peaks around this region exist for both oxides , and therefore , it is unsurprising that they merge in this instance . this suggestion of a mixture of oxides at the surface is consistent with the fitting of the magnetic moment seen for the pnr data . a small shoulder at 706.9 ev is attributed to the presence of unoxidized iron . the peak at 711.8 ev indicates that the main oxidation state is fe(iii ) , with a small amount of fe(ii ) at 709.9 ev , and some unoxidized iron present at 707.9 ev . ( b ) xps spectrum of the n 1s region for the amine - treated wafer . the fitted lines indicate the three different nitrogen environments at the peak positions shown . no change in the fe 2p region was observed for the sample treated with amine in dodecane ; this is unsurprising , because the proportion of iron affected by any reaction with the amine would be very small in comparison to the entire depth of iron analyzed ( on the order of around 100 ) . figure 6b , the n 1s spectrum for the sample treated with hexadecylamine / hexadecane , shows a broad peak that is not evident in the untreated substrate ; this may be resolved into three components , at 401.1 , 399.7 , and 397.2 ev ( with a ratio of intensities roughly 1:6:3 ) , which may be assigned by reference to expected peaks in various environments . the peak at 397.2 ev is presumed to result from amine that has not reacted with the surface but remains on the sample , free amine , because the lower binding energy indicates a higher electron density than the species that give rise to the remaining peaks . we were not able to completely remove any excess solution prior to measurement , and therefore , this free amine is taken to represent amine that was dried onto the surface during the xps measurements rather than amine adsorbed in the wet the peak at 399.7 ev corresponds well to what might be expected for a nitrogen atom donating electron density to the iron surface via its lone pair ( willenbruch et al . report a value of 399.8 ev for ammonia on an iron surface ) and , hence , is assumed to arise from the chemisorbed amine . the smallest peak , at 401.1 ev , has a rather high energy that indicates a more positively charged amine . this may arise from the interaction with negatively charged surface oxygen atoms ; weng et al . small water impurities present in the solvent may account for this protonation of the amine group . the low peak intensity relative to the other peaks indicates that this species would be present in far lower concentrations . experimental methods to characterize an iron / iron oxide surface and the adsorption behavior of an alkyl amine from oil thereupon have been presented in this work . all techniques used demonstrate the binding of the hexadecylamine species to the iron oxide surface . the xps data indicate chemisorbed amine , where the nitrogen is donating electron density to the surface iron(iii ) ions . the strong affinity of the amine for the surface is also observed in adsorption isotherms using solution depletion and pnr methods , in both of which the adsorbed amount is seen to increase steeply with the concentration , resulting in a high plateau coverage . the sfg spectra demonstrate that the methylene groups are in an all - trans configuration with the polar headgroup directed toward the surface . several parameters that characterize the adsorbed layer were identified ; from the pnr data fitting , a modest increase in layer thickness from 16 to 20 ( 3 ) was observed as the amine concentration was increased , slightly above the experimental precision . the molecular tilt angle was also calculated both using this pnr value and also from consideration of the sfg peak intensity ratios . however , the latter was considered unreliable because of the unexpected enhancement of the fermi peak intensity ; therefore , the tilt angle of the methyl group is considered most likely to fall into the 3050 range , relative to the surface normal . a depiction of the model for hexadecylamine adsorption to the iron oxide surface is shown in figure 7 . the molecules are considered to sit fully extended on the surface in a well - ordered layer , whereby the amine nitrogens donate electron density to the iron cations that sit on top of the iron oxide ( 0001 ) plane . from the xps nitrogen spectra , it seems possible that a small proportion of semi - positive amine molecules that interact electrostatically with the surface oxygen atoms may also be found in the adsorbed layer . the light gray circles represent surface oxygen atoms , and the smaller black circles represent the topmost layer of iron atoms .

the adsorption behavior of a model additive , hexadecylamine , onto an iron surface from hexadecane oil has been characterized using polarized neutron reflectometry , sum - frequency generation spectroscopy , solution depletion isotherm , and x - ray photoelectron spectroscopy ( xps ) . the amine showed a strong affinity for the metal surface , forming a dense monolayer at relatively low concentrations ; a layer thickness of 16 ( 3 ) at low concentrations , increasing to 20 ( 3 ) at greater amine concentrations , was determined from the neutron data . these thicknesses suggest that the molecules in the layer are tilted . adsorption was also indicated by sum - frequency generation spectroscopy and xps , the latter indicating that the most dominant amine surface interaction was via electron donation from the nitrogen lone pair to the positively charged iron ions . sum - frequency generation spectroscopy was used to determine the alkyl chain conformation order and orientation on the surface .

Introduction Experimental Section Results and Discussion Conclusion

in this work , we combine a number of surface - specific and surface - sensitive techniques to address the detailed adsorption behavior and surface molecular structure of a model additive , 1-hexadecylamine adsorbed from hexadecane onto an iron surface . in this approach , the generated reflectivity profile , obtained by reflecting a neutron beam at grazing angles close to the critical angle , has features that depend upon structural parameters of the adsorbed species , such as layer composition , thickness , and roughness . here , in a modification of the standard approach , polarized neutron reflectometry ( pnr ) has been used ; neutrons polarized either parallel ( + ) or antiparallel ( ) ( up- or down-spin ) to an external magnetic field interact differently with magnetic materials , such as iron ( magnetic moment , = 2.2 b ) . for magnetic materials that are magnetized in the plane of the surface using an external field , the neutron refractive index , n the measured reflectivity , r , is spin - dependent , and the refractive index must be written as a combination of the nuclear ( nnuclear ) and magnetic ( nmagnetic ) contributionswhere n is the number density , c is a constant ( 0.265 10 b cm ) , b is the scattering length , and is the moment per atom . a flat background is generally added to each point , and instrumental resolution may also be taken into consideration in the calculations . the calculated reflectivity is then compared to the experimental data , and the goodness of the fit is evaluated in terms of :where n is the number of data points , rf and rm are the fitted and measured reflectivities , respectively , and is the error bar associated with the measured reflectivity . from the fitted reflectivity data , the adsorbed amount in the layer , , may be calculated using the following formula : where ibi is the sum of the coherent scattering lengths for each species and na is avogadro s number . we have previously characterized the adsorption behavior of palmitic acid , the carboxylic acid analogue to the amine studied here , using pnr . a monolayer of the acid , with a layer thickness of 16 ( 5 ) was identified , with an additional diffuse layer in the surrounding oil . in sum - frequency generation spectroscopy ( sfg ) , another surface - specific technique , a vibrational spectrum of the interfacial molecules , is obtained by spatially and temporally overlapping a fixed visible and tunable infrared ( ir ) laser beam to generate an output laser beam with frequency equal to the sum of the two incident beams . hence , importantly for surface studies , there is no signal from the bulk material , and only the interface , where the symmetry is broken and has a non - zero value , is specifically investigated . the sfg technique can be used to extract several interfacial parameters , including the tilt angle of various moieties within the molecule ( most frequently methyl or methylene groups ) to the surface normal , molecular conformation , and polar orientation . depletion isotherms are used to quantitatively determine the adsorbed amount of a species as a function of the solution concentration . x - ray photoelectron spectroscopy ( xps ) was used to characterize the nature of the iron surface to determine the metal surface composition and oxidation state of the iron . samples were allowed to equilibrate over 24 h , before centrifuging to separate the solid from the supernatant , before analysis of the final concentration by integration of peaks in the c d stretching region of the ir spectra compared to a set of calibration standards . polarized neutrons were used to provide extra contrasts ; polarization was achieved by the use of a transmission polarizer , current sheet , and mirror polarizer . experimental setup for the pnr : ( a ) side view schematic of the neutron reflectometry cell and ( b ) top view of the polarized neutron reflection . the deuterated oil was pipetted directly onto a roughened silicon surface , and the sputtered iron surface [ cleaned using ultraviolet ( uv)/ozone ] was placed over this , separated from the base by a polytetrafluoroethylene ( ptfe ) o - ring , to create a thin oil layer and held in place by means of a heavy weight . solutions of hexadecylamine in the deuterated hexadecane were then added to the experimental cell in increasing concentrations , and reflectivity profiles were measured with both polarization states . ppp and ssp ( where the order is sum - frequency , visible , and ir ) polarization combinations over the c although the n h stretching region ( 35003300 cm ) was examined , no peak of significant intensity was observed and reference to the ftir and raman spectra of the amine showed overlap of only one peak at 3400 cm , which had significantly lower intensity in the raman compared to the c h stretches . a co - propagating geometry was used , with angles of 53 and 60 to the surface normal for the ir and visible laser beams , respectively . fe 2p and c 1s spectra were recorded from the bare substrate to characterize the oxide layer and cleanliness of the surface , respectively , and n 1s spectra were additionally recorded for the sample treated with the amine sample , to assess the n surface interactions , if any , resulting from the presence of the nitrogen - containing additive . bet measurements of the powder give a specific surface area of 4.15 m g. hence , the adsorption at the plateau in the isotherm corresponds to each amine molecule occupying 55 on the substrate surface . figure 2a presents the neutron reflection data from the substrate under deuterated hexadecane compared to the data from the surface when exposed to 1.25 10 mol dm hexadecylamine in deuterated hexadecane . the up - spin data exhibit a series of fringes , the spacing of which are inversely related to the thicknesses of the surface layers . from a careful fitting of these data using the polly software , the iron layer thickness of 202 corresponded to that estimated by xrr data provided by the helmholtz zentrum , berlin , germany , where the substrates were sputtered . the magnetic behavior of the iron and iron oxide layers is also contained in the reflectivity data and can be estimated by fitting . here , the magnetic moment , , of the oxide layer ( = 2.0 b ) was intermediate between fe2o3 ( non - magnetic , = 0.0 b ) and fe3o4 ( magnetic , = 4.1 b ) , suggesting that the layer consists of a mixture of the two oxides . the addition of increasing concentrations of hexadecylamine in hexadecane - d34 led to changes in the reflectivity profiles generated , indicating that the amine molecules were interacting with the iron surface and adsorbing . for the down - spin simulation , there is only one major contrast , related to the adsorbed layer , whereas the presence of a larger contrast ( between the iron and silicon ) for the up - spin simulation causes the up - spin neutrons to be less sensitive to changes in the adsorbed layer . ( a ) for the up - spin neutrons , the contrast relating to the thin adsorbed layer is swamped by that between the iron and underlying silicon , ( b ) whereas there is only one major contrast for the down - spin neutrons ( shown by the bold arrows ) . models fitted to the experimental data for each concentration gave a layer thickness for the adsorbed amine of 16 ( 3 ) at the two lower concentrations ( 1.03 10 and 4.18 10 mol dm ) and 20 ( 3 ) as the concentration was increased to 1.25 10 and 4.25 10 mol dm ( the error in the layer thickness was estimated from the minimum and maximum thicknesses over a range of slds that could be fitted to the data ) . the variation in layer thickness with concentration ( from 16 to 20 ) is of the order of the experimental uncertainty , and hence any changes on layer thickness are rather modest . the difference is proposed to arise from one or more of several possibilities ; the amine molecules may sit tilted on the surface or may extend into the porous oxide surface , or conformation disorder in the chains may exist , producing a slightly thinner layer than the fully extended chain . at higher concentrations , the increased adsorption appears to have led to some reorientation of the alkyl chains . it is expected that the ratio of amine to solvent in the adsorbed layer will increase with more amine adsorption , and the sld should therefore tend from that of hexadecane - d34 , 6.717 10 , toward that of hexadecylamine , 0.260 10 , as observed . the fitted values are shown in table 2 , also expressed as percent coverage of the two components ( deduced from the amine / solvent ratio needed to attain the fitted scattering length density ) , along with the corresponding adsorbed amounts ( calculated assuming the layer thickness of 16 and molecular volumes of 5.127 10 and 4.867 10 m for the amine and solvent , respectively ; these were calculated from molecular weights of 241.46 and 259.97 g mol and bulk densities of 0.782 and 0.887 g ml for the amine and solvent , respectively ) . the concentration dependence of the reflection indicates that the amine shows a relatively high affinity for the iron surface even at low concentrations , reaching a coverage of around 50% ( in terms of the amine / solvent ratio calculated from the fitted sld ) at saturation . while the curve shapes are very similar , with the half - maximum concentrations being the same for both sets of data , the pnr data show a plateau value significantly higher than that of the depletion isotherm , giving an area per amine molecule of around 30 , a value somewhat less than that given by the solution isotherms outlined above . these include the surface roughness of the iron oxide layer in the neutron reflection experiments , because the adsorbed amounts are calculated assuming a completely flat surface . however , as reported above , the surface roughness is significant ( approximately 10 ) , and hence the actual surface area is larger than the geometric area of the wafer . there are five resolvable bands in the ppp spectrum , which are assigned to the methylene symmetric stretch ( d ) at 2847 cm and its associated fermi resonance at ca . 2900 cm , the methyl symmetric stretch ( r ) at 2872 cm with its fermi resonance at 2936 cm , and the methyl antisymmetric stretch ( r ) at 2964 cm . in their paper , the increased intensity of the fermi resonance is attributed to the presence of iron oxide . because interpretation of the sfg spectra relies heavily on the relative intensities of the methyl and methylene bands , the ppp spectrum of deuterated hexadecylamine in the cd stretching region was also recorded ( figure 5b ) . from both qualitative inspection of the spectra and comparison of the modeled spectral intensities , it is clear that the methylene symmetric stretching peaks ( d , at 2846/2098 cm ) in the ppp spectra are of almost negligible intensity compared to the peaks that correspond to the terminal methyl group stretches ( at 2871/2070 , 2935/2123 , and 2964/2117 cm for the r , rfr+ , and r peaks , respectively , in the ch and cd regions ) . this occurs when the alkyl chain conformational order is predominately all trans and the methylene modes become locally centrosymmetric and , hence , sfg - inactive , and it is therefore concluded that , although a small number of gauche defects are present , the adsorbed layer shows a high degree of conformational order . this is most likely due to a combination of the reduced ir intensity on the metal surface because of absorption by the bulk oil and the presumed near all - trans conformational order of the interdigitated hexadecane , which would result in a centrosymmetric and , therefore , sfg - inactive hexadecane layer . the polar orientation of the molecules on the surface has been inferred from a comparison of the spectra presented here to spectra reported in the literature of odt on mild steel , in which the adsorbed monolayer has a known orientation ; the signals attributed to methyl group stretches all appear as peaks with a modeled phase of 43 in close agreement with the 45 previously reported , suggesting that the terminal methyl groups are pointing away from the surface , and hence , the adsorbed layer sits with the amine headgroup oriented toward the iron surface , a result that is supported by the xps data that show evidence of a nitrogen adsorption peak . the tilt angle of the terminal methyl group to the surface normal was calculated by comparison of the ratio of symmetric and antisymmetric peak intensities in the ppp spectra to the theoretically simulated value derived from the method proposed by zhang et al . , found according towhere l and k are the nonlinear and linear fresnel factors , respectively , is the second - order susceptibility , and ep / s is the surface electric field for the p- or s - polarized light . because can be directly related to the tilt angle , , this permits the plotting of a curve of the ratio of intensities for the two peaks as a function of , from which the experimentally derived ratio may be used to determine the methyl tilt angle relative to the surface normal . from simple geometric calculations using the layer thickness value of 20 ( 3 ) at saturation determined by pnr and a chain length of 21.5 , a tilt angle of 22 to the surface normal is predicted . this suggestion of a mixture of oxides at the surface is consistent with the fitting of the magnetic moment seen for the pnr data . the peak at 711.8 ev indicates that the main oxidation state is fe(iii ) , with a small amount of fe(ii ) at 709.9 ev , and some unoxidized iron present at 707.9 ev . ( b ) xps spectrum of the n 1s region for the amine - treated wafer . no change in the fe 2p region was observed for the sample treated with amine in dodecane ; this is unsurprising , because the proportion of iron affected by any reaction with the amine would be very small in comparison to the entire depth of iron analyzed ( on the order of around 100 ) . figure 6b , the n 1s spectrum for the sample treated with hexadecylamine / hexadecane , shows a broad peak that is not evident in the untreated substrate ; this may be resolved into three components , at 401.1 , 399.7 , and 397.2 ev ( with a ratio of intensities roughly 1:6:3 ) , which may be assigned by reference to expected peaks in various environments . the peak at 397.2 ev is presumed to result from amine that has not reacted with the surface but remains on the sample , free amine , because the lower binding energy indicates a higher electron density than the species that give rise to the remaining peaks . we were not able to completely remove any excess solution prior to measurement , and therefore , this free amine is taken to represent amine that was dried onto the surface during the xps measurements rather than amine adsorbed in the wet the peak at 399.7 ev corresponds well to what might be expected for a nitrogen atom donating electron density to the iron surface via its lone pair ( willenbruch et al . report a value of 399.8 ev for ammonia on an iron surface ) and , hence , is assumed to arise from the chemisorbed amine . experimental methods to characterize an iron / iron oxide surface and the adsorption behavior of an alkyl amine from oil thereupon have been presented in this work . the xps data indicate chemisorbed amine , where the nitrogen is donating electron density to the surface iron(iii ) ions . the strong affinity of the amine for the surface is also observed in adsorption isotherms using solution depletion and pnr methods , in both of which the adsorbed amount is seen to increase steeply with the concentration , resulting in a high plateau coverage . several parameters that characterize the adsorbed layer were identified ; from the pnr data fitting , a modest increase in layer thickness from 16 to 20 ( 3 ) was observed as the amine concentration was increased , slightly above the experimental precision . however , the latter was considered unreliable because of the unexpected enhancement of the fermi peak intensity ; therefore , the tilt angle of the methyl group is considered most likely to fall into the 3050 range , relative to the surface normal . the molecules are considered to sit fully extended on the surface in a well - ordered layer , whereby the amine nitrogens donate electron density to the iron cations that sit on top of the iron oxide ( 0001 ) plane . from the xps nitrogen spectra , it seems possible that a small proportion of semi - positive amine molecules that interact electrostatically with the surface oxygen atoms may also be found in the adsorbed layer .

ochratoxin a ( ota ) is a mycotoxin produced by several food - borne species of aspergillus and penicillium fungi . ota occurs in various food materials and therefore humans are continuously exposed to relatively small amounts of it . because of its wide occurrence and consequent exposure , together with a potent carcinogenic potential in animal models , ota has attracted significant public health attention over the last few years . several national and international food safety organizations and expert groups have conducted a thorough review of the situation as well as risk assessments in order to provide an insight on the health significance of ota in food . human epidemiology has been inconclusive : a number of studies have suggested a correlation between exposure to ota and balkan endemic nephropathy ( ben ) and mortality from urinary tract tumors [ 1 , 2 ] . epidemiological data were recently reviewed by several expert groups [ 36 ] . all concluded that causality between intake of ota and human nephropathy could not be established . therefore , the iarc statement that there is inadequate evidence for carcinogenicity in humans ( group 2b ) appears still valid . recently , other nephrotoxic agents have been put forward as the primary cause of ben [ 6 , 8 , 9 ] . in absence of adequate human data , kidney has been considered as the key target organ of ota toxicity . in all animal species studied , ota was found to produce renal toxicity , while in rodents renal carcinogenicity was clearly established . recently , ota renal and hepatic carcinogenicity was also observed in chicks . using a loael of 8 mcg / kg bw / day based on early markers of renal toxicity in pig ( the most sensitive animal species ) and applying an uncertainty factor of 450 , efsa allocated a tolerable weekly intake ( twi ) of 120 ng / kg bw . analysis of dietary exposure throughout europe revealed that the current average ota exposure ( 5060 ng / kg bw / week ) is well within the twi . the joint fao / who expert committee on food additives ( jecfa ) first evaluated ota at their 37th meeting . based on the loael in pig , and applying an uncertainty factor of 500 , jecfa allocated a provisional tolerable weekly intake ( ptwi ) of 112 ng / kg bw . this value was rounded to 100 ng / kg bw / week and confirmed in several subsequent meetings [ 12 , 13 ] . in 2008 , jecfa applied a benchmark dose ( bmd ) modeling approach using carcinogenicity data . the bmd is the dose estimated to cause a predefined increase ( e.g. , 10% for the bmd10 ) in tumor incidence over background . the bmdl is the lower limit of a one - sided 95% confidence interval of the bmd . the occurrence of combined adenomas and carcinomas in the kidneys of male rats was considered by jecfa to be the most appropriate data for modeling . values of 1833 mcg / kg bw / d and 1525 mcg / kg bw / day were calculated for , respectively , the bmd10 and bmdl10 . because the bmd approach did not provide a lower point of departure than the loael in pig , jecfa decided to reconfirm the ptwi of 100 ng / kg bw / day . a recent health risk assessment , performed by health canada recommends to regulate ota as a nonthreshold carcinogen , because of the uncertainties regarding the mode of action . the authors defined a negligible cancer risk intake ( ncri , risk level 1 : 100 000 ) using the tumorigenic dose at which 5% of rats are likely to develop tumors ( td05 , derived through modeling ) as point of departure . importantly , there is considerable convergence between the ncri established in this assessment and the tdi derived by efsa . in the risk assessment of carcinogenic substances , consideration of the mode of action ( moa ) is essential , determining the method to be applied in order to define levels of exposure below which a low safety concern is expected . the key events analysis framework of the moa has not yet been formally applied to ota . however , the approach used by most expert groups ( efsa , jecfa , ilsi ) to establish the safe level of exposure of ota ( based on uncertainty factors ) implies the consideration of key events compatible with a threshold effect . for these groups , amongst the mechanisms of action highlighted as possible , oxidative stress has been presented as one of the most probable [ 5 , 14 ] . over the last decades , studies aimed at elucidating the modes of action implicated in ota toxicity and carcinogenicity have been published . there has been considerable debate for many years over the genotoxicity of ota and its actual role in carcinogenicity [ 35 , 1115 , 17 , 18 ] . although genotoxicity is likely to play a role in ota carcinogenicity [ 3 , 4 ] , the actual molecular mechanism involved , either through covalent adduct formation , through other indirect modifications , or both is still unclear . the potential of ota to form covalent dna adducts has been subjected to debate due to conflicting data in the literature . using p - postlabelling analysis , large numbers of putative ota - derived dna adducts have been reported to be present in a wide range of tissues from ota - treated rats , mice as well as pigs [ 2 , 17 , 1922 ] . however , so far , these adducts have never been observed by any other highly specific techniques such as radioactivity measurements using h - labelled ota ( h - ota ) , accelerator mass spectrometry ( ams ) , or liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) . the present paper is not intended to provide a thorough review of the complex and controversial scientific literature on dna - adduct formation . however , it is important to keep in mind that dna adducts are increasingly considered as markers of exposures and not only of effects [ 26 , 27 ] and that dna - covalent binding does not necessarily determine the shape of the dose - response at low level of exposure [ 28 , 29 ] . according to mantle and coworkers [ 21 , 30 ] , experimental dose - response data for ota 's renal carcinogenesis makes a compelling case for ota being a thresholded carcinogen in male rat . in this context , it appears important to also consider other potential modes of action , which could potentially contribute to ota carcinogenicity . in the near future , the application of the mode of action framework will likely help to understand the individual contribution of all mechanisms described up to now for ota . the focus of the present short paper was to collect and highlight the evidence associated with a role for oxidative stress as a plausible mechanism to consider for ota . a list of the studies used to illustrate the main messages of the present paper is provided in table 1 . although not exhaustive , the list shows that over the last two decades , numerous investigators have documented the generation of oxidative stress as a result of ota treatment in both in vitro and in vivo model systems . production of reactive oxygen species ( ros ) leading to oxidative stress and macromolecular damage is known to contribute to the pathogenesis of age - related as well as chronic diseases including cancer [ 3135 ] . a number of studies are available documenting that ota is associated with the production of reactive oxygen species and resulting oxidative stress through various direct and indirect mechanisms . several oxido - reduction mechanisms elicited by ota have been proposed . in a reconstituted system consisting of phospholipid vesicles , the flavoprotein nadph - cytochrome p450 reductase and fe , ota was found to chelate ferric ions ( fe ) , facilitating their reduction to ferrous ions ( fe ) , which in the presence of oxygen , provided the active species initiating lipid peroxidation . results indicated that the hydroxyl radical was not involved in the process . a role for cytochrome p450 in this reaction in contrast , others found that ota induced oxidative damage through the generation of hydroxyl radicals . this reaction conducted with microsomes , in presence of nadph and o2 did not require exogenous fe . structure - activity studies have also suggested that the toxicity of ota may be attributable to its isocoumarin moiety and that the lactone carbonyl group may be involved in its toxicity . using a bacillus brevis model , hoehler 's et al . showed that ota behaved as a cell pro - oxidant through mobilization of fe and ca pathway leading to uncoupling oxidative phosphorylation and increased production of hydroxyl radical via the fenton reaction . however , in other studies using ota and structural analogs , a direct correlation between toxicity and iron chelating capacity was only partially supported . the generation of an ota hydroquinone / quinone couple from the oxidation of ota ( phenol oxidation ) by electrochemical , photochemical , and chemical processes was reported [ 39 , 40 ] . the quinone is thought to undergo reductions to form hydroquinone , postulated to be responsible for the formation of the glutathione conjugate of ota . such events are likely to result in redox cycling and in the generation of reactive oxygen species [ 20 , 40 , 41 ] . the formation of ota - derived quinones has been observed in cell cultures in vitro as well as in vivo [ 42 , 43 ] . ota was found to reduce the expression of several genes regulated by nuclear factor - erythroid 2 p45-relatetd factor ( nrf2 ) [ 4447 ] . this was observed at the rna and protein levels , both in vitro and in vivo test systems . nrf2 is involved in both the basal expression as well as in the induction of genes encoding detoxification , cytoprotective , and antioxidant enzymes [ 48 , 49 ] . a reduction of the expression of these genes is likely to result in decrease in antioxidant defenses leading to oxidative stress and macromolecular damage . this was confirmed with ota . through in vitro and in vivo studies , a correlation was observed between the ota - dependent reduction of the nrf2 pathway and an increased production of oxidative damage . in this context , it is interesting to note that ota was found to increase the expression of inducible nitric oxide synthase ( inos ) [ 5052 ] , an enzyme responsible for the production of nitric oxide ( no ) . an association between inos expression and the development of cancers was suggested in humans and animals in vivo . in excess , no may behave as a toxic radical producing nitrosative stress . no is known to react with oxygen anion radical superoxide to form the pro - oxidant peroxynitrite . under physiological conditions , peroxynitrite rapidly decomposes to generate a nitro radical intermediate leading to protein and dna nitration . ota was shown to stimulate protein and possibly dna nitration [ 52 , 53 ] indicating that ota exposure may be considered as a source of both oxygen and also reactive nitrogen radicals / species ( rns ) . ros , such as hydroxyl radicals and nitric oxide , are capable of forming oxidized dna bases that directly produce diverse types of dna damage [ 5456 ] . the oxidized dna bases appear to be capable of inducing mutations that are commonly observed in neoplasia . as illustrated in table 1 , diverse biomarkers have been analyzed showing that ota induces dna damage . in cell cultures , the ota - dependent production of ros was correlated with an increased formation of 8-oxoguanine [ 5759 ] . moreover , ota was shown to induce dna strand breaks as assessed by comet assay in liver , kidney , and spleen of f344 rats given 0 , 0.25 , 0.50 , 1.0 , and 2.0 mg / kg bw / day by gavage for 2 weeks . in liver and kidney , the extent of dna damage analyzed by comet assay was further enhanced in the presence of formamidopyrimidine glycosylase ( fpg ) , an enzyme involved in excision repair of oxidized dna bases [ 57 , 58 , 61 ] . another study detected dna - strand breaks ( using the comet assay ) in the kidney of female wistar rats treated intraperitoneally with ota ( 0.5 mg / kg bw / day for 7 , 14 , and 21 days ; n = 5 per group ) . the severity of the dna lesions in the kidney increased according to the ota dose and was at maximum after 21 days of treatment . other authors have observed oxidative dna - damage in various tissues of animals treated with a wide range of ota doses and treatment durations [ 46 , 60 , 63 , 64 ] . carbonylation of proteins occurs through a variety of oxidative pathways [ 54 , 6567 ] . carbonylation is an important protein modification associated with alterations of protein ( enzymes ) function , protein misfolding , protein fate , and proteolysis . an increase in protein carbonyl content of tissues has been associated with a number of pathological disorders . due to their abundance in mammalian cells , cytoskeletal proteins like actin are common targets for a variety of ros and low - molecular weight reactive carbonyl species . inconsistent data have been reported on the potential impact of ota on protein carbonylation [ 52 , 59 , 63 , 68 , 69 ] . no increase in protein oxidation was observed in liver and kidney of f344 rats treated with ota at 0.3 mg / kg bw per day for 4 weeks . the same finding was reported in liver of wistar rats treated with ota at 289 g / kg bw for 90 days . in contrast , in another in vivo study measuring protein oxidation in wistar rats treated with ota ( 0.5 mg / kg bw / day ) for 24 h , 7 , 14 and 21 days , a significant increase in protein carbonyls was found after 14 and 21 days of treatment in , respectively , the kidney and the liver . lipid peroxidation is among the most extensively investigated processes induced by free radicals . of these , the by - products , 4-hydroxy-2-nonenal ( hne ) , the tautomer malondialdehyde ( mda ) , acrolein and crotonaldehyde have been widely studied . the ability of these reactive electrophiles to modify dna bases , yielding promutagenic lesions , is considered to contribute to the mutagenic and carcinogenic effects associated with oxidative stress - induced lipid peroxidation . initially , rahimtula 's group observed that ota was able to stimulate lipid peroxidation when added to liver or kidney microsomes or when administered to rats in vivo . stimulation of lipid peroxidation by ota - iron complex facilitating the reduction of iron was further reported . moreover , additional studies indicated that ota induced lipid peroxidation accompanied by leakage of calcium from calcium - loaded microsomes . increased formation of mda was observed in animal models treated orally with different doses of ota [ 7579 ] . it is widely acknowledged that reactive oxygen and nitrosative species can trigger biological responses such as stimulation or inhibition of signal transduction and gene expression . such biological responses are considered to contribute to the expression of the carcinogenic potential of the reactive chemicals . a number of in vitro and in vivo studies are consistent with a role of oxidative and probably nitrosative stress as messengers involved in the adverse biological effects of ota ( table 1 ) . ros induces release of calcium from intracellular stores , resulting in the activation of kinases , such as protein kinase c ( pkc ) . ros species play also a critical role in the selective mobilization of other cell signaling responses . cell signaling phosphoproteins of mitogen - activated protein ( map ) kinases including erk , c - jun n - terminal kinases ( jnk ) , and p38 kinases are involved in proliferation , differentiation , and apoptosis . activation of these molecules has been observed in response to changes in the cellular redox balance and are considered as vectors of ros biological effects . in vitro experiments provided evidence of an effect of ota on intracellular calcium ( ca ) homeostasis [ 74 , 8082 ] . . an increased rate of microsomal ca uptake was observed after ota administration in vivo and in vitro . gene profiling analysis suggested a modulation of genes involved in calcium homeostasis by ota [ 47 , 85 ] . these data indicate that ca dependent signal transduction pathways may be affected by ota treatment . ota was shown to stimulate phosphorylation of erk1/2 , sap / jnk , and p38 using in vitro models [ 82 , 86 , 87 ] . using an in vivo model erk1/2 a specific signaling response was also observed with mobilization of the atypical protein kinase c ( pkc ) and the insulin - like growth factor-1 ( igf-1 ) system . gene profiling data supported a potential role of the ( igf)-pi3k - pkb pathway in ota - mediated renal toxicity in male rats . numerous reports have characterized interactions of ros and rns with activity of transcription factors [ 9092 ] . transcription factors contain a conserved redox sensitive cysteine residue ; the oxidation of this residue inactivates the dna - binding domain of the factor . several studies have observed an inactivation of transcription factors resulting from increased concentrations of ros [ 9395 ] . for example , it was shown that the complex ap-1 is a basic leucine zipper protein , which is highly sensitive to changes in redox environment due to a cysteine residue in the dna - binding domain . gene expression profile studies performed using in vivo models have shown that ota impairs the antioxidant defense system regulated by nrf2 . this effect was supported by further studies [ 4446 ] . using in vitro models together with electrophoretic mobility shift assay , an inhibition of nrf2 and ap-1 activity was shown as a result of ota treatment . interestingly , nrf2 binding site represents a bzip domain that interacts with the antioxidant response element ( are ) dna - binding site triggering the transcription of nrf2 regulated genes . the are motif ( 5-a / gtgac / tnnngca / g-3 ) shares structural similarity with ap-1 binding site ( 5-tgactca ) involving both cysteine rich residues , the target of ros oxidation [ 97 , 98 ] . these data strongly suggest a correlation between the generation of ros by ota and the subsequent inactivation of nrf2 as previously described for ap-1 . this molecular mechanism appears as a likely molecular component explaining the reduction of the defense response observed under ota treatment . in vivo experiments provided direct evidence that s - nitrosylation can interfere with transcription [ 91 , 92 ] . nitric oxide ( no ) induces the nitrosylation of cysteine residues ( thiol groups ) within or near the dna - binding domain and/or insertion of the zinc finger , which is a dna - binding motif , resulting in the inhibition of the dna - binding activity of transcription factors [ 99101 ] . this is illustrated by the example of the suppression of p450 gene expression by no . no - donors were found to suppress cyp2d6 promoter activity through inhibition of the transcription factor called hepatocyte nuclear factor 4 ( hnf4 ) [ 94 , 95 ] . interestingly , in a gene expression study performed by our group , ota was associated with a significant reduction of the expression of genes regulated by hnf4 suggesting an indirect evidence of the role oxidative stress and the transcription factors regulation by ota . a strong correlation between the ability of a compound to block cell - to - cell communication in cultured cells and its ability to induce rodent tumors through nongenotoxic mechanisms has been documented [ 33 , 128131 ] . disruption of gap junction intercellular communication was specifically reported in human renal cancer cell lines . for example , connexin 32 ( cx32 ) was discovered to be generally downregulated in human renal cell carcinoma ( rcc ) cell lines and in cancerous regions of the kidney . ros such as h2o2 , an established tumor promoter , is known to modulate cell - to - cell communication . likewise , certain chemicals inducing ros were shown to inhibit intercellular communication in a variety of cells in culture systems . kidney epithelial cells treated with ota resulted in modulation of gap junction - mediated intercellular communication , through a reduced expression of the gap - junction protein cx43 . in addition , ota strongly reduced the expression of other gap - junction proteins , cx26 , cx32 , and cx43 , in liver of rats treated with ota . in another study , even though a strong reduction of cx43 was found in renal cells in vitro , ota inhibited gjic only in liver but not in kidney . as mentioned earlier , ota was found to reduce the expression of antioxidant enzymes through inhibition of nrf2 activity . this reduction in antioxidant gene expression was correlated with increased oxidative damage of protein and dna . to further confirm the actual role of the reduction of defense mechanisms in the induction of cellular oxidative damage , reported ota effects at the levels of nrf2 activity , nrf2-regulated gene expression , and dna damage were prevented in cell pretreated with nrf2 inducers , strongly indicating a causal relationship between nrf2 effects and oxidative damage . several studies have been performed to try to counteract the adverse effects of oxygen radicals generated under ota - treatment . a number of molecules with various antioxidant properties were tested , including inula crithmoides , cyanidin 3-o - b - d - glucoside , catechins , melatonin , superoxide dismutase , catalase , and n - acetyl - l - cysteine ( nac ) , using in vivo or in vitro models . protection against ota - induced dna damage , lipid peroxidation as well as cytotoxicity was observed [ 75 , 79 , 102 , 105 , 106 , 116 , 118 , 127 ] further confirming the link between ota exposure and oxidative macromolecular damage . however , up to now , the application of chemicals known to possess antioxidant properties failed to prevent the development of ota - induced tumors in animal models . authors reported protection against nephrotoxicity but not carcinogenicity induced by ochratoxin a , implicating two separate pathways [ 122 , 123 ] . indomethacin and aspirin were found to prevent ota genotoxicity in the urinary bladder and kidney of mice . these data suggested the possible co - oxidation of ota by enzymes involved in arachidonic acid biotransformation , especially prostaglandin - h - synthase ( pghs ) and/or lipoxygenase . such reactions thought to produce activated ota metabolites are also known to generate ros which then may induce oxidation . the carcinogenic mycotoxin ota has been reviewed by a number of expert groups [ 35 , 14 , 137 ] . these expert groups identified the production of oxidative stress as an important event in the mode of action of ota - induced nephrocarcinogenicity . in the present paper , the actual evidence available supporting such hypothesis was collected and reviewed . it has been clearly shown that ota generates oxidative stress predominantly in kidney through potentially direct ( redox cycling ) and indirect ( reduction of cellular antioxidant defenses ) mechanisms . consequences resulting from the production of oxidative stress were observed at different levels . high kidney susceptibility to oxidative stress conditions oxidative stress has been incriminated in a number of kidney pathological pathways [ 138140 ] . as depicted in figure 1 , first , ota exposure was associated with increased levels of oxidative dna , lipid , and protein damage . second , various biological pathways known to be mobilized under oxidative stress were shown to be altered by ota . importantly , these effects were observed in both in vitro and in vivo test systems . these mechanisms are likely to be relevant for humans . in conclusion , the evidence for the induction of an oxidative stress response resulting from ota exposure can be considered strong . because the contribution of the oxidative stress response in the development of cancers is well established , a role in ota carcinogenicity altogether , the data reviewed above support the application of a threshold - based approach to establish safe level of dietary human exposure to ota .

the in vitro and in vivo evidence compatible with a role for oxidative stress in ota carcinogenicity has been collected and described . several potential oxido - reduction mechanisms have been identified in the past . more recently , the possibility of a reduction of cellular antioxidant defense has been raised as an indirect source of oxidative stress . consequences resulting from the production of oxidative stress are observed at different levels . first , ota exposure has been associated with increased levels of oxidative dna , lipid , and protein damage . second , various biological processes known to be mobilized under oxidative stress were shown to be altered by ota . these effects have been observed in both in vitro and in vivo test systems . in vivo , active doses were often within doses documented to induce renal tumors in rats . in conclusion , the evidence for the induction of an oxidative stress response resulting from ota exposure can be considered strong . because the contribution of the oxidative stress response in the development of cancers is well established , a role in ota carcinogenicity is plausible . altogether , the data reviewed above support the application of a threshold - based approach to establish safe level of dietary human exposure to ota .

1. Introduction 2. Sources of OTA-Mediated Oxygen-Species Generation 3. OTA-Mediated Oxidative Damage 4. OTA-Mediated Biological Response 5. Cell Signaling 6. Prevention of OTA-Induced Oxidative Stress 7. Conclusion

because of its wide occurrence and consequent exposure , together with a potent carcinogenic potential in animal models , ota has attracted significant public health attention over the last few years . several national and international food safety organizations and expert groups have conducted a thorough review of the situation as well as risk assessments in order to provide an insight on the health significance of ota in food . human epidemiology has been inconclusive : a number of studies have suggested a correlation between exposure to ota and balkan endemic nephropathy ( ben ) and mortality from urinary tract tumors [ 1 , 2 ] . therefore , the iarc statement that there is inadequate evidence for carcinogenicity in humans ( group 2b ) appears still valid . recently , other nephrotoxic agents have been put forward as the primary cause of ben [ 6 , 8 , 9 ] . in absence of adequate human data , kidney has been considered as the key target organ of ota toxicity . in all animal species studied , ota was found to produce renal toxicity , while in rodents renal carcinogenicity was clearly established . recently , ota renal and hepatic carcinogenicity was also observed in chicks . analysis of dietary exposure throughout europe revealed that the current average ota exposure ( 5060 ng / kg bw / week ) is well within the twi . based on the loael in pig , and applying an uncertainty factor of 500 , jecfa allocated a provisional tolerable weekly intake ( ptwi ) of 112 ng / kg bw . , 10% for the bmd10 ) in tumor incidence over background . the bmdl is the lower limit of a one - sided 95% confidence interval of the bmd . the occurrence of combined adenomas and carcinomas in the kidneys of male rats was considered by jecfa to be the most appropriate data for modeling . because the bmd approach did not provide a lower point of departure than the loael in pig , jecfa decided to reconfirm the ptwi of 100 ng / kg bw / day . a recent health risk assessment , performed by health canada recommends to regulate ota as a nonthreshold carcinogen , because of the uncertainties regarding the mode of action . in the risk assessment of carcinogenic substances , consideration of the mode of action ( moa ) is essential , determining the method to be applied in order to define levels of exposure below which a low safety concern is expected . the key events analysis framework of the moa has not yet been formally applied to ota . however , the approach used by most expert groups ( efsa , jecfa , ilsi ) to establish the safe level of exposure of ota ( based on uncertainty factors ) implies the consideration of key events compatible with a threshold effect . for these groups , amongst the mechanisms of action highlighted as possible , oxidative stress has been presented as one of the most probable [ 5 , 14 ] . over the last decades , studies aimed at elucidating the modes of action implicated in ota toxicity and carcinogenicity have been published . there has been considerable debate for many years over the genotoxicity of ota and its actual role in carcinogenicity [ 35 , 1115 , 17 , 18 ] . although genotoxicity is likely to play a role in ota carcinogenicity [ 3 , 4 ] , the actual molecular mechanism involved , either through covalent adduct formation , through other indirect modifications , or both is still unclear . the potential of ota to form covalent dna adducts has been subjected to debate due to conflicting data in the literature . using p - postlabelling analysis , large numbers of putative ota - derived dna adducts have been reported to be present in a wide range of tissues from ota - treated rats , mice as well as pigs [ 2 , 17 , 1922 ] . however , so far , these adducts have never been observed by any other highly specific techniques such as radioactivity measurements using h - labelled ota ( h - ota ) , accelerator mass spectrometry ( ams ) , or liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) . the present paper is not intended to provide a thorough review of the complex and controversial scientific literature on dna - adduct formation . however , it is important to keep in mind that dna adducts are increasingly considered as markers of exposures and not only of effects [ 26 , 27 ] and that dna - covalent binding does not necessarily determine the shape of the dose - response at low level of exposure [ 28 , 29 ] . in this context , it appears important to also consider other potential modes of action , which could potentially contribute to ota carcinogenicity . in the near future , the application of the mode of action framework will likely help to understand the individual contribution of all mechanisms described up to now for ota . the focus of the present short paper was to collect and highlight the evidence associated with a role for oxidative stress as a plausible mechanism to consider for ota . a list of the studies used to illustrate the main messages of the present paper is provided in table 1 . although not exhaustive , the list shows that over the last two decades , numerous investigators have documented the generation of oxidative stress as a result of ota treatment in both in vitro and in vivo model systems . production of reactive oxygen species ( ros ) leading to oxidative stress and macromolecular damage is known to contribute to the pathogenesis of age - related as well as chronic diseases including cancer [ 3135 ] . a number of studies are available documenting that ota is associated with the production of reactive oxygen species and resulting oxidative stress through various direct and indirect mechanisms . several oxido - reduction mechanisms elicited by ota have been proposed . in a reconstituted system consisting of phospholipid vesicles , the flavoprotein nadph - cytochrome p450 reductase and fe , ota was found to chelate ferric ions ( fe ) , facilitating their reduction to ferrous ions ( fe ) , which in the presence of oxygen , provided the active species initiating lipid peroxidation . a role for cytochrome p450 in this reaction in contrast , others found that ota induced oxidative damage through the generation of hydroxyl radicals . showed that ota behaved as a cell pro - oxidant through mobilization of fe and ca pathway leading to uncoupling oxidative phosphorylation and increased production of hydroxyl radical via the fenton reaction . however , in other studies using ota and structural analogs , a direct correlation between toxicity and iron chelating capacity was only partially supported . the generation of an ota hydroquinone / quinone couple from the oxidation of ota ( phenol oxidation ) by electrochemical , photochemical , and chemical processes was reported [ 39 , 40 ] . the quinone is thought to undergo reductions to form hydroquinone , postulated to be responsible for the formation of the glutathione conjugate of ota . such events are likely to result in redox cycling and in the generation of reactive oxygen species [ 20 , 40 , 41 ] . the formation of ota - derived quinones has been observed in cell cultures in vitro as well as in vivo [ 42 , 43 ] . this was observed at the rna and protein levels , both in vitro and in vivo test systems . nrf2 is involved in both the basal expression as well as in the induction of genes encoding detoxification , cytoprotective , and antioxidant enzymes [ 48 , 49 ] . a reduction of the expression of these genes is likely to result in decrease in antioxidant defenses leading to oxidative stress and macromolecular damage . through in vitro and in vivo studies , a correlation was observed between the ota - dependent reduction of the nrf2 pathway and an increased production of oxidative damage . in this context , it is interesting to note that ota was found to increase the expression of inducible nitric oxide synthase ( inos ) [ 5052 ] , an enzyme responsible for the production of nitric oxide ( no ) . an association between inos expression and the development of cancers was suggested in humans and animals in vivo . ota was shown to stimulate protein and possibly dna nitration [ 52 , 53 ] indicating that ota exposure may be considered as a source of both oxygen and also reactive nitrogen radicals / species ( rns ) . the oxidized dna bases appear to be capable of inducing mutations that are commonly observed in neoplasia . as illustrated in table 1 , diverse biomarkers have been analyzed showing that ota induces dna damage . in cell cultures , the ota - dependent production of ros was correlated with an increased formation of 8-oxoguanine [ 5759 ] . moreover , ota was shown to induce dna strand breaks as assessed by comet assay in liver , kidney , and spleen of f344 rats given 0 , 0.25 , 0.50 , 1.0 , and 2.0 mg / kg bw / day by gavage for 2 weeks . in liver and kidney , the extent of dna damage analyzed by comet assay was further enhanced in the presence of formamidopyrimidine glycosylase ( fpg ) , an enzyme involved in excision repair of oxidized dna bases [ 57 , 58 , 61 ] . another study detected dna - strand breaks ( using the comet assay ) in the kidney of female wistar rats treated intraperitoneally with ota ( 0.5 mg / kg bw / day for 7 , 14 , and 21 days ; n = 5 per group ) . the severity of the dna lesions in the kidney increased according to the ota dose and was at maximum after 21 days of treatment . other authors have observed oxidative dna - damage in various tissues of animals treated with a wide range of ota doses and treatment durations [ 46 , 60 , 63 , 64 ] . carbonylation of proteins occurs through a variety of oxidative pathways [ 54 , 6567 ] . carbonylation is an important protein modification associated with alterations of protein ( enzymes ) function , protein misfolding , protein fate , and proteolysis . an increase in protein carbonyl content of tissues has been associated with a number of pathological disorders . inconsistent data have been reported on the potential impact of ota on protein carbonylation [ 52 , 59 , 63 , 68 , 69 ] . no increase in protein oxidation was observed in liver and kidney of f344 rats treated with ota at 0.3 mg / kg bw per day for 4 weeks . in contrast , in another in vivo study measuring protein oxidation in wistar rats treated with ota ( 0.5 mg / kg bw / day ) for 24 h , 7 , 14 and 21 days , a significant increase in protein carbonyls was found after 14 and 21 days of treatment in , respectively , the kidney and the liver . of these , the by - products , 4-hydroxy-2-nonenal ( hne ) , the tautomer malondialdehyde ( mda ) , acrolein and crotonaldehyde have been widely studied . the ability of these reactive electrophiles to modify dna bases , yielding promutagenic lesions , is considered to contribute to the mutagenic and carcinogenic effects associated with oxidative stress - induced lipid peroxidation . initially , rahimtula 's group observed that ota was able to stimulate lipid peroxidation when added to liver or kidney microsomes or when administered to rats in vivo . stimulation of lipid peroxidation by ota - iron complex facilitating the reduction of iron was further reported . increased formation of mda was observed in animal models treated orally with different doses of ota [ 7579 ] . such biological responses are considered to contribute to the expression of the carcinogenic potential of the reactive chemicals . a number of in vitro and in vivo studies are consistent with a role of oxidative and probably nitrosative stress as messengers involved in the adverse biological effects of ota ( table 1 ) . ros induces release of calcium from intracellular stores , resulting in the activation of kinases , such as protein kinase c ( pkc ) . ros species play also a critical role in the selective mobilization of other cell signaling responses . cell signaling phosphoproteins of mitogen - activated protein ( map ) kinases including erk , c - jun n - terminal kinases ( jnk ) , and p38 kinases are involved in proliferation , differentiation , and apoptosis . activation of these molecules has been observed in response to changes in the cellular redox balance and are considered as vectors of ros biological effects . in vitro experiments provided evidence of an effect of ota on intracellular calcium ( ca ) homeostasis [ 74 , 8082 ] . an increased rate of microsomal ca uptake was observed after ota administration in vivo and in vitro . gene profiling analysis suggested a modulation of genes involved in calcium homeostasis by ota [ 47 , 85 ] . these data indicate that ca dependent signal transduction pathways may be affected by ota treatment . ota was shown to stimulate phosphorylation of erk1/2 , sap / jnk , and p38 using in vitro models [ 82 , 86 , 87 ] . using an in vivo model erk1/2 a specific signaling response was also observed with mobilization of the atypical protein kinase c ( pkc ) and the insulin - like growth factor-1 ( igf-1 ) system . gene profiling data supported a potential role of the ( igf)-pi3k - pkb pathway in ota - mediated renal toxicity in male rats . transcription factors contain a conserved redox sensitive cysteine residue ; the oxidation of this residue inactivates the dna - binding domain of the factor . several studies have observed an inactivation of transcription factors resulting from increased concentrations of ros [ 9395 ] . for example , it was shown that the complex ap-1 is a basic leucine zipper protein , which is highly sensitive to changes in redox environment due to a cysteine residue in the dna - binding domain . gene expression profile studies performed using in vivo models have shown that ota impairs the antioxidant defense system regulated by nrf2 . using in vitro models together with electrophoretic mobility shift assay , an inhibition of nrf2 and ap-1 activity was shown as a result of ota treatment . these data strongly suggest a correlation between the generation of ros by ota and the subsequent inactivation of nrf2 as previously described for ap-1 . this molecular mechanism appears as a likely molecular component explaining the reduction of the defense response observed under ota treatment . nitric oxide ( no ) induces the nitrosylation of cysteine residues ( thiol groups ) within or near the dna - binding domain and/or insertion of the zinc finger , which is a dna - binding motif , resulting in the inhibition of the dna - binding activity of transcription factors [ 99101 ] . no - donors were found to suppress cyp2d6 promoter activity through inhibition of the transcription factor called hepatocyte nuclear factor 4 ( hnf4 ) [ 94 , 95 ] . interestingly , in a gene expression study performed by our group , ota was associated with a significant reduction of the expression of genes regulated by hnf4 suggesting an indirect evidence of the role oxidative stress and the transcription factors regulation by ota . a strong correlation between the ability of a compound to block cell - to - cell communication in cultured cells and its ability to induce rodent tumors through nongenotoxic mechanisms has been documented [ 33 , 128131 ] . for example , connexin 32 ( cx32 ) was discovered to be generally downregulated in human renal cell carcinoma ( rcc ) cell lines and in cancerous regions of the kidney . likewise , certain chemicals inducing ros were shown to inhibit intercellular communication in a variety of cells in culture systems . kidney epithelial cells treated with ota resulted in modulation of gap junction - mediated intercellular communication , through a reduced expression of the gap - junction protein cx43 . in addition , ota strongly reduced the expression of other gap - junction proteins , cx26 , cx32 , and cx43 , in liver of rats treated with ota . in another study , even though a strong reduction of cx43 was found in renal cells in vitro , ota inhibited gjic only in liver but not in kidney . as mentioned earlier , ota was found to reduce the expression of antioxidant enzymes through inhibition of nrf2 activity . this reduction in antioxidant gene expression was correlated with increased oxidative damage of protein and dna . to further confirm the actual role of the reduction of defense mechanisms in the induction of cellular oxidative damage , reported ota effects at the levels of nrf2 activity , nrf2-regulated gene expression , and dna damage were prevented in cell pretreated with nrf2 inducers , strongly indicating a causal relationship between nrf2 effects and oxidative damage . several studies have been performed to try to counteract the adverse effects of oxygen radicals generated under ota - treatment . a number of molecules with various antioxidant properties were tested , including inula crithmoides , cyanidin 3-o - b - d - glucoside , catechins , melatonin , superoxide dismutase , catalase , and n - acetyl - l - cysteine ( nac ) , using in vivo or in vitro models . protection against ota - induced dna damage , lipid peroxidation as well as cytotoxicity was observed [ 75 , 79 , 102 , 105 , 106 , 116 , 118 , 127 ] further confirming the link between ota exposure and oxidative macromolecular damage . however , up to now , the application of chemicals known to possess antioxidant properties failed to prevent the development of ota - induced tumors in animal models . indomethacin and aspirin were found to prevent ota genotoxicity in the urinary bladder and kidney of mice . such reactions thought to produce activated ota metabolites are also known to generate ros which then may induce oxidation . the carcinogenic mycotoxin ota has been reviewed by a number of expert groups [ 35 , 14 , 137 ] . these expert groups identified the production of oxidative stress as an important event in the mode of action of ota - induced nephrocarcinogenicity . in the present paper , the actual evidence available supporting such hypothesis was collected and reviewed . it has been clearly shown that ota generates oxidative stress predominantly in kidney through potentially direct ( redox cycling ) and indirect ( reduction of cellular antioxidant defenses ) mechanisms . consequences resulting from the production of oxidative stress were observed at different levels . high kidney susceptibility to oxidative stress conditions oxidative stress has been incriminated in a number of kidney pathological pathways [ 138140 ] . as depicted in figure 1 , first , ota exposure was associated with increased levels of oxidative dna , lipid , and protein damage . second , various biological pathways known to be mobilized under oxidative stress were shown to be altered by ota . importantly , these effects were observed in both in vitro and in vivo test systems . these mechanisms are likely to be relevant for humans . in conclusion , the evidence for the induction of an oxidative stress response resulting from ota exposure can be considered strong . because the contribution of the oxidative stress response in the development of cancers is well established , a role in ota carcinogenicity altogether , the data reviewed above support the application of a threshold - based approach to establish safe level of dietary human exposure to ota .

although overall hiv prevalence is low ( 1.5%1.8%),1 ghana like other developing countries is still considered a high - risk country for several reasons : the presence of covert multi - partner sexual activity and denial , the low level of knowledge and low condom use , unsafe professional blood donation , high incidence of self - reported sexually transmitted infections among vulnerable groups , infected expatriates who infect their sexual partners when they return to ghana , and high levels of hiv / aids in the bordering countries all contribute to the spread of hiv.2 of all the various age groups affected by this pandemic , dehne and riedner3 identified the adolescent age group as that group that is most at risk of contracting hiv / aids and other sexually transmitted diseases ( stds ) . defined by the world health organization ( who)4 as persons between 10 and 19 years of age , many adolescents around the world are sexually active and many sexual contacts among them are unprotected . their vulnerability is further heightened by the fact that most parents do not discuss issues bordering on sex at home and hence many teens turn to peers and to the media and get inaccurate information . against the background that more than half of newly infected hiv adolescents are female,5 the knowledge and awareness of female adolescents on hiv / aids is of paramount concern in developing countries with high risks of transmission . however , a preliminary investigation revealed a paucity of information on the knowledge level on hiv / aids among high school girls in sub - saharan africa . further , there is a dearth of empirical research on hiv / aids among senior high school ( shs ) girls in ghana and other west african countries . this study was therefore undertaken to fill the knowledge gap on the subject matter and further arm health educators , peer counselors , and other stakeholders with the necessary information to address the information needs of shs girls in ghana on hiv / aids . particular reference is made by the study to causative factors , modes of transmission , prevention , and the sources of information on hiv / aids among shs girls . the study was carried out in the west african senior high school [ washs ] which was established in 1946 and is currently ranked a first class shs by the ghana education service ( ges ) . the school was selected primarily because of its regular participation in hiv / aids awareness interventions organized by the ghana aids commission ( gac ) . the study selected 260 female students from the total 1080 female students in the school . stratified sampling technique was employed in grouping female students into four strata according to their year of study ( that is shs 1 , 2 , 3 , and 4 ) . the stratified sampling technique was used in order to get a fair sample that is representative of the heterogeneous student population . the questionnaire was anonymous and solicited information on respondents background as well as their knowledge on the causes , mode of transmission , and prevention of hiv / aids . the first part was closed ended and contained questions on the background of the respondents . the second part contained questions on hiv / aids found in hiv / aids awareness leaflets used by the ghana aids commission and other hiv / aids materials used by the ghana education service . the final part of the questionnaire was open ended in nature and sought respondents views on how awareness creation on hiv / aids could be further enhanced in shss . appropriate permission was sought from the headmaster of the school who nominated a teacher to assist and facilitate the administration of the questionnaires . the teacher served primarily as a guide and helped in gaining access to students in class , introducing the research team and participants , ensuring that the research team did not obstruct normal student activities , as well as seeking permission from other teachers and staff encountered during the data collection . participation in the study was voluntary and prospective participants regardless of their choice to participate in the study or not were briefed on the purpose , use , and significance of the study . the data collected were analyzed using statistical package for the social sciences ( spss ; ibm corporation , armonk , ny , usa ) version 16 with the key issues being presented in summary frequency tables . the qualitative analysis was done using the thematic analysis approach based on braun and clarkes6 assertion that it offers an accessible and theoretically - flexible approach to analyzing qualitative data . using this approach , the data were reviewed and sorted out under the relevant themes based on the study objectives . subsequently , the data were discussed under each theme within the context of relevant literature and with the aim of identifying other subthemes and patterns under each theme . particular attention was also paid to comments and answers to follow up questions by respondents during the discussion . as shown in table 1 , more than two - thirds ( 70.4% ) of the respondents were between the ages of 16 and 19 . those between 12 and 15 years accounted for 16.5% while the remaining 13.1% of the respondents were above 19 years of age . the age variation of respondents in this study is consistent with adamchak7 which found that students in shs in ghana were more likely to be in their teens . this was explained by the 12-year primary and junior high school they had to attend before getting into shs . table 1 also shows that equal numbers of respondents were selected from each year group ( 65 students per year group ) . this was done to ensure that responses provided reflected the views of all female students from the first year through to the final year . respondents were also selected from across all programs offered by the school with 23.5% , 26.2% , 21.9% , and 28.4% being drawn from the science , general arts , business , and visual arts programs , respectively . respondents were also asked a number of questions bordering on the nature of hiv / aids , transmission of hiv / aids , prevention and cure of hiv / aids , as well as awareness creation on hiv / aids . governments all around africa have launched institutions and special initiatives to create awareness , reduce stigmatization and support people living with hiv / aids . this is in recognition of the fact that the best approach to addressing the hiv / aids pandemic remains prevention through awareness creation and sensitization since neither treatment nor vaccine is readily available . in ghana , this initiative is embodied in the creation and massive support for the ghana aids commission and the national hiv / aids control program ( nhacp ) by successive governments over the past decade . together with other key governmental and nongovernmental stakeholders , these institutions continue to design and implement several hiv / aids interventions tailored to suit particular vulnerable populations at risk in ghana . one such group that is often the focus of these interventions in ghana and other developing countries has been adolescent school girls . evidence from lal et al8 and gregson et al9 among others suggest that this group has become a key focus of hiv / aids interventions because of their relatively high sexual activity and high risk of contracting the disease . this is supported by bandawe and fosters10 findings that hiv / aids incidence was very high among females aged 1519 in the third world . additionally , studies by kaur et al11 in india conclude that most girls in developing countries become sexually aware and active in shs and generally after 15 years . knowledge and awareness on hiv / aids among adolescent school girls is therefore crucial not only in preventing the spread of the virus , but also in addressing the threats posed by hiv / aids to education for all goals12 and other interventions aimed at enhancing girl child education . the key to hiv / aids awareness therefore hinges on knowledge of what hiv and aids are , and what exposures and risky behaviors can potentially lead to contracting the virus . accordingly , substantial effort has been put in over the years to provide students in shs with the basic information on the nature of hiv / aids . against this background , and in spite of the numerous educational campaigns and advertisements on hiv / aids in the mass media , it was therefore surprising that only 73.3% of respondents could correctly mention the meaning of the acronym hiv . however , a relatively higher percentage of respondents ( 96.7% ) were able to identify the meaning of the acronym aids . these results confirm earlier studies by bauni and jarabi13 that explain that aids is more often used to represent hiv / aids in most african societies where no clear distinction is made among the general population between hiv and aids . almost all respondents ( 96.7% ) agreed that hiv is a virus that replicates in the body and can develop into aids . however , this percentage dropped significantly by 20% to 76.7% on the issue that the virus can potentially live in the human body for years ( sometimes 10 to 15 years ) before it is noticed or develops into aids . aside the 10% of respondents who disagreed , a further 13.3% were not sure whether the virus could live in the human body for a long time before detection . the fact that as much as 23.3% of respondents were not aware that hiv could live in the human body for years asks serious questions on hiv / aids prevention and control in ghana . as explained by seeley et al,14 ignorance or refusal to acknowledge that persons currently exhibiting signs and symptoms of aids may have acquired it years ago hinders hiv / aids prevention efforts since such persons are not able to alert their partners and other close ones who they may have transferred the virus to or contracted the virus from over the years . further , in mozambique , unaids15 states that most high school girls are stigmatized and seen as bad or immoral for testing positive to hiv / aids because of the misconception that they acquired the virus through teenage sexual activities . rather , these high school girls might have contracted the virus at birth , during immunization , or from other non - sexual causes earlier on in their lives . this arises from the lack of recognition that the virus could be contracted years before visible signs and symptoms are discovered and it explains why persons in whom signs and symptoms manifest are thus stigmatized and seen as promiscuous without considering that the virus may have been acquired years before . also , efforts at managing the source and further spread of hiv / aids are often centered on the immediate people around the victim neglecting those who may have had prior contact in earlier years with the victim . thus , the source of transmission is not easily detected . the key to hiv / aids awareness therefore hinges on knowledge of what hiv and aids are , and what exposures and risky behaviors can potentially lead to contracting the virus . accordingly , substantial effort has been put in over the years to provide students in shs with the basic information on the nature of hiv / aids . against this background , and in spite of the numerous educational campaigns and advertisements on hiv / aids in the mass media , it was therefore surprising that only 73.3% of respondents could correctly mention the meaning of the acronym hiv . however , a relatively higher percentage of respondents ( 96.7% ) were able to identify the meaning of the acronym aids . these results confirm earlier studies by bauni and jarabi13 that explain that aids is more often used to represent hiv / aids in most african societies where no clear distinction is made among the general population between hiv and aids . almost all respondents ( 96.7% ) agreed that hiv is a virus that replicates in the body and can develop into aids . however , this percentage dropped significantly by 20% to 76.7% on the issue that the virus can potentially live in the human body for years ( sometimes 10 to 15 years ) before it is noticed or develops into aids . aside the 10% of respondents who disagreed , a further 13.3% were not sure whether the virus could live in the human body for a long time before detection . the fact that as much as 23.3% of respondents were not aware that hiv could live in the human body for years asks serious questions on hiv / aids prevention and control in ghana . as explained by seeley et al,14 ignorance or refusal to acknowledge that persons currently exhibiting signs and symptoms of aids may have acquired it years ago hinders hiv / aids prevention efforts since such persons are not able to alert their partners and other close ones who they may have transferred the virus to or contracted the virus from over the years . further , in mozambique , unaids15 states that most high school girls are stigmatized and seen as bad or immoral for testing positive to hiv / aids because of the misconception that they acquired the virus through teenage sexual activities . rather , these high school girls might have contracted the virus at birth , during immunization , or from other non - sexual causes earlier on in their lives . this arises from the lack of recognition that the virus could be contracted years before visible signs and symptoms are discovered and it explains why persons in whom signs and symptoms manifest are thus stigmatized and seen as promiscuous without considering that the virus may have been acquired years before . also , efforts at managing the source and further spread of hiv / aids are often centered on the immediate people around the victim neglecting those who may have had prior contact in earlier years with the victim . it was disturbing to note that as much as 36.7% of the shs girls were either not sure or disagreed that , although symptoms may not be present , the virus was still transmittable . such persons therefore stood a higher chance of infection because this perception that only persons exhibiting symptoms of hiv / aids could transmit the virus will lead to them being less careful in their associations with seemingly healthy - looking persons . furthermore , studies on hiv / aids have shown that most relatives of persons living with hiv / aids ( plwha ) get infected because they failed to take precaution in their interactions and even shared personal items like blades , shaving sticks , tooth brushes , etc , with their seemingly healthy loved ones who though not currently exhibiting signs of the virus were infected . thus , problems could arise in schools simply because girls who are unaware that though symptoms may not be present , the virus is still transmissible may be sharing personal items with colleagues who may not be exhibiting signs or symptoms of the virus but may be infected . interestingly , follow up questions revealed that some students believed that the physical appearance of a person was an accurate measure of whether the person was infected with the hiv . in this regard , students were less careful , associated freely , and shared personal effects with persons who did not exhibit any physical sign or symptom of hiv / aids but might still be infected . one such student in response to whether she knew her hiv / aids status , was quick to respond with the following : see how i am healthy and fresh , do i look like i have hiv / aids ? respondents were however able to clearly point out that hiv / aids may be transmitted through having unprotected sex with an infected partner , being born to a mother who is infected with hiv , or through the infected mother s breast milk . the majority ( 83.3% ) of respondents were also aware that hiv / aids could be acquired through blood transfusion . this is a positive result considering bennell et al16 findings advised that even though hiv / aids transmission through blood transfusion is rare as blood donors are screened before they donate , and blood products are tested before they are used , there is still a likelihood of infection via this route . all the shs girls were also aware that using unsterilized instruments or sharing personal items like toothbrushes could lead to acquiring the virus . their awareness has a positive impact on prevention of hiv / aids considering that sharing items like blades , toothbrushes , pins and needles , cooking and pocket knives , hair and nail clippers , and other sharp objects is a major cause of transmission in less developed countries.1719 in the words of one respondent , until i learnt about hiv / aids transmission last term , i used to share nail clippers , shaving sticks and toothbrushes with my cousins every time they came to visit . aside from hiv / aids transmissions through sharing personal items , there have been cases of hiv / aids transmissions during mass immunizations , blood donations , and during clinical injections . even though this is a rare occurrence in most countries because the needles and instruments are only used once and disposed of or properly sterilized , the same does not apply to less developed countries where with underresourced health sectors , the misconception that merely washing items makes them safe for repetitive use and the acute lack of instruments significantly increases their level of risk . almost all the respondents ( 90% ) were also aware that hiv / aids could be transmitted through bodily fluids like blood , semen , vaginal secretions , and breast milk . follow up questions however revealed that some shs girls still believed that physical contact with infected persons or sharing public space with infected persons could be a source of transmission of hiv / aids . this might explain the relatively high stigmatization and discrimination against plwha by their colleagues in schools that was identified by asiimwe - okiror et al20 in uganda . the above is in sharp contrast to the content of educational materials and information provided by the ghana aids commission , national hiv / aids control program , and the mass media on hiv / aids transmission . these materials clearly explain that it is a scientifically proven fact that physical contact , without the exchange of bodily fluids with an infected person , can not transmit the virus . in fact , a lot of hiv / aids awareness messages in ghana focus on this point because of the realization that poor awareness of this fact leads to high stigmatization , seclusion , and discrimination of plwha . such perceptions also explain the case made by world bank21 that plwha in rural areas and less developed countries are sometimes denied access to public transport , swimming in public pools , fetching water from public standpipes , engaging in contact sports , and other activities that involve physical proximity . another contentious issue in the hiv / aids dialogue in sub - saharan africa is the question of whether hiv / aids could be caused by witchcraft or other spiritual factors . evidence provided by gregson and garnett22 suggest that in the early to mid 1990s when the causes , means of transmission , and prevention were still controversial , most traditional societies in africa blamed hiv / aids , like other inexplicable ailments , on witchcraft and other spiritual causes . however , as information on the virus became available over the years such misconceptions have become less commonplace . it is thus a serious setback for hiv / aids prevention that in the 21st century and in the modern information and communications technology ( ict ) era where information in hiv / aids abounds , as much as 30% of the respondents believed fully or were not sure that witchcraft and other spiritual factors caused hiv / aids . attention must also be paid to studies like that of bennell23 that have also unearthed unproven allegations of persons bewitching others with the virus or suffering from the virus as a result of a punishment for offending some spirits . if such perceptions are still being held by some people , then prevention and even management of new and existing cases of hiv / aids are seriously hindered . instead of getting attention from voluntary counseling and testing centers , hospitals , and other commissioned bodies , plwhas as well as persons suspected to be infected are sent to prayer camps for the virus to be exorcised . this not only increases victimization and stigmatization but also increases the risk of infecting church members , family , and friends as well as other victims in these prayer or witch camps . though trends in modern medicine are far advanced , the world is yet to possess a universally acceptable medically tested cure for the hiv virus.24 this is in spite of the numerous claims by both traditional and orthodox medicine practitioners as well as spiritual healers of curing hiv / aids patients . regardless of these claims and in the interest of public health , the ghana aids commission and the national hiv / aids control program maintains in ghana that no scientific cure exists for hiv / aids . rather , it supports medically approved ways including ( but not limited to ) the use of anti - retrovirals and regenerative health practices as the sanctioned means of managing the disease in order to prolong the life of infected persons , reduce the risk of transmission , and increase the quality of life of plwhas and their families . it was therefore surprising that as much as 50% of the shs girls believed that hiv / aids had a scientifically proven cure . even more controversial is the notion that herbal or traditional medicine as well as spiritual treatment could cure hiv / aids . though many hiv / aids patients or their relatives have come out to substantiate claims made by herbal , traditional , or spiritual healers of possessing a cure to the virus , such claims are yet to be scientifically confirmed . this notwithstanding , it is not uncommon to hear religious and herbal healers make open claims of curing hiv / aids in both print and electronic media adverts often with purportedly infected persons giving testimonies of their healing . this may explain why 43.3% of respondents believed that a herbal or spiritual treatment to hiv / aids existed . one of our relatives who had tested positive with hiv / aids was cured by dr togo ( a traditional herbal priest ) in kumasi . i know because after she came back from dr togo s place , she took another test which was negative . a number of girls also believed strongly that hiv / aids and in fact all the opportunistic infections arising thereof could be cured through anointed men of god . they were quick to share testimonies of several people they had seen on television or heard on radio who had been cured of hiv / aids . as put by one respondent : we have had several people in our church who have been cured of hiv / aids . they mostly come and give their testimonies and thanks - offering to god in church after being cured . however , some respondents disagreed on the veracity of the claims of being healed from hiv / aids accusing those pastors and traditional priests of being charlatans . they accused these pastors and traditional priests of conniving with hired people to give those testimonies in order to ensnare unsuspecting victims . i had a neighbor who tested hiv / aids positive , after going through a number of churches which claimed they had healed him , his conditions got worse and worse till he finally died from hiv / aids . further , it is impossible to tell that a person is infected with the hiv / aids virus by just looking at him / her . this is because persons with the virus show symptoms not of aids but of the opportunistic infections like tuberculosis that they suffer as a result of the deficiency in their immune system . physically assessing their behaviors as well as change in their bodies can not be used in any instance as a means of detecting the absence or presence of the virus in any individual . however , 35.4% of the respondents were not aware of this fact but rather believed or were not sure that you could tell that a person was hiv positive by merely looking at them . this is in spite of the hiv / aids educational materials that clearly point out that only testing can be used as a determinant of who has or has not . these persons are more likely to stigmatize those who exhibit any form of chronic ailment or who lose weight significantly as an hiv positive patient . further , as expressed in the previous sections , these persons are more at risk because of the tendency to relate carelessly with seemingly healthy looking persons who may be infected . additionally , it is now accepted globally that knowing your hiv / aids status can help prevent hiv / aids transmission . though knowing your status can not directly prevent infection from the virus , knowing your status is the key step in making decisions on your health . in other words , knowing your status means that you could make choices that can prevent persons around you from contracting the virus ( that is , if you are infected ) or the know - how to live healthily to prolong your life . it is therefore not surprising that 73.3% of respondents interviewed agreed that knowing their status could help prevent infection . when follow up questions were asked on how many had taken hiv / aids tests recently or knew their status , as much as 77% of respondents responded in the negative . thus , even though they knew that knowing their hiv status was key , only 13% of respondents had taken the test in recent times . an additional 10% had taken the test before but were not sure of their current status . this confirms the ghana aids commission campaign message that merely testing negative once in your life does not necessarily mean that your hiv status will always remain negative . rather , the tests must be continuous in so far as you engage in any form of risky behaviors . the fact that 77% of shs girls had not taken any hiv test before is worrying considering that most girls are sexually active and/or are exposed to other means of contracting hiv / aids in their teens in developing countries.25,26 as no scientifically proven cure exists for hiv / aids , the universally accepted means of handling the pandemic is prevention . prevention of hiv / aids aims to reduce the risk of infection by encouraging individuals to make healthy sexual life choices . in the 21st century , prevention of hiv / aids is seen in the context of abstinence , being faithful , and wearing a condom ( a , b , and c ) . as seeley and barnett14 explain , until a cure for the virus surfaces , a , b , and c remain the best way of handling hiv / aids . disappointingly , only 66.7% of respondents were aware of this fact with the remaining 33.3% disagreeing that a , b , and c helped reduce the risk of getting infected by the virus . in contrast however , 96.7% of respondents were aware that of a , b , and c , abstinence was the best means of preventing infection from the virus . the study also unearthed a worrying fact that some shs girls , even though aware of the likelihood of contracting the virus through unprotected sex , were still reluctant to use condoms and other forms of protection for various reasons . as shown in table 2 , as much as 51% of respondents were unlikely to use condoms during sexual intercourse . though few questions are raised about transmission of hiv / aids through the use of unsterilized instruments , condoms certainly remain highly controversial , since many churches and other religious groups believe strongly that condom use ( even among adults ) promotes promiscuity . the efficacy of condoms in preventing hiv transmission is also hotly debated , even among those without strong religious convictions.27 on one hand , it is argued that since condoms do not offer absolute protection ( because of improper use , reuse , inconsistent use , use while intoxicated , and manufacturing defects that lead to breakage or bursting ) , they should not be promoted as a method for preventing hiv transmission . the counterargument is that , since the only options to condom use are total sexual abstinence and having only one sexual partner ( which are not realistic options for many youth and adults ) , condom use must be actively promoted . bennell et al16 therefore advises , deriving from the above debate that recent efforts at creating awareness on hiv / aids , that prevention among the youth especially in developing countries must not focus merely on the use of condoms but on the correct use of condoms and also on the fact that condoms are not 100% safe but are subject to human error and manufacturing defects . the study reveals that shs girls in ghana generally had adequate knowledge on the basics of hiv / aids . the study also shows a positive impact of the hiv / aids awareness campaigns by the national hiv / aids control program , ghana aids commission , and other stakeholders in shs in ghana . however , knowledge on some vital information including the existence of herbal or scientifically proven cures , spiritual causes of hiv / aids , the transmission of hiv through body contact , and the determination of persons infected with hiv / aids by visual observation is deficient among segments of the students and needs further attention . particularly , the knowledge and awareness on hiv / aids related information did not translate to students decision to undergo voluntary testing with more than two - thirds of the student population not knowing their hiv status . the study therefore suggests that hiv / aids awareness campaigns among shs students in ghana in general must pay particular attention to specific issues on the transmission and management of hiv / aids . further , hiv / aids prevention drives must move beyond education into encouraging and enhancing voluntary counseling and testing services among students . hiv / aids awareness and prevention efforts must also pay close attention to addressing the social , cultural , and religious barriers to condom usage in ghana .

hiv / aids is recognized as a national priority health issue in ghana . consequently , the ghana aids commission and the national aids control programme were established , among other things , to enhance the knowledge and awareness on the nature , causes , effects and means of managing the spread of hiv / aids among populations at risk in ghana . through the efforts of these bodies and other stakeholders in health , several awareness creation and sensitization efforts have been targeted at teenage girls , a high risk group in ghana . this study therefore assesses the knowledge and awareness of hiv / aids among senior high school girls in their teens in ghana using a sample of 260 female students of west african senior high school . the data collected were analyzed and discussed under relevant themes and within the context of the literature . the study revealed that generally , senior high school girls were knowledgeable on the nature , modes of transmission , and prevention of hiv / aids . there were however some students who exhibited limited knowledge on some issues including the spiritual causes and treatment of hiv / aids , contacts and associations with infected persons , as well as determination of hiv infection from appearances rather than testing . the study also raised important concerns about the reluctance of senior high school girls to use condoms as a preventive measure and the need to reorient hiv / aids awareness interventions in ghana .

Introduction Methods Results Discussion Nature of HIV/AIDS awareness Transmission of HIV/AIDS Prevention and cure of HIV/AIDS Conclusion

although overall hiv prevalence is low ( 1.5%1.8%),1 ghana like other developing countries is still considered a high - risk country for several reasons : the presence of covert multi - partner sexual activity and denial , the low level of knowledge and low condom use , unsafe professional blood donation , high incidence of self - reported sexually transmitted infections among vulnerable groups , infected expatriates who infect their sexual partners when they return to ghana , and high levels of hiv / aids in the bordering countries all contribute to the spread of hiv.2 of all the various age groups affected by this pandemic , dehne and riedner3 identified the adolescent age group as that group that is most at risk of contracting hiv / aids and other sexually transmitted diseases ( stds ) . against the background that more than half of newly infected hiv adolescents are female,5 the knowledge and awareness of female adolescents on hiv / aids is of paramount concern in developing countries with high risks of transmission . however , a preliminary investigation revealed a paucity of information on the knowledge level on hiv / aids among high school girls in sub - saharan africa . further , there is a dearth of empirical research on hiv / aids among senior high school ( shs ) girls in ghana and other west african countries . this study was therefore undertaken to fill the knowledge gap on the subject matter and further arm health educators , peer counselors , and other stakeholders with the necessary information to address the information needs of shs girls in ghana on hiv / aids . particular reference is made by the study to causative factors , modes of transmission , prevention , and the sources of information on hiv / aids among shs girls . the study was carried out in the west african senior high school [ washs ] which was established in 1946 and is currently ranked a first class shs by the ghana education service ( ges ) . the school was selected primarily because of its regular participation in hiv / aids awareness interventions organized by the ghana aids commission ( gac ) . the study selected 260 female students from the total 1080 female students in the school . the questionnaire was anonymous and solicited information on respondents background as well as their knowledge on the causes , mode of transmission , and prevention of hiv / aids . the second part contained questions on hiv / aids found in hiv / aids awareness leaflets used by the ghana aids commission and other hiv / aids materials used by the ghana education service . the final part of the questionnaire was open ended in nature and sought respondents views on how awareness creation on hiv / aids could be further enhanced in shss . the teacher served primarily as a guide and helped in gaining access to students in class , introducing the research team and participants , ensuring that the research team did not obstruct normal student activities , as well as seeking permission from other teachers and staff encountered during the data collection . participation in the study was voluntary and prospective participants regardless of their choice to participate in the study or not were briefed on the purpose , use , and significance of the study . the data collected were analyzed using statistical package for the social sciences ( spss ; ibm corporation , armonk , ny , usa ) version 16 with the key issues being presented in summary frequency tables . using this approach , the data were reviewed and sorted out under the relevant themes based on the study objectives . subsequently , the data were discussed under each theme within the context of relevant literature and with the aim of identifying other subthemes and patterns under each theme . the age variation of respondents in this study is consistent with adamchak7 which found that students in shs in ghana were more likely to be in their teens . respondents were also asked a number of questions bordering on the nature of hiv / aids , transmission of hiv / aids , prevention and cure of hiv / aids , as well as awareness creation on hiv / aids . this is in recognition of the fact that the best approach to addressing the hiv / aids pandemic remains prevention through awareness creation and sensitization since neither treatment nor vaccine is readily available . in ghana , this initiative is embodied in the creation and massive support for the ghana aids commission and the national hiv / aids control program ( nhacp ) by successive governments over the past decade . together with other key governmental and nongovernmental stakeholders , these institutions continue to design and implement several hiv / aids interventions tailored to suit particular vulnerable populations at risk in ghana . one such group that is often the focus of these interventions in ghana and other developing countries has been adolescent school girls . evidence from lal et al8 and gregson et al9 among others suggest that this group has become a key focus of hiv / aids interventions because of their relatively high sexual activity and high risk of contracting the disease . knowledge and awareness on hiv / aids among adolescent school girls is therefore crucial not only in preventing the spread of the virus , but also in addressing the threats posed by hiv / aids to education for all goals12 and other interventions aimed at enhancing girl child education . the key to hiv / aids awareness therefore hinges on knowledge of what hiv and aids are , and what exposures and risky behaviors can potentially lead to contracting the virus . accordingly , substantial effort has been put in over the years to provide students in shs with the basic information on the nature of hiv / aids . against this background , and in spite of the numerous educational campaigns and advertisements on hiv / aids in the mass media , it was therefore surprising that only 73.3% of respondents could correctly mention the meaning of the acronym hiv . however , a relatively higher percentage of respondents ( 96.7% ) were able to identify the meaning of the acronym aids . these results confirm earlier studies by bauni and jarabi13 that explain that aids is more often used to represent hiv / aids in most african societies where no clear distinction is made among the general population between hiv and aids . the fact that as much as 23.3% of respondents were not aware that hiv could live in the human body for years asks serious questions on hiv / aids prevention and control in ghana . as explained by seeley et al,14 ignorance or refusal to acknowledge that persons currently exhibiting signs and symptoms of aids may have acquired it years ago hinders hiv / aids prevention efforts since such persons are not able to alert their partners and other close ones who they may have transferred the virus to or contracted the virus from over the years . further , in mozambique , unaids15 states that most high school girls are stigmatized and seen as bad or immoral for testing positive to hiv / aids because of the misconception that they acquired the virus through teenage sexual activities . rather , these high school girls might have contracted the virus at birth , during immunization , or from other non - sexual causes earlier on in their lives . also , efforts at managing the source and further spread of hiv / aids are often centered on the immediate people around the victim neglecting those who may have had prior contact in earlier years with the victim . thus , the source of transmission is not easily detected . the key to hiv / aids awareness therefore hinges on knowledge of what hiv and aids are , and what exposures and risky behaviors can potentially lead to contracting the virus . accordingly , substantial effort has been put in over the years to provide students in shs with the basic information on the nature of hiv / aids . against this background , and in spite of the numerous educational campaigns and advertisements on hiv / aids in the mass media , it was therefore surprising that only 73.3% of respondents could correctly mention the meaning of the acronym hiv . these results confirm earlier studies by bauni and jarabi13 that explain that aids is more often used to represent hiv / aids in most african societies where no clear distinction is made among the general population between hiv and aids . the fact that as much as 23.3% of respondents were not aware that hiv could live in the human body for years asks serious questions on hiv / aids prevention and control in ghana . as explained by seeley et al,14 ignorance or refusal to acknowledge that persons currently exhibiting signs and symptoms of aids may have acquired it years ago hinders hiv / aids prevention efforts since such persons are not able to alert their partners and other close ones who they may have transferred the virus to or contracted the virus from over the years . further , in mozambique , unaids15 states that most high school girls are stigmatized and seen as bad or immoral for testing positive to hiv / aids because of the misconception that they acquired the virus through teenage sexual activities . rather , these high school girls might have contracted the virus at birth , during immunization , or from other non - sexual causes earlier on in their lives . also , efforts at managing the source and further spread of hiv / aids are often centered on the immediate people around the victim neglecting those who may have had prior contact in earlier years with the victim . it was disturbing to note that as much as 36.7% of the shs girls were either not sure or disagreed that , although symptoms may not be present , the virus was still transmittable . such persons therefore stood a higher chance of infection because this perception that only persons exhibiting symptoms of hiv / aids could transmit the virus will lead to them being less careful in their associations with seemingly healthy - looking persons . furthermore , studies on hiv / aids have shown that most relatives of persons living with hiv / aids ( plwha ) get infected because they failed to take precaution in their interactions and even shared personal items like blades , shaving sticks , tooth brushes , etc , with their seemingly healthy loved ones who though not currently exhibiting signs of the virus were infected . in this regard , students were less careful , associated freely , and shared personal effects with persons who did not exhibit any physical sign or symptom of hiv / aids but might still be infected . respondents were however able to clearly point out that hiv / aids may be transmitted through having unprotected sex with an infected partner , being born to a mother who is infected with hiv , or through the infected mother s breast milk . this is a positive result considering bennell et al16 findings advised that even though hiv / aids transmission through blood transfusion is rare as blood donors are screened before they donate , and blood products are tested before they are used , there is still a likelihood of infection via this route . their awareness has a positive impact on prevention of hiv / aids considering that sharing items like blades , toothbrushes , pins and needles , cooking and pocket knives , hair and nail clippers , and other sharp objects is a major cause of transmission in less developed countries.1719 in the words of one respondent , until i learnt about hiv / aids transmission last term , i used to share nail clippers , shaving sticks and toothbrushes with my cousins every time they came to visit . aside from hiv / aids transmissions through sharing personal items , there have been cases of hiv / aids transmissions during mass immunizations , blood donations , and during clinical injections . even though this is a rare occurrence in most countries because the needles and instruments are only used once and disposed of or properly sterilized , the same does not apply to less developed countries where with underresourced health sectors , the misconception that merely washing items makes them safe for repetitive use and the acute lack of instruments significantly increases their level of risk . almost all the respondents ( 90% ) were also aware that hiv / aids could be transmitted through bodily fluids like blood , semen , vaginal secretions , and breast milk . follow up questions however revealed that some shs girls still believed that physical contact with infected persons or sharing public space with infected persons could be a source of transmission of hiv / aids . the above is in sharp contrast to the content of educational materials and information provided by the ghana aids commission , national hiv / aids control program , and the mass media on hiv / aids transmission . in fact , a lot of hiv / aids awareness messages in ghana focus on this point because of the realization that poor awareness of this fact leads to high stigmatization , seclusion , and discrimination of plwha . such perceptions also explain the case made by world bank21 that plwha in rural areas and less developed countries are sometimes denied access to public transport , swimming in public pools , fetching water from public standpipes , engaging in contact sports , and other activities that involve physical proximity . another contentious issue in the hiv / aids dialogue in sub - saharan africa is the question of whether hiv / aids could be caused by witchcraft or other spiritual factors . evidence provided by gregson and garnett22 suggest that in the early to mid 1990s when the causes , means of transmission , and prevention were still controversial , most traditional societies in africa blamed hiv / aids , like other inexplicable ailments , on witchcraft and other spiritual causes . however , as information on the virus became available over the years such misconceptions have become less commonplace . it is thus a serious setback for hiv / aids prevention that in the 21st century and in the modern information and communications technology ( ict ) era where information in hiv / aids abounds , as much as 30% of the respondents believed fully or were not sure that witchcraft and other spiritual factors caused hiv / aids . if such perceptions are still being held by some people , then prevention and even management of new and existing cases of hiv / aids are seriously hindered . instead of getting attention from voluntary counseling and testing centers , hospitals , and other commissioned bodies , plwhas as well as persons suspected to be infected are sent to prayer camps for the virus to be exorcised . this not only increases victimization and stigmatization but also increases the risk of infecting church members , family , and friends as well as other victims in these prayer or witch camps . though trends in modern medicine are far advanced , the world is yet to possess a universally acceptable medically tested cure for the hiv virus.24 this is in spite of the numerous claims by both traditional and orthodox medicine practitioners as well as spiritual healers of curing hiv / aids patients . regardless of these claims and in the interest of public health , the ghana aids commission and the national hiv / aids control program maintains in ghana that no scientific cure exists for hiv / aids . rather , it supports medically approved ways including ( but not limited to ) the use of anti - retrovirals and regenerative health practices as the sanctioned means of managing the disease in order to prolong the life of infected persons , reduce the risk of transmission , and increase the quality of life of plwhas and their families . it was therefore surprising that as much as 50% of the shs girls believed that hiv / aids had a scientifically proven cure . even more controversial is the notion that herbal or traditional medicine as well as spiritual treatment could cure hiv / aids . this notwithstanding , it is not uncommon to hear religious and herbal healers make open claims of curing hiv / aids in both print and electronic media adverts often with purportedly infected persons giving testimonies of their healing . they were quick to share testimonies of several people they had seen on television or heard on radio who had been cured of hiv / aids . as put by one respondent : we have had several people in our church who have been cured of hiv / aids . however , some respondents disagreed on the veracity of the claims of being healed from hiv / aids accusing those pastors and traditional priests of being charlatans . this is because persons with the virus show symptoms not of aids but of the opportunistic infections like tuberculosis that they suffer as a result of the deficiency in their immune system . physically assessing their behaviors as well as change in their bodies can not be used in any instance as a means of detecting the absence or presence of the virus in any individual . this is in spite of the hiv / aids educational materials that clearly point out that only testing can be used as a determinant of who has or has not . further , as expressed in the previous sections , these persons are more at risk because of the tendency to relate carelessly with seemingly healthy looking persons who may be infected . when follow up questions were asked on how many had taken hiv / aids tests recently or knew their status , as much as 77% of respondents responded in the negative . this confirms the ghana aids commission campaign message that merely testing negative once in your life does not necessarily mean that your hiv status will always remain negative . the fact that 77% of shs girls had not taken any hiv test before is worrying considering that most girls are sexually active and/or are exposed to other means of contracting hiv / aids in their teens in developing countries.25,26 as no scientifically proven cure exists for hiv / aids , the universally accepted means of handling the pandemic is prevention . prevention of hiv / aids aims to reduce the risk of infection by encouraging individuals to make healthy sexual life choices . in the 21st century , prevention of hiv / aids is seen in the context of abstinence , being faithful , and wearing a condom ( a , b , and c ) . as seeley and barnett14 explain , until a cure for the virus surfaces , a , b , and c remain the best way of handling hiv / aids . in contrast however , 96.7% of respondents were aware that of a , b , and c , abstinence was the best means of preventing infection from the virus . the study also unearthed a worrying fact that some shs girls , even though aware of the likelihood of contracting the virus through unprotected sex , were still reluctant to use condoms and other forms of protection for various reasons . as shown in table 2 , as much as 51% of respondents were unlikely to use condoms during sexual intercourse . though few questions are raised about transmission of hiv / aids through the use of unsterilized instruments , condoms certainly remain highly controversial , since many churches and other religious groups believe strongly that condom use ( even among adults ) promotes promiscuity . the efficacy of condoms in preventing hiv transmission is also hotly debated , even among those without strong religious convictions.27 on one hand , it is argued that since condoms do not offer absolute protection ( because of improper use , reuse , inconsistent use , use while intoxicated , and manufacturing defects that lead to breakage or bursting ) , they should not be promoted as a method for preventing hiv transmission . bennell et al16 therefore advises , deriving from the above debate that recent efforts at creating awareness on hiv / aids , that prevention among the youth especially in developing countries must not focus merely on the use of condoms but on the correct use of condoms and also on the fact that condoms are not 100% safe but are subject to human error and manufacturing defects . the study reveals that shs girls in ghana generally had adequate knowledge on the basics of hiv / aids . the study also shows a positive impact of the hiv / aids awareness campaigns by the national hiv / aids control program , ghana aids commission , and other stakeholders in shs in ghana . however , knowledge on some vital information including the existence of herbal or scientifically proven cures , spiritual causes of hiv / aids , the transmission of hiv through body contact , and the determination of persons infected with hiv / aids by visual observation is deficient among segments of the students and needs further attention . particularly , the knowledge and awareness on hiv / aids related information did not translate to students decision to undergo voluntary testing with more than two - thirds of the student population not knowing their hiv status . the study therefore suggests that hiv / aids awareness campaigns among shs students in ghana in general must pay particular attention to specific issues on the transmission and management of hiv / aids . hiv / aids awareness and prevention efforts must also pay close attention to addressing the social , cultural , and religious barriers to condom usage in ghana .

ninety - nine purpose - bred male and female rm ( macaca mulatta ) of indian genetic background were used with the approval of the oregon national primate research center animal care and use committee , under the standards of the us national institutes of health guide for the care and use of laboratory animals . these animals were specific - pathogen free ( spf ) as defined by being free of cercopithicine herpesvirus 1 , d - type simian retrovirus , simian t - lymphotrophic virus type 1 , rhesus rhadinovirus , and mycobacterium tuberculosis . mhc-1 genotyping for common mamu alleles such as mamu - a*01/-a*02 and -b*08/-b*17 was performed by sequence - specific priming pcr , as described . the 99 rm include 15 rhcmv / siv vector - vaccinated rm ( 710 years of age ) with aviremic control of siv infection ( 14 with sivmac239 , 1 with sivmac251 ) after ir challenge ( 5 with short - term follow up ; 10 with medium- to long - term follow up ) , 52 rm ( 419 years of age ) vaccinated with rhcmv / siv or control rhcmv vectors or left unvaccinated prior to sivmac239 challenge ( 42 ivag , 10 iv ) , 12 siv+ rm ( 512 years of age ; 8 with progressive sivmac239 infection , 1 with spontaneously controlled sivmac239 infection , 3 with art - suppressed sivmac251 infection ) , and 20 siv - nave rm ( 414 years of age ) used as negative controls ( these include 4 rm vaccinated with rhcmv / siv vectors ) or as siv and rhcmv vector - nave recipients in the adoptive transfer experiments . early ( < 1 year ) follow up of 8 of the rhcmv / siv vector - vaccinated rm with long - term , aviremic control of siv infection was previously reported , with this study extending that follow - up from 1 to > 3 years . rhcmv / gag , rev / tat / nef , env and pol-1 and pol-2 vectors were administered subcutaneously at a dose of 5 10 plaque forming units per vector . the control ag - expressing rhcmv vector was used at a total dose of 2.5 10 plaque forming units to match the total dose of the rhcmv / siv vectors . all siv challenges ( ir , ivag , iv ) used a repeated limiting dose protocol using dosing designed to require > 1 challenge for infection of > 60% of challenged rm , and to infect all or nearly all challenged rm with 10 ( weekly ) challenges for ir or ivag inoculation ( 300 focus forming units ) and 3 ( every 3 week ) challenges for iv inoculation ( 0.2 focus - forming units ) . rm were considered siv - infected ( and challenge discontinued ) with the onset of plasma viral load 30 c. eq./ml and the de novo development of cd4 + and cd8 + t cell responses to sivvif , an siv ag not included in the rhcmv / siv vectors . rm were considered controllers if plasma viral load became undetectable ( < 30 c. eq./ml ) within 2 weeks of the initial positive plasma viral load and was then maintained below threshold for 3 consecutive determinations . rm with progressive siv infection were followed for 20 weeks post - infection , or if progression was rapid , until the onset of aids . art consisted of 2 reverse transcriptase inhibitors ( 20 mg / day tenofovir , 50 mg / day emtricitabine ) , an integrase inhibitor ( 240 mg / day raltegravir ) and a protease inhibitor ( 600 mg twice daily darunavir boosted with 100 mg twice daily ritonavir ) . selected rm were depleted of cd8 + lymphocytes by administration of 10 , 5 , 5 , and 5 mg per kg body weight of the cd8 monoclonal antibody ( mab ) m - t807r1 , a modified version of the cm - t807 humanized anti - cd8 mab with rhesus constant and variable framework regions ( http://nhpreagents.bidmc.harvard.edu ) , administered on days 0 , 3 , 7 and 10 , respectively . tissues obtained by biopsy or at necropsy were processed for mononuclear cell preparation , virologic , and/or immunohistologic analysis as previously described . for adoptive transfer experiments , freshly obtained peripheral blood and bm buffy coats were prepared by centrifugation ( 400xg for 20 minutes ) . these buffy coats and/or freshly obtained whole ln cell or splenocyte preparations were washed 3 times in saline prior to iv infusion with each rm receiving 3 10 peripheral blood leukocytes + 3 10 ln cells ( or in 1 rm , 3 10 bm leukocytes + 3 10 splenocytes ) over 1 hour . the construction and characterization of the strain 68 - 1-derived rhcmv / siv vectors , including rhcmv(gag ) , rhcmv(rtn ) , rhcmv(env ) and rhcmv(pol-1 ) and rhcmv(pol-2 ) , has been previously described . a control rhcmv vector expressing an m. tuberculosis ag85b - esat6 fusion protein under the control of the ef1- promoter was constructed with the same e / t recombination approach and rhcmv ( 68 - 1 ) bacterial artificial chromosome ( bac ) used for rhcmv / siv construction . the pathogenic siv challenge stocks used in these experiments were generated by expanding the sivmac239 clone ( or sivmac251 swarm ) in rm pbmc , and were titered using the smagi cell assay . ultrasensitive determinations of plasma viral loads at necropsy were achieved by concentrating virus from the larger volumes of material available by ultracentrifugation ( # 6041 10 ml tubes and # 4018 crown assembly , seton scientific ; t1270 rotor , thermo - sorvall scientific ) at 170,000 g for 30 min prior to processing rna . reactions were also run in triplicate and followed the analysis recommendations in palmer , et al . permitting per reaction determinations of 1 copy ( 2 of 3 positive amplifications ) and threshold sensitivities correspondingly lower , dependent on the amount of plasma input . plasma viral sequencing of rh20363 post - rebound was performed by synthesis of cdna with siv gene specific primers followed by sequencing using the single genome amplification strategy ( genbank accession # s kf439057 , kf439058 , kf439059 ) . quantitative assessment of siv dna and rna in isolated cells and tissues were determined by the quantitative hybrid real - time / digital rt - pcr and pcr assays , essentially as previously described , but with modifications to allow more efficient processing of samples larger than ~100 mg and to increase sample throughput . with respect to the former , the large tissue samples were directly disrupted in trireagent ( molecular research center , inc . ) utilizing two 7/16 stainless steel balls over 1015 stainless steel hex nuts ( 5.5 mm wide ) as grinding media , rather than first attempting to cryogenically pulverize the tissue . with respect to the latter , the rt - pcr and pcr assay conditions were also modified to reduce reaction volumes to allow use of 384-well plates . the cdna reactions were reduced to 15 l comprised of 10 l sample plus 5 l concentrated reaction cocktail and contained 2 mm sivnestr01 primer , 10 units rnasin , and 50 units momlv reverse transcriptase ( promega ) . the cdna synthesis stage of the thermal profile was optimal for momlv reverse transcription at 37c for 60 min , as opposed to 42c for 40 min . the rt - pcr pre - amplification reactions were 25 l in volume with 1.25 units of platinumtaq polymerase ( life technologies , inc . ) and 2.5 l of this reaction was transferred to 20 l of real - time pcr reaction mix with 1 unit of platinumtaq polymerase . for dna determinations , the preamplification reactions were 20 l in volume , comprised of 10 l sample and 10 l reaction cocktail ; 2 l of this nested reaction was transferred to 20 l of real - time pcr reaction mix . as previously described , for both rna and dna determinations , 12 replicate reactions were tested per sample including a spike of rna or dna internal control sequence standard in two of the 12 reactions to assess overall amplification efficiency and assess potential inhibition of the pcr or rt - pcr . the amount of dna or rna standard added to replicate reactions to monitor inhibition and pcr performance was typically 10 to 100 copies , depending on the anticipated level of siv sequences present . samples showing greater than a 5 cycle shift in amplification of the spiked standard , compared to amplification in the absence of specimen nucleic acid , corresponding to less than 74% overall amplification efficiency , were diluted and re - assayed . quantitative determinations for samples showing amplification in all replicates were derived directly with reference to a standard curve . quantitative determinations for samples showing fewer than 10 positive amplifications in replicates were derived from the frequency of positive amplifications , corresponding to the presence of at least one target copy in a reaction , according to a poisson distribution of a given median copy number per reaction . to avoid false positives in biopsy material , where the specimen size and total number of specimens is limited , we required a minimum of 2 positive reactions out of 10 for a sample to be considered positive . the presence of inducible , replication - competent siv in mononuclear cell preparations derived from different tissue sites at necropsy was detected by co - cultivation of 2.5 10 unfractionated cells from each tissue with 2 10 cemx174 cells ( x 20 replicates per tissue , cell numbers permitting ; cemx174 cells obtained from nih aids research & reference reagent program ) . after 18 days , each culture was stained for cd3 , cd4 and intracellular sivgag - p27 ( mab 55 - 2f12 ) with positive cultures based on 0.5% cemx174 cells with intracellular sivgag expression over background by flow cytometry . siv - specific cd4 + and cd8 + t cell responses were measured in blood and tissues by flow cytometric intracellular cytokine analysis , as previously described in detail . to determine t cell responses to siv peptide mixes or individual peptides , mononuclear cells were incubated with mixes of overlapping 15mer peptides comprising siv proteins or individual epitopic 8 - 10mer peptides ( with every individual peptide always at 2g / ml ) and the co - stimulatory molecules cd28 and cd49d ( bd biosciences ) for 1 hour , followed by addition of brefeldin a ( sigma - aldrich ) for an additional 8 hrs . to determine responses to autologous siv - infected cells , siv+ and siv target cells were produced by spinoculating ( or not spinoculating ) activated cd4 + t cells with sucrose - purified sivmac239 , followed by culturing the cells for 4 days and then purifying the cd4 + cells with cd4 microbeads and ls columns ( miltenyi biotec ) , as described . these cell preparations were > 95% cd4 + t cells and the siv - infected preparations were > 50% siv+ following enrichment . siv+ vs. siv t cells were then incubated with microbead - purified cd8 + t cells at an effector : target ratio of 40:1 under the same conditions used for peptide - specific flow cytometric intracellular cytokine analysis . following incubation , stimulated cells were stored at 4c until staining with combinations of fluorochrome - conjugated mabs including : sp34 - 2 ( cd3 ; pacific blue , percp - cy5.5 ) , l200 ( cd4 ; amcyan ) , sk-1 ( cd8 ; apc , percp - cy5.5 ) , cd28.2 ( cd28 ; pe , pe - texasred ) , dx2 ( cd95 ; apc , pe ) , 15053 ( ccr7 ; pacific blue ) , b56 ( ki-67 ; fitc ) , mab11 ( tnf- ; apc , fitc , pe ) , b27 ( ifn- ; apc , fitc ) , and fn50 ( cd69 ; pe , pe - texasred ) . analysis was performed using flowjo software ( tree star ) . in all analyses , gating on the lymphocyte population was followed by the separation of the cd3 t cell subset and progressive gating on cd4 + and cd8 + t cell subsets . antigen - responding cells in both cd4 + and cd8 + t cell populations were determined by their intracellular expression of cd69 and either or both of the ifn- and tnf cytokines . after subtracting background , the raw response frequencies were memory corrected , as previously described . in selected experiments , cells responding to siv peptides by production of either or both of ifn- and tnf titres of sivenv - specific abs were determined by neutralization of tissue culture - adapted sivmac251 using a luciferase reporter gene assay . immunohistochemistry was performed using a biotin - free polymer approach ( golden bridge international , inc . ) on 5m tissue sections mounted on glass slides , which were dewaxed and rehydrated with double - distilled h2o . heat induced epitope retrieval ( hier ) was performed by heating sections in 0.01% citraconic anhydride containing 0.05% tween-20 in a pressure cooker set at 122125c for 30 sec . slides were incubated with blocking buffer ( tbs with 0.05% tween-20 and 0.5% casein ) for 10 min . for apobec3 g , slides were incubated with rabbit anti - apobec3 g slides were washed in 1x tbs with 0.05% tween-20 , endogenous peroxidases blocked using 1.5% ( v / v ) h2o2 in tbs ( ph 7.4 ) for 10 min . , incubated with rabbit polink-2 hrp and developed with impact dab ( 3,3-diaminobenzidine ; vector laboratories ) . for isg15 staining , after the hier step , the slides were loaded on an intellipath autostainer ( biocare medical ) and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer ( tbs with 0.05% tween-20 and 0.5% casein ) for 10 min . and an endogenous peroxidase block using 1.5% ( v / v ) h2o2 in tbs ( ph 7.4 ) for 10 min . rabbit anti - isg ( 1:250 ; sigma hpa004627 ) was diluted in blocking buffer and incubated for 1h at room temperature . tissue sections were washed and developed using the rabbit polink-1 hrp staining system ( golden bridge international , inc . ) according to manufacturer s recommendations . all slides were washed in h2o , counterstained with hematoxylin , mounted in permount ( fisher scientific ) , and scanned at high magnification ( x200 ) using the scanscope cs system ( aperio technologies ) yielding high - resolution data from the entire tissue section . representative regions of interest ( 500 mm ) were identified and high - resolution images extracted from these whole - tissue scans . the percent area of the t cell zone that stained for apobec3 g and isg15 were quantified using photoshop cs5 and fovea tools . the rm used in the vaccine efficacy analysis were randomly assigned to vaccine groups with randomization stratified to balance groups for expression of protective mhc alleles . the criteria for categorizing post - challenge rm into protected vs. unprotected groups were established previously . experimenters were not explicitly blinded to the treatment assignments of the rm , nor were the analyses conducted by blinded investigators . all statistical analyses were conducted using nonparametric and model - independent analysis procedures either for the main analysis or as a sensitivity analysis , and in every sensitivity analysis the result was qualitatively consistent with the main analysis . the only exceptions were the time series analyses , which were conducted with two model - based ( regression ) approaches ; the residuals of these analyses were evaluated and found to be consistent with homoschedasticity and normality requirements , and the results were consistent across approaches . for comparisons of continuous - valued data from independent samples , we applied bivariate mann - whitney u tests , also known as wilcoxon rank sum tests . for comparisons of dichotomous values across groups , we applied fisher s exact tests . we estimated confidence bounds for binomial proportions using the wilson score method , as described in agresti and coull . we compared group means of positivity frequencies for which we have repeated binary measures on individual rm using mixed effects logistic regression ( with individual rm mean deviations from group means modeled as a normally - distributed random effect ) . we compared confidence intervals for rm groups to confidence intervals for individual rm ( from other groups ) by directly determining overlap of the intervals ( and we used the estimated random effect variance and estimated group means to conduct z - tests in sensitivity analyses , which yielded consistent results ) . we conducted time series analyses using standard linear regression with time as the primary predictor , and we used gaussian first - order autoregressive process models in sensitivity analyses , which yielded consistent results . all tests were conducted as two - tailed tests with a type - i error rate of 5% . ninety - nine purpose - bred male and female rm ( macaca mulatta ) of indian genetic background were used with the approval of the oregon national primate research center animal care and use committee , under the standards of the us national institutes of health guide for the care and use of laboratory animals . these animals were specific - pathogen free ( spf ) as defined by being free of cercopithicine herpesvirus 1 , d - type simian retrovirus , simian t - lymphotrophic virus type 1 , rhesus rhadinovirus , and mycobacterium tuberculosis . mhc-1 genotyping for common mamu alleles such as mamu - a*01/-a*02 and -b*08/-b*17 was performed by sequence - specific priming pcr , as described . the 99 rm include 15 rhcmv / siv vector - vaccinated rm ( 710 years of age ) with aviremic control of siv infection ( 14 with sivmac239 , 1 with sivmac251 ) after ir challenge ( 5 with short - term follow up ; 10 with medium- to long - term follow up ) , 52 rm ( 419 years of age ) vaccinated with rhcmv / siv or control rhcmv vectors or left unvaccinated prior to sivmac239 challenge ( 42 ivag , 10 iv ) , 12 siv+ rm ( 512 years of age ; 8 with progressive sivmac239 infection , 1 with spontaneously controlled sivmac239 infection , 3 with art - suppressed sivmac251 infection ) , and 20 siv - nave rm ( 414 years of age ) used as negative controls ( these include 4 rm vaccinated with rhcmv / siv vectors ) or as siv and rhcmv vector - nave recipients in the adoptive transfer experiments . early ( < 1 year ) follow up of 8 of the rhcmv / siv vector - vaccinated rm with long - term , aviremic control of siv infection was previously reported , with this study extending that follow - up from 1 to > 3 years . rhcmv / gag , rev / tat / nef , env and pol-1 and pol-2 vectors were administered subcutaneously at a dose of 5 10 plaque forming units per vector . the control ag - expressing rhcmv vector was used at a total dose of 2.5 10 plaque forming units to match the total dose of the rhcmv / siv vectors . all siv challenges ( ir , ivag , iv ) used a repeated limiting dose protocol using dosing designed to require > 1 challenge for infection of > 60% of challenged rm , and to infect all or nearly all challenged rm with 10 ( weekly ) challenges for ir or ivag inoculation ( 300 focus forming units ) and 3 ( every 3 week ) challenges for iv inoculation ( 0.2 focus - forming units ) . rm were considered siv - infected ( and challenge discontinued ) with the onset of plasma viral load 30 c. eq./ml and the de novo development of cd4 + and cd8 + t cell responses to sivvif , an siv ag not included in the rhcmv / siv vectors . rm were considered controllers if plasma viral load became undetectable ( < 30 c. eq./ml ) within 2 weeks of the initial positive plasma viral load and was then maintained below threshold for 3 consecutive determinations . rm with progressive siv infection were followed for 20 weeks post - infection , or if progression was rapid , until the onset of aids . art consisted of 2 reverse transcriptase inhibitors ( 20 mg / day tenofovir , 50 mg / day emtricitabine ) , an integrase inhibitor ( 240 mg / day raltegravir ) and a protease inhibitor ( 600 mg twice daily darunavir boosted with 100 mg twice daily ritonavir ) . selected rm were depleted of cd8 + lymphocytes by administration of 10 , 5 , 5 , and 5 mg per kg body weight of the cd8 monoclonal antibody ( mab ) m - t807r1 , a modified version of the cm - t807 humanized anti - cd8 mab with rhesus constant and variable framework regions ( http://nhpreagents.bidmc.harvard.edu ) , administered on days 0 , 3 , 7 and 10 , respectively . tissues obtained by biopsy or at necropsy were processed for mononuclear cell preparation , virologic , and/or immunohistologic analysis as previously described . for adoptive transfer experiments , freshly obtained peripheral blood and bm buffy coats were prepared by centrifugation ( 400xg for 20 minutes ) . these buffy coats and/or freshly obtained whole ln cell or splenocyte preparations were washed 3 times in saline prior to iv infusion with each rm receiving 3 10 peripheral blood leukocytes + 3 10 ln cells ( or in 1 rm , 3 10 bm leukocytes + 3 10 splenocytes ) over 1 hour . the construction and characterization of the strain 68 - 1-derived rhcmv / siv vectors , including rhcmv(gag ) , rhcmv(rtn ) , rhcmv(env ) and rhcmv(pol-1 ) and rhcmv(pol-2 ) , has been previously described . a control rhcmv vector expressing an m. tuberculosis ag85b - esat6 fusion protein under the control of the ef1- promoter was constructed with the same e / t recombination approach and rhcmv ( 68 - 1 ) bacterial artificial chromosome ( bac ) used for rhcmv / siv construction . the pathogenic siv challenge stocks used in these experiments were generated by expanding the sivmac239 clone ( or sivmac251 swarm ) in rm pbmc , and were titered using the smagi cell assay . ultrasensitive determinations of plasma viral loads at necropsy were achieved by concentrating virus from the larger volumes of material available by ultracentrifugation ( # 6041 10 ml tubes and # 4018 crown assembly , seton scientific ; t1270 rotor , thermo - sorvall scientific ) at 170,000 g for 30 min prior to processing rna . reactions were also run in triplicate and followed the analysis recommendations in palmer , et al . permitting per reaction determinations of 1 copy ( 2 of 3 positive amplifications ) and threshold sensitivities correspondingly lower , dependent on the amount of plasma input . plasma viral sequencing of rh20363 post - rebound was performed by synthesis of cdna with siv gene specific primers followed by sequencing using the single genome amplification strategy ( genbank accession # s kf439057 , kf439058 , kf439059 ) . quantitative assessment of siv dna and rna in isolated cells and tissues were determined by the quantitative hybrid real - time / digital rt - pcr and pcr assays , essentially as previously described , but with modifications to allow more efficient processing of samples larger than ~100 mg and to increase sample throughput . with respect to the former , the large tissue samples were directly disrupted in trireagent ( molecular research center , inc . ) utilizing two 7/16 stainless steel balls over 1015 stainless steel hex nuts ( 5.5 mm wide ) as grinding media , rather than first attempting to cryogenically pulverize the tissue . with respect to the latter , the rt - pcr and pcr assay conditions were also modified to reduce reaction volumes to allow use of 384-well plates . the cdna reactions were reduced to 15 l comprised of 10 l sample plus 5 l concentrated reaction cocktail and contained 2 mm sivnestr01 primer , 10 units rnasin , and 50 units momlv reverse transcriptase ( promega ) . the cdna synthesis stage of the thermal profile was optimal for momlv reverse transcription at 37c for 60 min , as opposed to 42c for 40 min . the rt - pcr pre - amplification reactions were 25 l in volume with 1.25 units of platinumtaq polymerase ( life technologies , inc . ) and 2.5 l of this reaction was transferred to 20 l of real - time pcr reaction mix with 1 unit of platinumtaq polymerase . for dna determinations , the preamplification reactions were 20 l in volume , comprised of 10 l sample and 10 l reaction cocktail ; 2 l of this nested reaction was transferred to 20 l of real - time pcr reaction mix . as previously described , for both rna and dna determinations , 12 replicate reactions were tested per sample including a spike of rna or dna internal control sequence standard in two of the 12 reactions to assess overall amplification efficiency and assess potential inhibition of the pcr or rt - pcr . the amount of dna or rna standard added to replicate reactions to monitor inhibition and pcr performance was typically 10 to 100 copies , depending on the anticipated level of siv sequences present . samples showing greater than a 5 cycle shift in amplification of the spiked standard , compared to amplification in the absence of specimen nucleic acid , corresponding to less than 74% overall amplification efficiency , were diluted and re - assayed . quantitative determinations for samples showing amplification in all replicates were derived directly with reference to a standard curve . quantitative determinations for samples showing fewer than 10 positive amplifications in replicates were derived from the frequency of positive amplifications , corresponding to the presence of at least one target copy in a reaction , according to a poisson distribution of a given median copy number per reaction . to avoid false positives in biopsy material , where the specimen size and total number of specimens is limited , we required a minimum of 2 positive reactions out of 10 for a sample to be considered positive . the presence of inducible , replication - competent siv in mononuclear cell preparations derived from different tissue sites at necropsy was detected by co - cultivation of 2.5 10 unfractionated cells from each tissue with 2 10 cemx174 cells ( x 20 replicates per tissue , cell numbers permitting ; cemx174 cells obtained from nih aids research & reference reagent program ) . after 18 days , each culture was stained for cd3 , cd4 and intracellular sivgag - p27 ( mab 55 - 2f12 ) with positive cultures based on 0.5% cemx174 cells with intracellular sivgag expression over background by flow cytometry . siv - specific cd4 + and cd8 + t cell responses were measured in blood and tissues by flow cytometric intracellular cytokine analysis , as previously described in detail . to determine t cell responses to siv peptide mixes or individual peptides , mononuclear cells were incubated with mixes of overlapping 15mer peptides comprising siv proteins or individual epitopic 8 - 10mer peptides ( with every individual peptide always at 2g / ml ) and the co - stimulatory molecules cd28 and cd49d ( bd biosciences ) for 1 hour , followed by addition of brefeldin a ( sigma - aldrich ) for an additional 8 hrs . co - stimulation without antigen served as a background control . to determine responses to autologous siv - infected cells , siv+ and siv target cells were produced by spinoculating ( or not spinoculating ) activated cd4 + t cells with sucrose - purified sivmac239 , followed by culturing the cells for 4 days and then purifying the cd4 + cells with cd4 microbeads and ls columns ( miltenyi biotec ) , as described . these cell preparations were > 95% cd4 + t cells and the siv - infected preparations were > 50% siv+ following enrichment . siv+ vs. siv t cells were then incubated with microbead - purified cd8 + t cells at an effector : target ratio of 40:1 under the same conditions used for peptide - specific flow cytometric intracellular cytokine analysis . following incubation , stimulated cells were stored at 4c until staining with combinations of fluorochrome - conjugated mabs including : sp34 - 2 ( cd3 ; pacific blue , percp - cy5.5 ) , l200 ( cd4 ; amcyan ) , sk-1 ( cd8 ; apc , percp - cy5.5 ) , cd28.2 ( cd28 ; pe , pe - texasred ) , dx2 ( cd95 ; apc , pe ) , 15053 ( ccr7 ; pacific blue ) , b56 ( ki-67 ; fitc ) , mab11 ( tnf- ; apc , fitc , pe ) , b27 ( ifn- ; apc , fitc ) , and fn50 ( cd69 ; pe , pe - texasred ) . analysis was performed using flowjo software ( tree star ) . in all analyses , gating on the lymphocyte population was followed by the separation of the cd3 t cell subset and progressive gating on cd4 + and cd8 + t cell subsets . antigen - responding cells in both cd4 + and cd8 + t cell populations were determined by their intracellular expression of cd69 and either or both of the ifn- and tnf cytokines . after subtracting background , the raw response frequencies were memory corrected , as previously described . in selected experiments , cells responding to siv peptides by production of either or both of ifn- and tnf titres of sivenv - specific abs were determined by neutralization of tissue culture - adapted sivmac251 using a luciferase reporter gene assay . immunohistochemistry was performed using a biotin - free polymer approach ( golden bridge international , inc . ) on 5m tissue sections mounted on glass slides , which were dewaxed and rehydrated with double - distilled h2o . heat induced epitope retrieval ( hier ) was performed by heating sections in 0.01% citraconic anhydride containing 0.05% tween-20 in a pressure cooker set at 122125c for 30 sec . slides were incubated with blocking buffer ( tbs with 0.05% tween-20 and 0.5% casein ) for 10 min . for apobec3 g , slides were incubated with rabbit anti - apobec3 g ( 1:100 ; sigma hpa001812 ) diluted in blocking buffer overnight at 4c . slides were washed in 1x tbs with 0.05% tween-20 , endogenous peroxidases blocked using 1.5% ( v / v ) h2o2 in tbs ( ph 7.4 ) for 10 min . , incubated with rabbit polink-2 hrp and developed with impact dab ( 3,3-diaminobenzidine ; vector laboratories ) . for isg15 staining , after the hier step , the slides were loaded on an intellipath autostainer ( biocare medical ) and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer ( tbs with 0.05% tween-20 and 0.5% casein ) for 10 min . and an endogenous peroxidase block using 1.5% ( v / v ) h2o2 in tbs ( ph 7.4 ) for 10 min . rabbit anti - isg ( 1:250 ; sigma hpa004627 ) was diluted in blocking buffer and incubated for 1h at room temperature . tissue sections were washed and developed using the rabbit polink-1 hrp staining system ( golden bridge international , inc . ) according to manufacturer s recommendations . all slides were washed in h2o , counterstained with hematoxylin , mounted in permount ( fisher scientific ) , and scanned at high magnification ( x200 ) using the scanscope cs system ( aperio technologies ) yielding high - resolution data from the entire tissue section . representative regions of interest ( 500 mm ) were identified and high - resolution images extracted from these whole - tissue scans . the percent area of the t cell zone that stained for apobec3 g and isg15 were quantified using photoshop cs5 and fovea tools . the rm used in the vaccine efficacy analysis were randomly assigned to vaccine groups with randomization stratified to balance groups for expression of protective mhc alleles . the criteria for categorizing post - challenge rm into protected vs. unprotected groups were established previously . experimenters were not explicitly blinded to the treatment assignments of the rm , nor were the analyses conducted by blinded investigators . all statistical analyses were conducted using nonparametric and model - independent analysis procedures either for the main analysis or as a sensitivity analysis , and in every sensitivity analysis the result was qualitatively consistent with the main analysis . the only exceptions were the time series analyses , which were conducted with two model - based ( regression ) approaches ; the residuals of these analyses were evaluated and found to be consistent with homoschedasticity and normality requirements , and the results were consistent across approaches . for comparisons of continuous - valued data from independent samples , we applied bivariate mann - whitney u tests , also known as wilcoxon rank sum tests . for comparisons of dichotomous values across groups , we applied fisher s exact tests . we estimated confidence bounds for binomial proportions using the wilson score method , as described in agresti and coull . we compared group means of positivity frequencies for which we have repeated binary measures on individual rm using mixed effects logistic regression ( with individual rm mean deviations from group means modeled as a normally - distributed random effect ) . we compared confidence intervals for rm groups to confidence intervals for individual rm ( from other groups ) by directly determining overlap of the intervals ( and we used the estimated random effect variance and estimated group means to conduct z - tests in sensitivity analyses , which yielded consistent results ) . we conducted time series analyses using standard linear regression with time as the primary predictor , and we used gaussian first - order autoregressive process models in sensitivity analyses , which yielded consistent results . all tests were conducted as two - tailed tests with a type - i error rate of 5% .

established infections with the human and simian immunodeficiency viruses ( hiv , siv ) are thought to be permanent with even the most effective immune responses and anti - retroviral therapies ( art ) only able to control , but not clear , these infections14 . whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of hiv - infected individuals . we recently reported that ~50% of rhesus macaques ( rm ) vaccinated with siv protein - expressing rhesus cytomegalovirus ( rhcmv / siv ) vectors manifest durable , aviremic control of infection with highly pathogenic sivmac2395 . here , we demonstrate that regardless of route of challenge , rhcmv / siv vector - elicited immune responses control sivmac239 after demonstrable lymphatic and hematogenous viral dissemination , and that replication - competent siv persists in multiple sites for weeks to months . however , over time , protected rm lost signs of siv infection , showing a consistent lack of measurable plasma or tissue - associated virus using ultrasensitive assays , and loss of t cell reactivity to siv determinants not in the vaccine . extensive ultrasensitive rt - pcr and pcr analysis of tissues from rhcmv / siv vector - protected rm necropsied 69172 weeks after challenge did not detect siv rna or dna over background , and replication - competent siv was not detected in these rm by extensive co - culture analysis of tissues or by adoptive transfer of 60 million hematolymphoid cells to nave rm . these data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection , and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory t cell - mediated immune surveillance elicited and maintained by cmv vectors .

FULL METHODS Rhesus macaques Vectors and viruses Viral detection assays Immunologic assays Immunohistology Statistical analysis Supplementary Material

the 99 rm include 15 rhcmv / siv vector - vaccinated rm ( 710 years of age ) with aviremic control of siv infection ( 14 with sivmac239 , 1 with sivmac251 ) after ir challenge ( 5 with short - term follow up ; 10 with medium- to long - term follow up ) , 52 rm ( 419 years of age ) vaccinated with rhcmv / siv or control rhcmv vectors or left unvaccinated prior to sivmac239 challenge ( 42 ivag , 10 iv ) , 12 siv+ rm ( 512 years of age ; 8 with progressive sivmac239 infection , 1 with spontaneously controlled sivmac239 infection , 3 with art - suppressed sivmac251 infection ) , and 20 siv - nave rm ( 414 years of age ) used as negative controls ( these include 4 rm vaccinated with rhcmv / siv vectors ) or as siv and rhcmv vector - nave recipients in the adoptive transfer experiments . early ( < 1 year ) follow up of 8 of the rhcmv / siv vector - vaccinated rm with long - term , aviremic control of siv infection was previously reported , with this study extending that follow - up from 1 to > 3 years . rhcmv / gag , rev / tat / nef , env and pol-1 and pol-2 vectors were administered subcutaneously at a dose of 5 10 plaque forming units per vector . the control ag - expressing rhcmv vector was used at a total dose of 2.5 10 plaque forming units to match the total dose of the rhcmv / siv vectors . all siv challenges ( ir , ivag , iv ) used a repeated limiting dose protocol using dosing designed to require > 1 challenge for infection of > 60% of challenged rm , and to infect all or nearly all challenged rm with 10 ( weekly ) challenges for ir or ivag inoculation ( 300 focus forming units ) and 3 ( every 3 week ) challenges for iv inoculation ( 0.2 focus - forming units ) . rm were considered siv - infected ( and challenge discontinued ) with the onset of plasma viral load 30 c. eq./ml and the de novo development of cd4 + and cd8 + t cell responses to sivvif , an siv ag not included in the rhcmv / siv vectors . rm with progressive siv infection were followed for 20 weeks post - infection , or if progression was rapid , until the onset of aids . selected rm were depleted of cd8 + lymphocytes by administration of 10 , 5 , 5 , and 5 mg per kg body weight of the cd8 monoclonal antibody ( mab ) m - t807r1 , a modified version of the cm - t807 humanized anti - cd8 mab with rhesus constant and variable framework regions ( http://nhpreagents.bidmc.harvard.edu ) , administered on days 0 , 3 , 7 and 10 , respectively . the construction and characterization of the strain 68 - 1-derived rhcmv / siv vectors , including rhcmv(gag ) , rhcmv(rtn ) , rhcmv(env ) and rhcmv(pol-1 ) and rhcmv(pol-2 ) , has been previously described . a control rhcmv vector expressing an m. tuberculosis ag85b - esat6 fusion protein under the control of the ef1- promoter was constructed with the same e / t recombination approach and rhcmv ( 68 - 1 ) bacterial artificial chromosome ( bac ) used for rhcmv / siv construction . the pathogenic siv challenge stocks used in these experiments were generated by expanding the sivmac239 clone ( or sivmac251 swarm ) in rm pbmc , and were titered using the smagi cell assay . plasma viral sequencing of rh20363 post - rebound was performed by synthesis of cdna with siv gene specific primers followed by sequencing using the single genome amplification strategy ( genbank accession # s kf439057 , kf439058 , kf439059 ) . quantitative assessment of siv dna and rna in isolated cells and tissues were determined by the quantitative hybrid real - time / digital rt - pcr and pcr assays , essentially as previously described , but with modifications to allow more efficient processing of samples larger than ~100 mg and to increase sample throughput . with respect to the former , the large tissue samples were directly disrupted in trireagent ( molecular research center , inc . ) with respect to the latter , the rt - pcr and pcr assay conditions were also modified to reduce reaction volumes to allow use of 384-well plates . the cdna reactions were reduced to 15 l comprised of 10 l sample plus 5 l concentrated reaction cocktail and contained 2 mm sivnestr01 primer , 10 units rnasin , and 50 units momlv reverse transcriptase ( promega ) . the rt - pcr pre - amplification reactions were 25 l in volume with 1.25 units of platinumtaq polymerase ( life technologies , inc . ) as previously described , for both rna and dna determinations , 12 replicate reactions were tested per sample including a spike of rna or dna internal control sequence standard in two of the 12 reactions to assess overall amplification efficiency and assess potential inhibition of the pcr or rt - pcr . the amount of dna or rna standard added to replicate reactions to monitor inhibition and pcr performance was typically 10 to 100 copies , depending on the anticipated level of siv sequences present . quantitative determinations for samples showing fewer than 10 positive amplifications in replicates were derived from the frequency of positive amplifications , corresponding to the presence of at least one target copy in a reaction , according to a poisson distribution of a given median copy number per reaction . to avoid false positives in biopsy material , where the specimen size and total number of specimens is limited , we required a minimum of 2 positive reactions out of 10 for a sample to be considered positive . the presence of inducible , replication - competent siv in mononuclear cell preparations derived from different tissue sites at necropsy was detected by co - cultivation of 2.5 10 unfractionated cells from each tissue with 2 10 cemx174 cells ( x 20 replicates per tissue , cell numbers permitting ; cemx174 cells obtained from nih aids research & reference reagent program ) . to determine t cell responses to siv peptide mixes or individual peptides , mononuclear cells were incubated with mixes of overlapping 15mer peptides comprising siv proteins or individual epitopic 8 - 10mer peptides ( with every individual peptide always at 2g / ml ) and the co - stimulatory molecules cd28 and cd49d ( bd biosciences ) for 1 hour , followed by addition of brefeldin a ( sigma - aldrich ) for an additional 8 hrs . to determine responses to autologous siv - infected cells , siv+ and siv target cells were produced by spinoculating ( or not spinoculating ) activated cd4 + t cells with sucrose - purified sivmac239 , followed by culturing the cells for 4 days and then purifying the cd4 + cells with cd4 microbeads and ls columns ( miltenyi biotec ) , as described . antigen - responding cells in both cd4 + and cd8 + t cell populations were determined by their intracellular expression of cd69 and either or both of the ifn- and tnf cytokines . in selected experiments , cells responding to siv peptides by production of either or both of ifn- and tnf titres of sivenv - specific abs were determined by neutralization of tissue culture - adapted sivmac251 using a luciferase reporter gene assay . for isg15 staining , after the hier step , the slides were loaded on an intellipath autostainer ( biocare medical ) and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer ( tbs with 0.05% tween-20 and 0.5% casein ) for 10 min . the percent area of the t cell zone that stained for apobec3 g and isg15 were quantified using photoshop cs5 and fovea tools . the rm used in the vaccine efficacy analysis were randomly assigned to vaccine groups with randomization stratified to balance groups for expression of protective mhc alleles . all statistical analyses were conducted using nonparametric and model - independent analysis procedures either for the main analysis or as a sensitivity analysis , and in every sensitivity analysis the result was qualitatively consistent with the main analysis . the only exceptions were the time series analyses , which were conducted with two model - based ( regression ) approaches ; the residuals of these analyses were evaluated and found to be consistent with homoschedasticity and normality requirements , and the results were consistent across approaches . for comparisons of continuous - valued data from independent samples , we applied bivariate mann - whitney u tests , also known as wilcoxon rank sum tests . we conducted time series analyses using standard linear regression with time as the primary predictor , and we used gaussian first - order autoregressive process models in sensitivity analyses , which yielded consistent results . the 99 rm include 15 rhcmv / siv vector - vaccinated rm ( 710 years of age ) with aviremic control of siv infection ( 14 with sivmac239 , 1 with sivmac251 ) after ir challenge ( 5 with short - term follow up ; 10 with medium- to long - term follow up ) , 52 rm ( 419 years of age ) vaccinated with rhcmv / siv or control rhcmv vectors or left unvaccinated prior to sivmac239 challenge ( 42 ivag , 10 iv ) , 12 siv+ rm ( 512 years of age ; 8 with progressive sivmac239 infection , 1 with spontaneously controlled sivmac239 infection , 3 with art - suppressed sivmac251 infection ) , and 20 siv - nave rm ( 414 years of age ) used as negative controls ( these include 4 rm vaccinated with rhcmv / siv vectors ) or as siv and rhcmv vector - nave recipients in the adoptive transfer experiments . early ( < 1 year ) follow up of 8 of the rhcmv / siv vector - vaccinated rm with long - term , aviremic control of siv infection was previously reported , with this study extending that follow - up from 1 to > 3 years . the control ag - expressing rhcmv vector was used at a total dose of 2.5 10 plaque forming units to match the total dose of the rhcmv / siv vectors . all siv challenges ( ir , ivag , iv ) used a repeated limiting dose protocol using dosing designed to require > 1 challenge for infection of > 60% of challenged rm , and to infect all or nearly all challenged rm with 10 ( weekly ) challenges for ir or ivag inoculation ( 300 focus forming units ) and 3 ( every 3 week ) challenges for iv inoculation ( 0.2 focus - forming units ) . rm were considered siv - infected ( and challenge discontinued ) with the onset of plasma viral load 30 c. eq./ml and the de novo development of cd4 + and cd8 + t cell responses to sivvif , an siv ag not included in the rhcmv / siv vectors . rm with progressive siv infection were followed for 20 weeks post - infection , or if progression was rapid , until the onset of aids . selected rm were depleted of cd8 + lymphocytes by administration of 10 , 5 , 5 , and 5 mg per kg body weight of the cd8 monoclonal antibody ( mab ) m - t807r1 , a modified version of the cm - t807 humanized anti - cd8 mab with rhesus constant and variable framework regions ( http://nhpreagents.bidmc.harvard.edu ) , administered on days 0 , 3 , 7 and 10 , respectively . the construction and characterization of the strain 68 - 1-derived rhcmv / siv vectors , including rhcmv(gag ) , rhcmv(rtn ) , rhcmv(env ) and rhcmv(pol-1 ) and rhcmv(pol-2 ) , has been previously described . a control rhcmv vector expressing an m. tuberculosis ag85b - esat6 fusion protein under the control of the ef1- promoter was constructed with the same e / t recombination approach and rhcmv ( 68 - 1 ) bacterial artificial chromosome ( bac ) used for rhcmv / siv construction . the pathogenic siv challenge stocks used in these experiments were generated by expanding the sivmac239 clone ( or sivmac251 swarm ) in rm pbmc , and were titered using the smagi cell assay . plasma viral sequencing of rh20363 post - rebound was performed by synthesis of cdna with siv gene specific primers followed by sequencing using the single genome amplification strategy ( genbank accession # s kf439057 , kf439058 , kf439059 ) . quantitative assessment of siv dna and rna in isolated cells and tissues were determined by the quantitative hybrid real - time / digital rt - pcr and pcr assays , essentially as previously described , but with modifications to allow more efficient processing of samples larger than ~100 mg and to increase sample throughput . with respect to the latter , the rt - pcr and pcr assay conditions were also modified to reduce reaction volumes to allow use of 384-well plates . the cdna reactions were reduced to 15 l comprised of 10 l sample plus 5 l concentrated reaction cocktail and contained 2 mm sivnestr01 primer , 10 units rnasin , and 50 units momlv reverse transcriptase ( promega ) . the rt - pcr pre - amplification reactions were 25 l in volume with 1.25 units of platinumtaq polymerase ( life technologies , inc . ) as previously described , for both rna and dna determinations , 12 replicate reactions were tested per sample including a spike of rna or dna internal control sequence standard in two of the 12 reactions to assess overall amplification efficiency and assess potential inhibition of the pcr or rt - pcr . the amount of dna or rna standard added to replicate reactions to monitor inhibition and pcr performance was typically 10 to 100 copies , depending on the anticipated level of siv sequences present . samples showing greater than a 5 cycle shift in amplification of the spiked standard , compared to amplification in the absence of specimen nucleic acid , corresponding to less than 74% overall amplification efficiency , were diluted and re - assayed . quantitative determinations for samples showing fewer than 10 positive amplifications in replicates were derived from the frequency of positive amplifications , corresponding to the presence of at least one target copy in a reaction , according to a poisson distribution of a given median copy number per reaction . to avoid false positives in biopsy material , where the specimen size and total number of specimens is limited , we required a minimum of 2 positive reactions out of 10 for a sample to be considered positive . the presence of inducible , replication - competent siv in mononuclear cell preparations derived from different tissue sites at necropsy was detected by co - cultivation of 2.5 10 unfractionated cells from each tissue with 2 10 cemx174 cells ( x 20 replicates per tissue , cell numbers permitting ; cemx174 cells obtained from nih aids research & reference reagent program ) . after 18 days , each culture was stained for cd3 , cd4 and intracellular sivgag - p27 ( mab 55 - 2f12 ) with positive cultures based on 0.5% cemx174 cells with intracellular sivgag expression over background by flow cytometry . to determine t cell responses to siv peptide mixes or individual peptides , mononuclear cells were incubated with mixes of overlapping 15mer peptides comprising siv proteins or individual epitopic 8 - 10mer peptides ( with every individual peptide always at 2g / ml ) and the co - stimulatory molecules cd28 and cd49d ( bd biosciences ) for 1 hour , followed by addition of brefeldin a ( sigma - aldrich ) for an additional 8 hrs . co - stimulation without antigen served as a background control . to determine responses to autologous siv - infected cells , siv+ and siv target cells were produced by spinoculating ( or not spinoculating ) activated cd4 + t cells with sucrose - purified sivmac239 , followed by culturing the cells for 4 days and then purifying the cd4 + cells with cd4 microbeads and ls columns ( miltenyi biotec ) , as described . following incubation , stimulated cells were stored at 4c until staining with combinations of fluorochrome - conjugated mabs including : sp34 - 2 ( cd3 ; pacific blue , percp - cy5.5 ) , l200 ( cd4 ; amcyan ) , sk-1 ( cd8 ; apc , percp - cy5.5 ) , cd28.2 ( cd28 ; pe , pe - texasred ) , dx2 ( cd95 ; apc , pe ) , 15053 ( ccr7 ; pacific blue ) , b56 ( ki-67 ; fitc ) , mab11 ( tnf- ; apc , fitc , pe ) , b27 ( ifn- ; apc , fitc ) , and fn50 ( cd69 ; pe , pe - texasred ) . for apobec3 g , slides were incubated with rabbit anti - apobec3 g ( 1:100 ; sigma hpa001812 ) diluted in blocking buffer overnight at 4c . for isg15 staining , after the hier step , the slides were loaded on an intellipath autostainer ( biocare medical ) and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer ( tbs with 0.05% tween-20 and 0.5% casein ) for 10 min . all slides were washed in h2o , counterstained with hematoxylin , mounted in permount ( fisher scientific ) , and scanned at high magnification ( x200 ) using the scanscope cs system ( aperio technologies ) yielding high - resolution data from the entire tissue section . the rm used in the vaccine efficacy analysis were randomly assigned to vaccine groups with randomization stratified to balance groups for expression of protective mhc alleles . experimenters were not explicitly blinded to the treatment assignments of the rm , nor were the analyses conducted by blinded investigators . all statistical analyses were conducted using nonparametric and model - independent analysis procedures either for the main analysis or as a sensitivity analysis , and in every sensitivity analysis the result was qualitatively consistent with the main analysis . the only exceptions were the time series analyses , which were conducted with two model - based ( regression ) approaches ; the residuals of these analyses were evaluated and found to be consistent with homoschedasticity and normality requirements , and the results were consistent across approaches . for comparisons of continuous - valued data from independent samples , we applied bivariate mann - whitney u tests , also known as wilcoxon rank sum tests . for comparisons of dichotomous values across groups , we applied fisher s exact tests .

the major reactive aldehyde produced during peroxidation of polyunsaturated fatty acids , malondialdehyde ( mda ) , reacts with nucleophilic sites of dna and protein to form oxopropenyl adducts . reaction of mda with dna produces primarily 3-(2-deoxy--d - erythro - pentofuranosyl)pyrimido[1,2-]purine-10(3h)-one ( m1dg ) and lesser amounts of 2-oxopropenyl - deoxyadenosine ( figure 1 ) . m1dg is mutagenic in vitro and in vivo , and 2-oxopropenyl - deoxyadenosine alters dna polymerase activity . both species can react with da or dg , resulting in oxypropenylation of the base . the reaction of mda with protein occurs predominantly at lysine residues under physiological conditions . protein adduct formation is rudimentary , as the exact targets and sites of adduction are largely unknown . however , a particularly interesting effect of mda exposure in human cells is the inhibition of nucleotide excision repair ( ner ) , which is associated with increased sensitivity to the mutagenicity of ultraviolet light and benzo[a]pyrene dihydrodiolepoxide . ner is the primary process for repair of bulky dna adducts , and in humans , ner deficiencies result in xeroderma pigmentosum , a spectrum of disorders characterized by hypersensitivity to sunlight , neurological degeneration , and a greatly increased incidence of cancer . although not verified experimentally , inhibition of ner by mda was proposed to arise from loss of function of mda - modified ner proteins . a potential mda target is the ner scaffold protein , xeroderma pigmentosum complementation group a ( xpa ) , which binds to the damaged dna substrate via a c - terminal basic cleft comprising multiple lysine residues . xpa coordinates recruitment and assembly of the enzymes that repair the damaged dna , and mutations in its dna - binding domain are associated with the most severe clinical xp phenotypes . oxopropenyl adducts to dna and protein can also be formed through reaction with base propenal , a reactive aldehyde produced by oxidant - mediated hydrogen abstraction from the 4-position of dna bases ( figure 1 ) . base propenals are approximately 100-fold more reactive to dna than mda , and experiments in which escherichia coli strains of varying membrane composition were exposed to oxidants demonstrated that base propenals are more important than mda as a source of m1dg . currently , very little is known about potential protein targets of base propenal adduction ; however , as a highly reactive form of dna damage , base propenals are ideally situated to oxopropenylate dna - binding proteins such as xpa . this has led us to hypothesize that base propenal - mediated protein damage contributes to toxicity and mutagenicity associated with oxidative or electrophile stress . to test this hypothesis , a comprehensive evaluation of base propenal s ability to directly damage proteins adenine propenal is known to react with glutathione via michael addition to form glutathionylpropenal and a glutathione adenine propenal cross - link , but , to date , no detailed investigation of the reaction of any base propenal with amino acids or proteins has been reported . one approach to evaluate the protein adduction potential of an electrophile is to identify all sites of modification and the structures of those modifications using a model protein . a frequently used target for this purpose is human serum albumin ( hsa ) . albumin is highly abundant and has affinity for a broad range of ligands , a relatively long half - life , and the potential for noninvasive sampling . thus , it is an ideal candidate for the global qualitative and quantitative evaluation of exposure to electrophiles of endogenous or exogenous origin ( the exposome ) . in fact , in a large multilaboratory comparison of biomarkers of in vivo oxidant generation , the level of carbonylated plasma protein ( i.e. , albumin ) was one of only two markers that correlated with oxidant exposure and tissue pathology , and it was the only marker that was effective longer than 6 h after exposure . to exploit albumin s potential as a biomarker , however , requires a more complete understanding of its major sites of adduction and determinants of their reactivity to a range of electrophiles . for example , whereas much attention has been directed to hsa s highly reactive cys-34 as a sensor of oxidant damage , the major site of oxopropenylation by mda is lys-525 . here , we report the application of a recently described mass spectrometric approach to identify adenine propenal modifications of hsa and xpa . our data demonstrate that , like mda , adenine propenal primarily reacts at protein lysine residues ; however , the major site of attack on hsa is distinct from that of mda . we also report that adenine propenal modifies lysine residues of xpa , including residues within its dna - binding domain and residues reported to be sites of regulation via sirt1-mediated deacetylation . biophysical analysis confirms that modification of xpa by adenine propenal decreases its affinity for damaged dna . n--acetyllysine , trichloroacetic acid ( tca ) , dithiothreitol , iodoacetate ( sigma ultra ) , and nacnbh3 were obtained from sigma - aldrich ( st . nmr experiments were acquired using a 14.0 t bruker magnet equipped with a bruker av - iii console operating at 600.13 mhz . all spectra were acquired in 3 mm nmr tubes using a bruker 5 mm tci cryogenically cooled nmr probe . chemical shifts were referenced internally to d2o ( 4.70 ppm ) , which also served as the h lock solvent . for 1d h nmr , experiments were acquired with presaturation in order to suppress the residual h2o signal , and typical experimental conditions included 32k data points , 13 ppm sweep width , a recycle delay of 1.5 s , and 32 scans . for 2d h h cosy , presaturation was also performed , and experimental conditions included a 2048 1024 data matrix , 13 ppm sweep width , recycle delay of 1.5 s , and 8 scans per increment . the data were processed using squared sinebell window function , symmetrized , and displayed in magnitude mode . c hsqc experiments were acquired using a 1024 128 data matrix , a j(c h ) value of 145 hz , which resulted in a multiplicity selection delay of 34 ms , a recycle delay of 1.5 s , and 80 scans per increment along with garp decoupling on c during the acquisition time ( 150 ms ) . the data were processed using a p/2 shifted squared sine window function and displayed with ch / ch3 signals phased positive and ch2 signals phased negative . c hmbc experiments were acquired using a 2048 128 data matrix , a j(c h ) value of 9 hz for detection of long - range couplings resulting in an evolution delay of 55 ms , j1(c h ) filter delay of 145 hz ( 34 ms ) for the suppression of one - bond couplings , a recycle delay of 1.5 s , and 144 scans per increment . the hmbc data were processed using a p/2 shifted squared sine window function and displayed in magnitude mode . compounds were greater than 90% pure , and mass spectral analysis was accomplished on an applied biosystems 3200 q trap mass spectrometer ( mds sciex ) . the mass spectrometer was equipped with an electrospray source and operated in positive ion mode . h nmr ( dmso - d6 ) : ( ppm ) 7.17 ( dd , j1 = 7.88 hz , j2 = 14.4 hz , 1h ) , 7.55 ( br s , 2h ) , 8.27 ( s , 1h ) , 8.35 ( d , j = 14.4 hz , 1h ) , 8.63 ( s , 1h ) , 9.67 ( d , j = 7.88 hz , 1h ) . c nmr ( dmso - d6 ) : ( ppm ) 119.2 , 120.5 , 136.4 , 142.8 , 147.6 , 151.4 , 158.2 , 190.8 . h nmr ( d2o ) : ( ppm ) 9.15 ( d , j = 10.0 hz , 2h ) , 5.73 ( t , j = 10.0 hz , 1h ) . the sodium salt of malondialdehyde ( mda ) ( 100 mg , 1.06 mmol ) was added to a solution of n--acetyllysine ( 200 mg , 1.06 mmol ) in 5 ml of ammonium acetate buffer ( ph 4.5 ) . the reaction was then stirred at room temperature for 15 h. the reaction mixture was lyophilized , and the residue was purified by column chromatography ( etoac / meoh , 5:1 ) on silica gel ( 60 porosity , 4063 m particle size ) . h nmr ( d2o ) : ( ppm ) 1.33 ( m , 2h ) , 1.561.61 ( m , 3h ) , 1.71 ( m , 1h ) , 1.93 ( s , 3h ) , 2.91 ( t , j = 7.3 hz , 2h ) , 4.05 ( m , 1h ) , 5.31 ( m , 1h ) , 7.44 ( d , j = 13.0 hz , 1h ) , 8.59 ( d , j = 8.5 hz , 1h ) . c nmr ( d2o ) : ( ppm ) 22.0 , 23.4 , 27.2 , 32.0 , 39.3 , 55.3 , 86.6 , 163.7 , 173.7 , 179.1 , 191.2 . the sodium salt of mda ( 60 mg , 0.319 mmol ) was added to a solution of n--acetyllysine ( 15 mg , 0.159 mmol ) in 2 ml of anhydrous methanol . the mixture was heated at reflux , and the progress of the reaction was monitored by uv vis spectroscopy . the product was then purified by column chromatography ( etoac / meoh , 5:1 ) on silica gel ( 60 porosity , 4063 m particle size ) and hplc . hplc chromatography was carried out on a luna c18 column ( 250 4.60 mm , 5 m ) at a flow rate of 1.25 ml / min . the eluting solvents included solvent a ( 10 mm ammonium acetate ) and solvent b ( acetonitrile ) . the following solvent gradient was used : 010.5 min linear gradient from 5% b to 20% b , followed by a 2 min linear gradient to 80% b , hold for 6.50 min at 80% b , followed by a 1 min linear gradient to the initial conditions of 5% b. the product was observed with a retention time of 6.1 min . h nmr ( d2o ) : ( ppm ) 1.30 ( m , 4h ) , 1.591.61 ( m , 6h ) , 1.73 ( m , 2h ) , 1.88 ( s , 6h ) , 3.22 ( m , 2h ) , 3.30 ( t , j = 7.3 hz , 2h ) , 4.08 ( m , 2h ) , 5.45 ( m , 1h ) , 7.49 ( d , j = 11.5 hz , 1h ) , 7.56 ( d , j = 11.5 hz , 1h ) . c nmr ( d2o ) : ( ppm ) 21.3 , 21.8 , 25.7 , 30.2 , 42.0 , 53.7 , 88.5 , 158.9 , 160.5 , 177.5 , 178.9 . n--acetyllysine ( various concentrations ) was mixed with 10 mm adenine propenal in 50 l reaction mixtures containing 10 mm nacl and 10 mm 4-morpholinepropanesulfonic acid ( mops ) ( ph 6.5 ) . at 1 h intervals , aliquots of each reaction mixture were injected onto a waters hplc system equipped with a 717 plus autosampler , 1525 binary hplc pump , and 2996 photodiode array detector . analytes were separated using a phenomenex polar - rp synergi hplc column , 75 cm 2 mm with 4 m particle size , at a flow rate of 0.6 ml / min . the mobile phase solvents were h2o + 0.05% acetic acid ( v / v , solvent a ) and 3:1 meoh / acn + 0.05% acetic acid ( v / v , solvent b ) delivered in a 19 min gradient consisting of the following : 010.5 min , 520% b ; 10.512.5 min , 2080% b ; 12.516 min , 80% b ; 1619 min , 5% b. data were analyzed using empower software , and peak areas were manually integrated . reactions were performed as described above and analyzed using a thermo accela hplc pump ( thermo fisher scientific ) and htc pal autosampler ( leap technologies ) in - line with a thermo quantum triple quadrupole mass spectrometer ( thermo fisher scientific ) . the quantum was equipped with an electrospray source operated in q1 full scan , positive ion mode . q1 was scanned from 100600 amu in 0.45 s. analytes were chromatographically separated on a synergi polar - rp hplc column ( phenomenex , 7.5 0.2 cm , 4 m particle size ) using the following gradient : 010.5 min , 520% b ; 10.512.5 min , 2080% b ; 12.516 min , 80% b ; 1617 min , 5% b. the mobile phase solvents were h2o + 0.05% acetic acid ( v / v , solvent a ) and acetonitrile + 0.05% acetic acid ( v / v , solvent b ) , and they were delivered at a flow rate of 0.6 ml / min . all data were acquired and analyzed using thermo xcalibur software . solutions containing known concentrations of synthetic 3 and 4 were used to measure extinction coefficients of each compound . the value obtained for both compounds ( 33 600 200 mcm ) was the same within experimental error ; however , the wavelengths of maximal absorbance were different ( 280 nm for 3 and 300 nm for 4 ) ( figure s1 ) . adenine propenal ( 10 mm ) was incubated with increasing concentrations of n--acetyllysine ( 1 , 2 , 5 , 10 , 50 , and 100 mm ) in reaction mixtures containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . the mixtures were incubated for 2 h at room temperature and then diluted 1:1000 in h2o . the absorbance at 300 nm of each dilution was then obtained using a du800 spectrophotometer ( beckman coulter ) , and the experimental extinction coefficient of 4 was used to estimate its concentration in the reaction mixture . note that , for reaction mixtures containing > 10 mm n--acetyllysine , complete consumption of adenine propenal eliminates concerns of its contribution to the absorbance of the final reaction mixture . the adenine product does not absorb significantly at 300 nm ( figure s1 ) . on the basis of its full uv spectrum ( figure s1 ) , the extinction coefficient of 3 at 300 nm is estimated to be 7.8 10 m cm . the presence of this product at a concentration equal to approximately 10% of the concentration of 4 , as found in the reaction mixtures , indicates that its contribution to the absorbance at 300 nm will be no more than 3% . consequently , the absorbance at 300 nm can be used to estimate the concentration of 4 with reasonable accuracy . n--acetyllysine ( 10 mm ) was incubated with adenine propenal ( 10 mm ) in 10 mm mops ( ph 6.5 ) containing 10 mm nacl in a 200 l reaction mixture for 30 min . after incubation , 50 mm nacnbh3 ( in h2o ) ( sigma - aldrich ) was added for an additional 30 min , and samples were then analyzed using lc - ms as described below . lc - ms analysis was performed on the thermo quantum triple quadrupole mass spectrometer . here , the instrument was operated in q1 full - scan negative ion mode using electrospray ionization . q1 was scanned over the range 100400 amu over 0.5 s. chromatography was carried out on a synergi max - rp column ( phenomenex , 7.5 0.2 cm , 5 m particle size ) using the following gradient : 05 min linear gradient from 2% b to 50% b , 57 min at 50% b , 710 min 50% b to 2% b. the mobile phase solvents were h2o + 0.05% formic acid ( v / v , solvent a ) and 3:1 ch3cn / meoh + 0.05% formic acid ( v / v , solvent b ) , and they were delivered at a flow rate of 400 l / min . all data were acquired and analyzed using thermo xcalibur software . using this chromatographic system , the reduced intermediate eluted at approximately 2.9 min . n - acetylcysteine ( 200 m ) was combined with 50 m adenine propenal in 50 mm sodium phosphate buffer , ph 7.4 , and incubated for 6 h at 37 c . nabh4 ( 50 mm ) ( sigma - aldrich ) was used to reduce samples as indicated . analytes were separated on a supelco ascentis c18 column ( 50 2.1 mm , 3 m ) with buffers a and b comprising 0.1% formic acid in water and 0.1% formic acid in acetonitrile , respectively . the gradient was as follows : 13 min , 1% b ; 37 min , 98% b ; 711 min , 98% b ; 1117 min , 1% b. samples were analyzed on a thermofinnigan tsq quantum mass spectrometer with esi source interfaced to a thermofinnigan ms pump plus and autosampler plus ( thermo , san jose , ca ) . n - acetylcysteine - adducted species were analyzed with data - dependent scanning enabled in negative ion mode , scanning from m / z 150650 over 0.5 s. the top two m / z peaks from each ms scan were fragmented with 10 ev collision energy for 0.5 s. hsa ( 15 m ) ( abcam , cambridge , ma ) was incubated with increasing amounts of adenine propenal ( 0.15 , 0.38 , 1.5 , 3 , and 7 mm ) , representing a molar excess of adenine propenal to hsa of 10 , 25 , 100 , 250 , and 500 , respectively . reactions were incubated for 6 h at room temperature in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . following incubation , samples were precipitated with 25% trichloroacetic acid ( sigma - aldrich ) on ice for 1 h , washed with cold acetone , dried , and reconstituted in 50 mm tris ( ph 8.0 ) containing 50% 2,2,2-trifluoroethanol ( acros organics , pittsburgh , pa ) . samples were reduced with dtt ( sigma - aldrich ) , carbamidomethylated with iodoacetamide ( sigma - alrich ) , and diluted 5-fold with 100 mm tris ( to obtain a final solution containing 10% 2,2,2-trifluoroethanol ) . following dilution , samples were digested with sequencing - grade trypsin ( promega , madison , wi ) overnight , acidified , and diluted in 0.1% formic acid . the resulting solutions of trypsin - generated peptides were loaded onto a capillary reverse - phase analytical column ( 360 m o.d . 100 m i.d . ) using an eksigent nanolc ultra hplc and autosampler . the 20 cm analytical column was directly packed into a laser - pulled emitter tip using jupiter c18 reverse - phase medium ( 3 m beads , 300 pore size , phenomenex ) . mobile phase solvents consisting of 0.1% formic acid in water ( solvent a ) and 0.1% formic acid in acetonitrile ( solvent b ) at a flow rate of 500 nl / min were used to elute the peptides . the 90 min gradient consisted of the following : 010 min , 2% b ; 1050 min , 235% b ; 5060 min , 3595% b ; 6065 min , 95% b ; 6570 min 952% b ; 7090 min , 2% b. mass analysis of eluting peptides was performed on an ltq orbitrap xl mass spectrometer ( thermo scientific ) , equipped with a nanoelectrospray ionization source with detection in positive ion mode . the ltq orbitrap was operated using a data - dependent method , enabling dynamic exclusion . full scan ( m / z 4002000 ) spectra were acquired with the orbitrap ( resolution 60 000 ) , and the five most abundant ions in each ms scan were selected for fragmentation in the ltq . an isolation width of 2 m / z , 30 ms activation time , and collision energy normalized to 35% were used to generate ms spectra . dynamic exclusion settings allowed for a repeat count of 2 within a 10 s duration , with exclusion duration time set to 15 s. for identification of modified peptides , tandem mass spectra were searched with sequest ( thermo fisher scientific ) against a human subset database created from the uniprotkb protein database ( www.uniprot.org ) . variable modifications of + 57.0214 on cys ( carbamidomethylation ) , + 15.9949 on met ( oxidation ) , and + 54.0105 on lys or cys ( oxypropenylation ) were included for database searching . search results were assembled with scaffold 3.0 ( proteome software ) using threshold filtering criteria consisting of 95% peptide probability to achieve a peptide false discovery rate estimate of 0.2% . sites of modification were validated by manual interrogation of tandem mass spectra using xcalibur 2.1 qual browser software ( thermo scientific ) . calculations of mass errors ( in ppm ) of precursor ions mass analyzed in the orbitrap were performed using the following equation : ( ( theoretical mass observed mass)/theoretical mass ) 10 . to obtain theoretical masses , analysis of cross - links was performed in a similar manner with inclusion of + 36 on lys for database searching . purified hsa ( 0.75 m ) was incubated with increasing concentrations of adenine propenal in reaction mixtures containing 10 mm nacl and 10 mm mops ( ph 6.5 ) for 6 h at room temperature . following incubation , the protein was heated at 95 c for 10 min , cooled to room temperature , and then treated with 1 mg of pronase ( calbiochem , gibbstown , nj ) per mg of protein at 37 c for 24 h. pronase was inactivated by heating at 95 c for 10 min , followed by cooling to room temperature , and treating with 1 l of aminopeptidase m ( calbiochem , gibbstown , nj ) at 37 c for 24 h. a final incubation at 95 c for 10 min inactivated the aminopeptidase m , and the samples were then cooled to room temperature in preparation for adduct purification . for use as an internal standard , [ h]-[c]-4 was prepared by reaction of mda with [ h]-lysine containing [ c]-lysine as a tracer and quantified using a method similar to that described for the [ h]-[c]-lysine - lactam internal standard as described . an oasis hlb cartridge ( 1 cm ) ( waters , milford , ma ) was equilibrated with 2 ml of methanol followed by 2 ml of h2o . following addition of [ h]-[c]-4 , the sample was loaded onto the cartridge using gravity flow . the cartridge was washed with 1 ml of h2o , and 4 was eluted with 3 ml 1:1 methanol / ethyl acetate and dried to 100 l under n2 gas . the sample was then diluted in 0.1% formic acid solution to a final volume of 1 ml and filtered using a 0.22 m nylon spin - x centrifuge tube spun at 6000 rpm for 5 min . samples were purified using a thermo scientific aquasil c18 5 m reverse - phase column using liquid scintillation counting to monitor elution of the radioactive [ h]-[c]-lysine lysine cross - link standard . the internal standard [ h]-[c]-lysine lysine cross - link was prepared by reaction of mda with [ h]-lysine containing [ c]-lysine as a tracer and quantified using a method similar to that described for the [ h]-[c]-lysine - lactam internal standard . for quantitation , the transition between m / z 341 and m / z 306 was monitored ; it corresponds to the loss of one molecule of water and one molecule of ammonia . portions of samples containing the standard were then concentrated using 1 cc oasis hlb columns and dried under nitrogen . the purified samples were analyzed using a thermo scientific tsq vantage quadrupole mass spectrometer equipped with xcalibur software for data collection and manipulation . all measured mda - adduct signal in each sample was divided by the signal for the [ h]-[c]-lysine lysine cross - link standard of known concentration to quantify experimental results . xpa was expressed and purified as described . to modify xpa , 3.2 m of purified protein was incubated with 500 ( 1.6 mm ) of adenine propenal for 6 h at room temperature in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . a high - throughput fluorescence anisotropy assay was used to measure the dna - binding activity of xpa . the substrate for this assay was a fluorescein - labeled y - shaped ssdna dsdna junction substrate containing 8 base pairs of duplex and 12 nucleotide noncomplementary ssdna overhangs at both the 3 and 5 ends . xpa was modified with nhs biotin as described previously . modified or mock - treated xpa protein and the dna substrate were diluted in binding buffer ( 20 mm hepes , ph 7.9 , 75 m kcl , 5 mm mgcl2 , 5% glycerol , 1 mm dtt ) . fluorescence anisotropy was measured using a synergy h1 plate reader ( ex = 485 nm , em = 528 nm ) . apparent dissociation constants ( kd ) were determined for each individual titration by plotting fluorescence anisotropy against protein concentration and fitting to a simple two - state binding model with kaleidagraph ( v4.03 ) software . adenine propenal was reacted with n--acetyllysine , n--acetylhistidine , n--acetylarginine , or n--acetylcysteine in 10 mm nacl buffered with 10 mm mops ( ph 6.5 ) . reaction progress was monitored hourly by hplc using a c18 reverse - phase column for product separation . adenine propenal eluted at 8 min and displayed a maximum absorbance of 260 nm ( figures 2 and s1 ) . reaction with n--acetyllysine resulted in the appearance of three new chromatographic peaks at 1.0 , 4.8 , and 5.3 min , which were analyzed by uv / vis , nmr , and ms ( figures 2 and s1 ) . the peak eluting at 1 min displayed a uv absorbance maximum at 260 nm and an m / z of 136 , corresponding to protonated adenine ( [ m + h ] ) ( 1 ) . on the basis of nmr analysis , this product was identified as 3 , and lc - ms analysis gave an m / z of 243 , which corresponds to the protonated species ( [ m + h ] ) . the 5.3 min peak , with a maximum absorbance at 300 nm and an m / z of 413 , which corresponds to the protonated species ( [ m + h ] ) , was identified as 4 by nmr analysis . the total amount of product formed was unaffected by variations in ph over a range of 6.5 to 9.5 . n--acetyllysine ( 50 mm ) was incubated with adenine propenal ( 10 mm ) in a 50 l reaction mixture for 2 h at room temperature . the column eluate was monitored at 260 nm ( a ) or 300 nm ( b ) . analytes present in each peak were subjected to ms , uv , and nmr spectrometry . chemical synthesis of 3 and 4 yielded standards that verified the product structure assignments . the molar extinction coefficients of the pure compounds ( = 33.6 10 m cm at 280 nm for 3 and = 33.6 10 m cm at 300 nm for 4 ) were similar to that of adenine propenal ( = 32.5 10 m cm at 260 nm ) . hplc analysis of reaction mixtures of n--acetyllysine and adenine propenal using diode - array detection enabled direct comparison of peak areas for each compound measured at its optimal wavelength ( figure 3 ) . the essentially identical absorbance coefficients of 3 and 4 allowed peak areas to be used to estimate relative product quantity . these comparisons indicated that the major product formed from the reaction was 4 , with much lower quantities of 3 . hplc analysis of product formation in the reaction of adenine propenal and n--acetyllysine . the indicated amounts of n--acetyllysine ( nal ) were incubated with 10 mm adenine propenal , and reactions were monitored hourly for 910 h. peaks corresponding to 3 and 4 were monitored at 280 and 300 nm , respectively , by diode - array detection . variation of the amount of n--acetyllysine ( from 2 to 100 mm ) in reaction mixtures while holding the amount of adenine propenal ( 10 mm ) fixed demonstrated that the amount of 3 formed increased with n--acetyllysine concentrations from 2 to 10 mm and then decreased somewhat at the higher concentrations . in contrast , the amount of 4 produced increased with n--acetyllysine concentrations up to 50 mm . on the basis of the relative peak area , the ratio of 4 to 3 in most reaction mixtures was approximately 10:1 . estimation of the concentration of 4 using absorbance at 300 nm indicated a maximum of 9 mm formed in reaction mixtures containing 50100 mm n--acetyllysine ( figure s2 ) . our initial hypothesis for the production of 4 was that it arose by reaction of n--acetyllysine with 3 , but this reaction did not occur under conditions comparable to those used for the reaction of adenine propenal with n--acetyllysine . an alternative reaction scheme , in which an enaminoimine cross - link between adenine propenal and n--acetyllysine is first formed and then reacts with h2o to produce 3 or another equivalent of n--acetyllysine to form 4 , is shown in figure 4 . if this scheme is correct , then our data indicate that , at the concentrations of n--acetyllysine and adenine propenal used here , reaction of the proposed intermediate with another molecule of n--acetyllysine is more favorable than its reaction with water . hplc analysis with uv or in - line nmr was unable to detect the putative intermediate 5 ( figure 4 ) . therefore , we attempted to trap it by reduction with nacnbh3 . adenine propenal was incubated with n--acetyllysine in 10 mm nacl/10 mm mops ( ph 6.5 ) in the presence of nacnbh3 . we utilized lc - ms analysis of the reaction mixture to detect ions with m / z 360 , which corresponds to the partially reduced intermediate ( 6 ) . a peak corresponding to this mass eluted at 2.9 min , consistent with the transient formation of the adenine propenal adduct to n--acetyllysine ( figure 5 ) . ( a ) representative lc - ms chromatogram of 6 . in this example , 5 mm n--acetyllysine was reacted with 10 mm adenine propenal for 30 min followed by reduction with 50 mm nacnbh3 . ( b ) mass spectrum of the parent ion of the reduced intermediate displaying m / z 360 with detection in negative ion mode . reactions of adenine propenal with n--acetylhistidine and n--acetylarginine did not result in the formation of new chromatographic peaks . reaction with n--acetylcysteine resulted in the formation of an oxopropenylated product , which displayed m / z 216 , corresponding to the deprotonated molecular ion , and its major fragmentation product , m / z 86.9 . treatment of the reaction mixture with nabh4 yielded the partially reduced product ( m / z 218 ) and its major fragmentation product ( m / z 89 ) ( figure s3 ) . to ascertain adenine propenal s ability to react with protein , we incubated increasing amounts of the electrophile with a fixed amount of purified hsa for 6 h , and the samples were prepared for lc - ms / ms by trichloroacetic acid precipitation , resolubilization of the precipitated protein pellet , reduction and carbamidomethylation of cysteine residues , and proteolytic digestion with sequencing - grade trypsin . the resultant proteolytic peptides were analyzed via lc - ms / ms using an ltq - orbitrap velos mass spectrometer with collision - induced dissociation . sequence coverage for unmodified hsa was found to be 90% , but exposure to increasing concentrations of adenine propenal reduced coverage to a minimum value of 62% ( table s1 ) . hsa peptides were interrogated for the presence of a + 54.0105 mass increase , as would be expected from the addition of an oxopropenyl group . to verify positively identified residues , extracted ion chromatograms were generated from precursor peptide masses , and theoretical masses calculated from protein prospector were compared to observed masses to calculate ppm mass error . for modified precursor peptides displaying a ppm mass error less than 10 , ms / ms spectra were interrogated with xcalibur software . at least 40% of each theoretical b- and y - product ion series was present in the observed product ion spectra of positively identified peptides , and 50% sequence coverage was obtained for each peptide validated . to validate the site of adduction , at least two ions within a product ion series with a + 54.0105 adduction mass shift were required . multiple lysine residues were found to be modified in adenine propenal - exposed hsa , including lys26 , lys36 , lys161 , lys183 , lys214 , lys219 , lys236 , lys249 , lys257 , lys347 , lys375 , lys402 , lys413 , lys438 , lys549 , lys569 , and lys598 . residues verified to have a + 54.0105 adduction mass were mapped to the crystal structure of hsa ( pdb i d : 1ao6 ) and were found to be largely confined to the protein surface ( figure 6a ) . the 17 modified lysine residues represented 28% of the total lysines within the protein ( table 1 ) . although we determined that adenine propenal can also form oxopropenylated cysteine , searching for adducts on cysteine residues within hsa did not result in the identification of any modified sites . s - oxopropenal - n - acetylcysteine was stable to treatment with tca as well a dtt , which are reagents used for proteomic analysis , so any oxopropenylated cysteine residues on hsa - derived peptides should have survived the workup prior to mass spectrometry . therefore , adenine propenal is acting as a lysine - specific modifier of hsa . ( a ) modified lysine residues identified in table 1 were mapped to the crystal structure of hsa ( 1ao6 ) . ( b ) the most highly modified residues by individual electrophiles are highlighted with the modifying electrophile noted . hsa was treated at the indicated molar ratios of adenine propenal and analyzed for lysine residues modified with + 54.0105 . the presence of each modified lysine in a sample is indicated with . the modified lysine in each peptide sequence is indicated with an asterisk . equivalent lysine residues within the crystal structure of hsa are provided in parentheses . the use of hsa as a biomarker has primarily been described in the context of electrophilic modifications at cys34 . adenine propenal did not modify cys34 , so we sought to identify the most reactive lysine residue(s ) that might be used as a biomarker for modification by adenine propenal . we reduced the levels of adenine propenal relative to protein to a ratio of 10:1 , which was the lowest ratio at which modification was observed . using the interrogation methods described above to verify adduction sites , two lysine residues , k190 and k351 , displayed the highest reactivity ( table 1 ) . mapping of those residues to the crystal structure of hsa revealed that both are located within domain ii , a recognized binding region for many compounds ( figure 6a ) . decreased hsa sequence coverage observed with increasing concentrations of adenine propenal suggested that adducts impede protein digestion and/or fragmentation required for peptide identification . lysine cross - links are formed from the reaction of adenine propenal with n--acetyllysine , we hypothesized that at high concentrations of adenine propenal , lysine lysine cross - links may be induced in hsa . to investigate the presence of cross - links , we interrogated previously acquired proteomics data for a + 36 adduct mass , corresponding to an unreduced 3-carbon linkage between lysine residues . lysine cross - links , which was not surprising considering the decreased sequence coverage and potential hindrance of peptide fragmentation by cross - links . although cross - linked sites were not identified with these algorithm - based data searching methods , we manually interrogated the data to look for the presence of precursor peptides containing cross - linked residues . to predict lysine residues likely to participate in cross - link formation , we performed in silico analysis of the hsa crystal structure ( 1ao6 ) . carbon bond is approximately 1.5 in length , we hypothesized that the 3-carbon cross - link would be 57 long . flexible , dynamic protein movements in solution may result in cross - link formation between lysines at a distance greater than 7 . we therefore examined the crystal structure to identify lysine residue pairs separated by a distance of less than or equal to 20 as potential sites of cross - link formation . following identification of these residues , tryptic peptide sequences were determined from the primary sequence , and theoretical peptide masses containing the cross - link mass of + 36 were calculated using protein prospector . we then thoroughly interrogated the data manually for the presence of ions of the theoretical precursor peptide masses . no evidence for the presence of cross - link - containing peptides was obtained . as proteomics - based mass spectrometric analyses were unsuccessful , we employed a different analytical approach to search for lysine lysine cross - links , in which modified hsa was degraded to the level of amino acids , and the resultant hydrolysate was analyzed by ms . this methodology employed selected reaction monitoring to identify analytes exhibiting a mass transition from m / z 329 to m / z 294 , as exhibited by analysis of the lysine lysine cross - link standard . addition of a [ h]-[c]-4 internal standard ( m / z 341 to m / z 306 transition ) enabled identification of analyte - containing fractions during purification by scintillation counting and quantification by stable isotope dilution . application of this approach to hsa that had been incubated with adenine propenal resulted in the identification of 30 12 ng cross - link / mg protein in vehicle - treated hsa and 57 17 ng cross - link / mg protein in adenine propenal - treated hsa . these differences were not statistically significant , supporting the conclusion that adenine propenal did not induce intramolecular cross - links in hsa . having confirmed adenine propenal s capacity to modify lysine residues in hsa , we investigated its reaction with xpa . adenine propenal was incubated with xpa for 6 h at room temperature , and the samples were prepared for and analyzed by lc - ms / ms using the same methods as described for hsa . as for hsa , peptides were interrogated for the presence of a + 54.0105 mass addition , and rigorous criteria were applied to peptide and site of adduction identification . of note , three of these , k145 , k183 , and k204 , are located in the proposed dna - binding domain of xpa ( figure 7a ) , and two , k63 and k67 , are known sites of acetylation that reduces xpa s interaction with replication protein a. adenine propenal modifies xpa and reduces its dna - binding activity . ( a ) lysine residues modified with an adduct mass of + 54.0105 were mapped to the xpa nmr solution structure ( 1d4u ) . ( b ) plots of the change in fluorescence anisotropy for a y - shaped 8/12 ssdna dsdna junction substrate ( 50 nm ) versus added protein treated with dmso , adenine propenal , or nhs - biotin . purified xpa was incubated with a 500-fold molar excess of adenine propenal and analyzed for lysine residues modified with + 54.0105 . modification of residues within the xpa dna - binding domain has the potential to affect dna - binding activity . using a plate - based fluoresence anisotropy assay , the effect of adenine propenal - mediated oxopropenylation on xpa s affinity for a y - shaped ssdna fitting of the data for binding of unmodified xpa to the fluorescein - labeled 20-nucleotide substrate provided a dissociation constant ( kd ) of 300 30 nm ( figure s4 ) . binding affinities for the 20-nucleotide substrate were measured for purified xpa incubated with vehicle ( dmso ) , adenine propenal , or nhs biotin ( biotin n - hydroxysuccinimide ester ) ( table 3 ) . as expected , incubation of xpa with dmso had no effect on its dna - binding activity . in contrast , exposure to nhs biotin , which efficiently modifies lysine residues and disrupts the dna - binding activity of many proteins , resulted in a nearly complete loss of xpa s dna - binding affinity . xpa modified with adenine propenal bound to the substrate , but with a greater than 3-fold reduction in affinity as compared to that of the unmodified protein ( figure 7b ) . in fact , oxopropenylation of the protein reduced the affinity to such an extent that it could not be accurately measured ; thus , the kd value reported in table 3 ( 1 0.1 m ) is an estimate . dissociation constants ( kd ) were determined by fitting fluorescence anisotropy data to a simple two - state binding model . the biotin - modification of xpa causes such a large loss of dna - binding affinity that there is no significant change in fluorescence anisotropy ; hence , a kd value could not be determined . covalent modifications of dna and proteins by electrophiles produced during oxidative stress have multiple consequences on cellular function , including mutagenesis , stress responses , cellular dysfunction , and ultimately cell death . all of these may contribute to pathological processes such as carcinogenesis , neurodegeneration , atherosclerosis , and chronic inflammation . however , elucidation of the exact mechanisms by which specific macromolecular damage leads to pathology requires a better understanding of the sites and nature of that damage . the most frequently used biomarker of electrophile damage to protein during oxidative stress is the change in global protein carbonylation . although useful as a general indicator that stress has occurred , this approach does not identify the exact type of modification or the modifying electrophile . recent developments in highly sensitive ms - based proteomics techniques have allowed for expansion of this approach to the investigation of the specific types of modifications induced and the impact of these modifications on protein activity . the resultant increased ability to identify specific protein modifications induced by a single electrophile enables the search for biomarkers that distinguish between different mechanisms of oxidant damage . the generation of adenine propenal occurs predominantly in intact dna , suggesting that most of its targets will likely be dna - associated proteins in the nucleus or mitochondria . nevertheless , we initiated our investigation of adenine propenal s protein - modifying profile , using hsa as the model protein . hsa s reactivity with electrophilic agents has been studied extensively in the past , allowing us to place adenine propenal - dependent modifications in the context of those existing data . for example , teucrin a was found to preferentially modify lys351 and lys545 , while 4-hydroxynonenal modifies lys195 , lys199 , lys233 , and lys525 , and polycyclic aromatic hydrocarbon epoxides modify lys137 . mda has been shown to modify multiple lysine residues including lys136 , lys174 , lys240 , lys281 , lys525 , and lys541 . as mda and adenine propenal both produce oxopropenyl adducts on lysine residues , it is interesting to note that the major sites of adduction for the two electrophiles are different : lys190 and lys351 for adenine propenal versus lys525 for mda . these findings suggest that distinct hsa modification patterns may be used as markers for different electrophiles ( figure 6b ) . clearly , the use of hsa as a biomarker of electrophile damage is attractive , given its high concentration in the blood and ease of sampling . however , as noted above , adenine propenal generation is largely confined to intact dna , so it remains to be seen if enough of the electrophile reaches the circulation to allow the modification of hsa to serve as a biomarker of exposure . the impact of electrophiles has long been studied in the context of covalent modification of dna and its potential to lead directly to mutations . however , adenine propenal produced in the nucleus also has the potential to modify dna - associated proteins , including those involved in dna repair . we report here that adenine propenal covalently modifies the ner protein xpa in vitro and that this modification leads to a reduction in xpa s dna - binding activity . oxopropenylation of residues known to be acetylated further suggests that adenine propenal - dependent modification could result in failure of regulation of xpa s activity through sirt-1-dependent deacetylation . xpa is a critical component of the ner machinery , without which repair activity ceases . mutations in xpa , particularly in the dna - binding domain , are associated with severe xp clinical phenotypes . thus , it is possible that decreased xpa - binding activity resulting from adenine propenal - dependent modification could result in reduced ner activity in cells and increased potential for mutagenesis and carcinogenesis . it should be noted that , in this model , the modification of repair proteins may have a synergistic effect on mutagenesis by preventing the repair of the original dna lesion . in this case , even moderate reductions in the activity of the repair proteins have the potential to result in significant increases in genomic instability . further analysis will be required to determine whether reactive base propenal species modify dna repair proteins in cells under oxidative stress and to characterize the effects of modification on dna repair activity in vivo .

base propenals are products of the reaction of dna with oxidants such as peroxynitrite and bleomycin . the most reactive base propenal , adenine propenal , is mutagenic in escherichia coli and reacts with dna to form covalent adducts ; however , the reaction of adenine propenal with protein has not yet been investigated . a survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts , whereas histidine and arginine do not . n-oxopropenyllysine , a lysine lysine cross - link , and s - oxopropenyl cysteine are the major products . comprehensive profiling of the reaction of adenine propenal with human serum albumin and the dna repair protein , xpa , revealed that the only stable adduct is n-oxopropenyllysine . the most reactive sites for modification in human albumin are k190 and k351 . three sites of modification of xpa are in the dna - binding domain , and two sites are subject to regulatory acetylation . modification by adenine propenal dramatically reduces xpa s ability to bind to a dna substrate .

Introduction Experimental Procedures Results Discussion

the major reactive aldehyde produced during peroxidation of polyunsaturated fatty acids , malondialdehyde ( mda ) , reacts with nucleophilic sites of dna and protein to form oxopropenyl adducts . m1dg is mutagenic in vitro and in vivo , and 2-oxopropenyl - deoxyadenosine alters dna polymerase activity . the reaction of mda with protein occurs predominantly at lysine residues under physiological conditions . however , a particularly interesting effect of mda exposure in human cells is the inhibition of nucleotide excision repair ( ner ) , which is associated with increased sensitivity to the mutagenicity of ultraviolet light and benzo[a]pyrene dihydrodiolepoxide . ner is the primary process for repair of bulky dna adducts , and in humans , ner deficiencies result in xeroderma pigmentosum , a spectrum of disorders characterized by hypersensitivity to sunlight , neurological degeneration , and a greatly increased incidence of cancer . xpa coordinates recruitment and assembly of the enzymes that repair the damaged dna , and mutations in its dna - binding domain are associated with the most severe clinical xp phenotypes . oxopropenyl adducts to dna and protein can also be formed through reaction with base propenal , a reactive aldehyde produced by oxidant - mediated hydrogen abstraction from the 4-position of dna bases ( figure 1 ) . base propenals are approximately 100-fold more reactive to dna than mda , and experiments in which escherichia coli strains of varying membrane composition were exposed to oxidants demonstrated that base propenals are more important than mda as a source of m1dg . currently , very little is known about potential protein targets of base propenal adduction ; however , as a highly reactive form of dna damage , base propenals are ideally situated to oxopropenylate dna - binding proteins such as xpa . to test this hypothesis , a comprehensive evaluation of base propenal s ability to directly damage proteins adenine propenal is known to react with glutathione via michael addition to form glutathionylpropenal and a glutathione adenine propenal cross - link , but , to date , no detailed investigation of the reaction of any base propenal with amino acids or proteins has been reported . one approach to evaluate the protein adduction potential of an electrophile is to identify all sites of modification and the structures of those modifications using a model protein . albumin is highly abundant and has affinity for a broad range of ligands , a relatively long half - life , and the potential for noninvasive sampling . to exploit albumin s potential as a biomarker , however , requires a more complete understanding of its major sites of adduction and determinants of their reactivity to a range of electrophiles . for example , whereas much attention has been directed to hsa s highly reactive cys-34 as a sensor of oxidant damage , the major site of oxopropenylation by mda is lys-525 . our data demonstrate that , like mda , adenine propenal primarily reacts at protein lysine residues ; however , the major site of attack on hsa is distinct from that of mda . we also report that adenine propenal modifies lysine residues of xpa , including residues within its dna - binding domain and residues reported to be sites of regulation via sirt1-mediated deacetylation . biophysical analysis confirms that modification of xpa by adenine propenal decreases its affinity for damaged dna . c hmbc experiments were acquired using a 2048 128 data matrix , a j(c h ) value of 9 hz for detection of long - range couplings resulting in an evolution delay of 55 ms , j1(c h ) filter delay of 145 hz ( 34 ms ) for the suppression of one - bond couplings , a recycle delay of 1.5 s , and 144 scans per increment . the reaction was then stirred at room temperature for 15 h. the reaction mixture was lyophilized , and the residue was purified by column chromatography ( etoac / meoh , 5:1 ) on silica gel ( 60 porosity , 4063 m particle size ) . the mixture was heated at reflux , and the progress of the reaction was monitored by uv vis spectroscopy . the value obtained for both compounds ( 33 600 200 mcm ) was the same within experimental error ; however , the wavelengths of maximal absorbance were different ( 280 nm for 3 and 300 nm for 4 ) ( figure s1 ) . the absorbance at 300 nm of each dilution was then obtained using a du800 spectrophotometer ( beckman coulter ) , and the experimental extinction coefficient of 4 was used to estimate its concentration in the reaction mixture . note that , for reaction mixtures containing > 10 mm n--acetyllysine , complete consumption of adenine propenal eliminates concerns of its contribution to the absorbance of the final reaction mixture . the presence of this product at a concentration equal to approximately 10% of the concentration of 4 , as found in the reaction mixtures , indicates that its contribution to the absorbance at 300 nm will be no more than 3% . n - acetylcysteine - adducted species were analyzed with data - dependent scanning enabled in negative ion mode , scanning from m / z 150650 over 0.5 s. the top two m / z peaks from each ms scan were fragmented with 10 ev collision energy for 0.5 s. hsa ( 15 m ) ( abcam , cambridge , ma ) was incubated with increasing amounts of adenine propenal ( 0.15 , 0.38 , 1.5 , 3 , and 7 mm ) , representing a molar excess of adenine propenal to hsa of 10 , 25 , 100 , 250 , and 500 , respectively . full scan ( m / z 4002000 ) spectra were acquired with the orbitrap ( resolution 60 000 ) , and the five most abundant ions in each ms scan were selected for fragmentation in the ltq . sites of modification were validated by manual interrogation of tandem mass spectra using xcalibur 2.1 qual browser software ( thermo scientific ) . following incubation , the protein was heated at 95 c for 10 min , cooled to room temperature , and then treated with 1 mg of pronase ( calbiochem , gibbstown , nj ) per mg of protein at 37 c for 24 h. pronase was inactivated by heating at 95 c for 10 min , followed by cooling to room temperature , and treating with 1 l of aminopeptidase m ( calbiochem , gibbstown , nj ) at 37 c for 24 h. a final incubation at 95 c for 10 min inactivated the aminopeptidase m , and the samples were then cooled to room temperature in preparation for adduct purification . samples were purified using a thermo scientific aquasil c18 5 m reverse - phase column using liquid scintillation counting to monitor elution of the radioactive [ h]-[c]-lysine lysine cross - link standard . the internal standard [ h]-[c]-lysine lysine cross - link was prepared by reaction of mda with [ h]-lysine containing [ c]-lysine as a tracer and quantified using a method similar to that described for the [ h]-[c]-lysine - lactam internal standard . all measured mda - adduct signal in each sample was divided by the signal for the [ h]-[c]-lysine lysine cross - link standard of known concentration to quantify experimental results . to modify xpa , 3.2 m of purified protein was incubated with 500 ( 1.6 mm ) of adenine propenal for 6 h at room temperature in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . a high - throughput fluorescence anisotropy assay was used to measure the dna - binding activity of xpa . modified or mock - treated xpa protein and the dna substrate were diluted in binding buffer ( 20 mm hepes , ph 7.9 , 75 m kcl , 5 mm mgcl2 , 5% glycerol , 1 mm dtt ) . the molar extinction coefficients of the pure compounds ( = 33.6 10 m cm at 280 nm for 3 and = 33.6 10 m cm at 300 nm for 4 ) were similar to that of adenine propenal ( = 32.5 10 m cm at 260 nm ) . these comparisons indicated that the major product formed from the reaction was 4 , with much lower quantities of 3 . hplc analysis of product formation in the reaction of adenine propenal and n--acetyllysine . the indicated amounts of n--acetyllysine ( nal ) were incubated with 10 mm adenine propenal , and reactions were monitored hourly for 910 h. peaks corresponding to 3 and 4 were monitored at 280 and 300 nm , respectively , by diode - array detection . variation of the amount of n--acetyllysine ( from 2 to 100 mm ) in reaction mixtures while holding the amount of adenine propenal ( 10 mm ) fixed demonstrated that the amount of 3 formed increased with n--acetyllysine concentrations from 2 to 10 mm and then decreased somewhat at the higher concentrations . our initial hypothesis for the production of 4 was that it arose by reaction of n--acetyllysine with 3 , but this reaction did not occur under conditions comparable to those used for the reaction of adenine propenal with n--acetyllysine . an alternative reaction scheme , in which an enaminoimine cross - link between adenine propenal and n--acetyllysine is first formed and then reacts with h2o to produce 3 or another equivalent of n--acetyllysine to form 4 , is shown in figure 4 . if this scheme is correct , then our data indicate that , at the concentrations of n--acetyllysine and adenine propenal used here , reaction of the proposed intermediate with another molecule of n--acetyllysine is more favorable than its reaction with water . reactions of adenine propenal with n--acetylhistidine and n--acetylarginine did not result in the formation of new chromatographic peaks . to ascertain adenine propenal s ability to react with protein , we incubated increasing amounts of the electrophile with a fixed amount of purified hsa for 6 h , and the samples were prepared for lc - ms / ms by trichloroacetic acid precipitation , resolubilization of the precipitated protein pellet , reduction and carbamidomethylation of cysteine residues , and proteolytic digestion with sequencing - grade trypsin . sequence coverage for unmodified hsa was found to be 90% , but exposure to increasing concentrations of adenine propenal reduced coverage to a minimum value of 62% ( table s1 ) . therefore , adenine propenal is acting as a lysine - specific modifier of hsa . adenine propenal did not modify cys34 , so we sought to identify the most reactive lysine residue(s ) that might be used as a biomarker for modification by adenine propenal . we reduced the levels of adenine propenal relative to protein to a ratio of 10:1 , which was the lowest ratio at which modification was observed . lysine cross - links are formed from the reaction of adenine propenal with n--acetyllysine , we hypothesized that at high concentrations of adenine propenal , lysine lysine cross - links may be induced in hsa . to predict lysine residues likely to participate in cross - link formation , we performed in silico analysis of the hsa crystal structure ( 1ao6 ) . carbon bond is approximately 1.5 in length , we hypothesized that the 3-carbon cross - link would be 57 long . we therefore examined the crystal structure to identify lysine residue pairs separated by a distance of less than or equal to 20 as potential sites of cross - link formation . following identification of these residues , tryptic peptide sequences were determined from the primary sequence , and theoretical peptide masses containing the cross - link mass of + 36 were calculated using protein prospector . as proteomics - based mass spectrometric analyses were unsuccessful , we employed a different analytical approach to search for lysine lysine cross - links , in which modified hsa was degraded to the level of amino acids , and the resultant hydrolysate was analyzed by ms . this methodology employed selected reaction monitoring to identify analytes exhibiting a mass transition from m / z 329 to m / z 294 , as exhibited by analysis of the lysine lysine cross - link standard . application of this approach to hsa that had been incubated with adenine propenal resulted in the identification of 30 12 ng cross - link / mg protein in vehicle - treated hsa and 57 17 ng cross - link / mg protein in adenine propenal - treated hsa . adenine propenal was incubated with xpa for 6 h at room temperature , and the samples were prepared for and analyzed by lc - ms / ms using the same methods as described for hsa . of note , three of these , k145 , k183 , and k204 , are located in the proposed dna - binding domain of xpa ( figure 7a ) , and two , k63 and k67 , are known sites of acetylation that reduces xpa s interaction with replication protein a. adenine propenal modifies xpa and reduces its dna - binding activity . ( b ) plots of the change in fluorescence anisotropy for a y - shaped 8/12 ssdna dsdna junction substrate ( 50 nm ) versus added protein treated with dmso , adenine propenal , or nhs - biotin . modification of residues within the xpa dna - binding domain has the potential to affect dna - binding activity . using a plate - based fluoresence anisotropy assay , the effect of adenine propenal - mediated oxopropenylation on xpa s affinity for a y - shaped ssdna fitting of the data for binding of unmodified xpa to the fluorescein - labeled 20-nucleotide substrate provided a dissociation constant ( kd ) of 300 30 nm ( figure s4 ) . binding affinities for the 20-nucleotide substrate were measured for purified xpa incubated with vehicle ( dmso ) , adenine propenal , or nhs biotin ( biotin n - hydroxysuccinimide ester ) ( table 3 ) . as expected , incubation of xpa with dmso had no effect on its dna - binding activity . in contrast , exposure to nhs biotin , which efficiently modifies lysine residues and disrupts the dna - binding activity of many proteins , resulted in a nearly complete loss of xpa s dna - binding affinity . the biotin - modification of xpa causes such a large loss of dna - binding affinity that there is no significant change in fluorescence anisotropy ; hence , a kd value could not be determined . the generation of adenine propenal occurs predominantly in intact dna , suggesting that most of its targets will likely be dna - associated proteins in the nucleus or mitochondria . as mda and adenine propenal both produce oxopropenyl adducts on lysine residues , it is interesting to note that the major sites of adduction for the two electrophiles are different : lys190 and lys351 for adenine propenal versus lys525 for mda . however , as noted above , adenine propenal generation is largely confined to intact dna , so it remains to be seen if enough of the electrophile reaches the circulation to allow the modification of hsa to serve as a biomarker of exposure . the impact of electrophiles has long been studied in the context of covalent modification of dna and its potential to lead directly to mutations . however , adenine propenal produced in the nucleus also has the potential to modify dna - associated proteins , including those involved in dna repair . we report here that adenine propenal covalently modifies the ner protein xpa in vitro and that this modification leads to a reduction in xpa s dna - binding activity . oxopropenylation of residues known to be acetylated further suggests that adenine propenal - dependent modification could result in failure of regulation of xpa s activity through sirt-1-dependent deacetylation . mutations in xpa , particularly in the dna - binding domain , are associated with severe xp clinical phenotypes . it should be noted that , in this model , the modification of repair proteins may have a synergistic effect on mutagenesis by preventing the repair of the original dna lesion . further analysis will be required to determine whether reactive base propenal species modify dna repair proteins in cells under oxidative stress and to characterize the effects of modification on dna repair activity in vivo .

it clinically diagnoses with recurrent oral and/or genital aphthosis , mucocutaneous lesions , central nervous system manifestation , and ocular , vascular , articular , gastrointestinal , and inflammatory eye lesions . autoinflammatory conditions are a group of heritable inflammatory - mediated conditions characterized by idiopathic attacks of systemic inflammation with lack of known - specific antigens and antibodies or immune response . these conditions share in some clinical aspects including uveitis , fever , arthritis , and mucocutanous lesions as seen in hyperimmunoglobulinemia d syndrome , periodic fever syndrome ( hids ) , familial mediterranean fever ( fmf ) , and tumor necrosis factor receptor - associated fever syndrome ( traps ) . both genders at the third and fourth decade of life can be affected [ 2 , 3 ] . the highest prevalence was reported in turkey ( 40370 per 100000 ) , while this was 80 per 100000 in iran and 7 to 8.5 per 100000 in japan . by contrast , the disease is less common in north america , northern europe , usa , uk , and germany ( 0.12 to 0.64 ) . vascular involvement has been reported in 7.7 to 38% of bd cases , with mortality of about 20% in severe cases . despite lower sensitivity of this manifestation in bd , its higher specificity with respect to eye and skin involvement merits special consideration for behcetologists . venous thrombosis could be as the initial presentation of bd . both veins and arteries with any size can be affected . most authors believed that venous manifestation is more common in bd . according to hamza classification the leading cause of sudden death in patients with bd is usually ruptured of a large aortic or arterial aneurysm . we performed a comprehensive search through medline , web of science , scopus , and google scholar for recently published articles , using the following keywords : behcet 's disease , behcet 's syndrome , cardiovascular , thrombosis , aneurysm , pseudoaneurysm , deep vein thrombosis , embolism , heart , vessel , biological therapy , thrombophilia , infliximab , etanercept , rituximab , atherosclerosis , platelet , and aspirin . the aim of this study is to review the main new concepts on pathophysiology , clinical manifestation , and current and potential future treatment in cardiovascular bd . the real etiopathogenesis of bd is not well understood . as in most autoinflammatory and autoimmune conditions , genetic predisposition and environmental factors may play some role in pathophysiology of bd . hla - b51expression is believed to be associated with more severe disease in several studies and is related to familial bd . in various studies from iran , tunisia , turkey , israel , france , and canada , it was shown that ( tumor necrosis factor ) tnf - alpha-1031c allele and polymorphism in il-21 , 10 , and 8 genes are related to pathogenesis of bd [ 1117 ] . the role of il-23/il-17 axis is recently introduced in inducing bd [ 18 , 19 ] . delayed - type hypersensitivity to infections such as streptococcus species ( especially sanguis ) are supposed in part by some authors to be an important factor in inducing bd . these bacterial antigens along with heat shock protein ( hsp ) 60/65 kda activate t cells to promote inflammatory response in bd [ 9 , 21 ] . besides from antiinflammatory activities of antibiotics , favorable response of joint , and mucucutaneous manifestations of bd to benzathine penicillin may potentiate the hypothesis of some role for infection [ 2224 ] . coagulation abnormalities and platelet overactivity may have some additional role on endothelial damage , inflammation and vascular pathologies in bd ( figure 1 ) [ 2527 ] . increased levels of nitric oxide ( no ) after induction by interferon - gamma are seen in active bd . in addition , hypovitaminosis d was shown to be related to immune aberrancy leading to bd . it has been postulated that platelet - neutrophils complexes ( pnc ) can play an important role in thrombosis - inflammation which is seen in some critical vascular events including bd [ 3035 ] . also it has been shown that inhibition of pnc formation led to marked reduction in pulmonary injuries and improved pulmonary function . khler et al . found that pnc formation facilitates transendothelial movement of platelets involving in process of thrombosis inflammation in vitro and in vivo . elevation in some markers of endothelial dysfunction ( thrombomodulin and adhesion molecules ) and low - grade inflammation ( proinflammatory cytokines , c - reactive protein , and serum amyloid a ) disclose their important role in pathophysiology of most immune - mediated cardiovascular events in bd . impaired hypothalamus - pituitary - adrenal ( hpa ) axis which is an important background in most autoimmune disease may be involved in etiopathogenesis of bd , while this issue has been less addressed in the literatures . the main reason for this is due to the fact that frequently symptoms of patients will appear after a major or even minor emotional stresses . this incompetent hpa axis may lead to overexpression of proinflammatory cytokines definitely involved in pathogenesis of bd . in addition , similar to the other disorders with increased risk of thrombosis formation , there is endothelial cell injury and hypercoagulable state in bd . endothelial dysfunction due to antiendothelial cell antibodies and elevated plasma vascular endothelial growth factor ( vegf ) levels lead to microcirculation disturbance and increased blood viscosity and abnormal blood flow [ 3739 ] . furthermore , selectins , a group of adhesion molecules consist of p and e - selectins that mediate leukocyte adhesion to platelets and endothelium and have a role in thrombogenesis . e - selectin is expressed by activated endothelial cells and p - selectin is synthesized and stored in platelets and endothelial cells . increased p and e - selectin level in bd in addition , it has been reported that factor v leiden [ 4548 ] may be a factor in the development of thrombotic events in bd . also other factors which may play some role in the hypercoagulability are protein c or protein s deficiency [ 49 , 50 ] , impaired fibrinolytic activity , increased homocysteine levels [ 51 , 52 ] , decreased thrombomodulin , elevated lipoprotein ( a ) [ 54 , 55 ] , platelet hyperactivity , high von willebrand factor antigen , and the presence of antiphospholipid antibodies [ 5658 ] . dyslipidaemia and predominantly hypertriglyceridemia might also be risk factors . besides inherent factors inducing thrombophilia in bd ; vasculitis can lead to the enhancement of platelet aggregation , and an impairment of fibrinolysis resulting in thrombosis . it is well known that platelet is one of the most important components in hemostasis . following endothelial injury , the first step in thrombosis , formation is the platelet adherence , aggregation and activation . increased mean platelet volume ( mpv ) , a marker of platelet function , is associated with thrombosis in bd [ 60 , 61 ] . increased aggregation of platelet in vitro in response to adenosine diphosphate ( adp ) and collagen is reported . necrotizing leukocytoclastic or polymorphonuclear ( pmn ) , obliterative perivasculitis and lymphoplasma cell infiltration of capillaries , veins , and arteries , and vasa vasorum ( and not vasa nervorum ) are the main pathologic features in affected tissues . infiltration of pmns is a distinctive pathologic feature of bd addressing to therapeutic role of pmn inhibiting agents such as colchicines and dapson . vascular wall destruction due to inflammatory cell infiltration leads to arterial wall weakening and aneurysmal dilatation . in pseudoaneurysm all layers of intima , media , and adventitia are not involved as seen in aneurysmal lesion ( figure 2 ) . although accelerated atherosclerosis is a major contributor to cardiovascular events in most autoimmune disorders like systemic lupus erythematous and rheumatoid arthritis , several studies showed that atherosclerosis is characteristically not increased in bd [ 64 , 65 ] . despite nonincreased incidence of overt atherosclerosis in bd , intima / media thickness ( imt ) is shown to be increased in some studies that may be due to nonatherosclerotic causes of endothelial hyperactivity . they include pericarditis , coronary artery stenosis or aneurysm , myocarditis , cardiomyopathy , congestive heart failure , valvular pathology , endocarditis , intracardiac thrombosis , and aneurysm of aorta or its branches . the incidence and nature of the cardiovascular involvement in bd are not yet clearly documented . it was reported 1%5% in a case series while it was 16.5% in a registry of behet autopsy cases in japan [ 69 , 70 ] . acute pericarditis , tamponade , constrictive pericarditis , and even only pericardial effusion without any symptom have been reported [ 7275 ] . cad can lead to clinical manifestation such as myocardial infarction , silent ischemia , and stable or unstable angina . aneurysm , stenosis , occlusion , and arteritis are the most common etiologies for cad in bd . coronary aneurysms are more frequent than the stenosis and can present as acute coronary syndrome and myocardial infarction , but sometimes are symptomatic . two forms of aneurysm can be detected by angiography : saccular and fusiform . in general population , acute myocardial infarction ( ami ) is always associated with atherosclerotic coronary artery disease . in young adults with myocardial infarction , nonatherosclerotic etiologies such as embolization , trauma , arteritis , spasm , dissection , and congenital abnormalities should be considered . however , in bd , coronary arteritis has been supposed as an independent pathophysiological mechanism for ami . coronary arteritis may lead to myocardial infarction , but in some of patients with mi , coronary artery is normal . it was shown that occlusion is developed as a result of thrombosis formation in cad , consequently producing ami . understanding the etiology of ami in bd may be important for determination of best treatment strategy . silent myocardial infarction ( smi ) has also been reported in bd [ 81 , 82 ] . the etiology of smi is not clear , although autonomic nervous dysfunction has been suggested as an etiology . reported high prevalence of smi on their studies ( 25% and 19.5% among 36 and 41 patients , resp . ) . myocardial perfusion scintigraphy was recommended by trklmez et al . in patients with long lasted bd for screening of smi a few cases with intracardiac thrombus which often preceding other manifestations of bd have been reported . these thrombi , found mainly in the right ventricle , are often associated with pulmonary artery aneurysm . based on autopsy findings , it looks that endomyocardial fibrosis plays a role in the intracardiac thrombus development in some patients , but it is not clear whether they are secondary to underlying endocarditis or endomyocardial fibrosis , because in some case reports , a normal underlying myocardium was described . due to high specificity of right heart thrombus in bd , in any patient with this finding , diagnosis of bd should be considered [ 86 , 88 , 89 ] . intracardiac thrombus is the major differential diagnosis when a young patient presents with an intracardiac mass . intracardiac thrombus can lead to superior vena cava syndrome [ 90 , 92 , 93 ] and pulmonary embolism [ 83 , 9496 ] . endocardial involvement may present with mitral and aortic valve prolapse , mitral or aortic insufficiency , aneurysm of sinus valsalva , and endocarditis mimicking bacterial endocarditis [ 67 , 97 , 98 ] . the fibrosis secondary to endocardial involvement in bd may predispose to intracardiac thrombus formation . it was reported that valvular prolapse including mitral valve prolapse can be related to vasculitis and tissue derangement . most of the aneurysm of the sinus valsalva has been found in right coronary sinus which project into the right atrium or ventricle . usually they occurred in active phase of bd and were enlarging progressively . heart failure due to conductive abnormalities were also reported in several papers in the past [ 100 , 101 ] that they could directly be attributable or even nonrelated to bd per se . thrombosis is one of the most common and important features in bd with prevalence ranging 1030% in different studies [ 102107 ] . in most case series , fever is frequently found in vascular thrombosis along with constitutional symptoms and high acute phase response . deep vein thrombosis of the lower extremities is the most frequent site for thrombosis in bd ( 6080% of vascular lesion ) [ 103 , 109 ] . this thrombosis has been described at any sites such as cerebral sinuses , intracardiac , renal , ulnar , subclavian , hepatic ( budd - chiari syndrome ) or portal veins , and vena cava . one of important clinical entities in vasculo - bd is venous claudication due to iliac and/or femoral vein thrombosis . classic vessel - related lesions in central nervous system ( cns ) are related to thrombosis , aneurysms , and frank vasculitis . both arteries ( ischemic and embolic stroke - like picture ) and veins ( dural sinus thrombosis ) are vulnerable in bd . in addition , cerebral venous thrombosis ( cvt ) is a serious manifestation of bd . it was reported that cvt represents 730% of all cns lesions of bd in different studies [ 115119 ] . frequently cvt is seen in a young male patient approximately 5 years after initiation of bd . however , cvt might be the presenting feature in up to 20% of patients and could be subacute or chronic . persistent headache , papilledema , nausea , vomiting , and focal deficits are the main complaint among the patients . in 60% of cases , superior sagittal sinus and transverse sinus are the most common sites of cvt ( figure 3 ) . association between major vessel disorders and cvt has also been described . while parenchymal lesions in cns are categorized as a nonvascular lesions , but even parenchymal lesions are resulted from some microvascular events . although it has been reported that venous involvement is the most common vasculo - behcet complication but jamoussi reported that arterial involvement is more frequently seen than the venous complication . abdominal aorta is the common site of aneurysm and approximately 60% of reported arterial lesions associated with bd is aneurysms . aorta is among the most common affected arteries followed by the pulmonary artery . in thoracoabdominal aorta unusual aneurysms and pseudoaneurysms like ulnar , celiac , subclavian , left anterior descending , tibioperoneal , iliac , and superior mesenteric artery aneurysm were reported as rare manifestations of bd [ 124129 ] . pulmonary artery aneurysm ( paa ) with an associated rate of 1%10% of patients in bd is the most common pulmonary lesion . yekeler and goueffic believed that bd is the only vasculitis which could be associated with paa . perihilar or parenchymal opacities can be seen on chest radiographs ; however , these radiographic features are not specific and pathognomonic . occurrence of paa along with deep vein thrombosis is defined as hughes - stovin syndrome ( hss ) , which may be a variant of bd . the patients usually present with cough , dyspnea , fever , chest pain , and hemoptysis . the occurrence of intracranial aneurysm in bd is very rare ( e.g. , only one case in 316 and 119 patients was reported in benamour and al - dalaan studies , resp . ) . middle cerebral artery is the most common affected vessel and subarachnoid hemorrhage is the most presenting manifestation secondary to aneurysm rupture . association of extracranial and intracranial aneurysm was reported in several bd cases , also multiplicity almost always is seen in vasculo - behcet ; thus more investigation in a patient with extracranial aneurysm is recommended . the prognosis of aneurysm has been differed due to severity of the hemorrhage and bd activity . most of the time , aortic aneurysms show rapid progression to rupture , and as a result , huge retroperitoneal hematoma or hemoperitoneum is developed as initial manifestations . aortic aneurysms are often a pseudoaneurysm and usually originate from defects located in the posterior or lateral walls of normally sized aorta and then extend into the adjacent retroperitoneal space . angiography is the best method in diagnosis of aneurysm and pseudoanurysm , but in bd the risk of new aneurysm formation is high . therefore noninvasive methods such as digital subtraction angiography , contrast - enhanced tomography and magnetic resonance angiography are the other methods which have been used safely . inflammatory obliteration of the vasa vasorum and consequent transmural necrosis may develop a pseudoanurysm or rupture an arterial wall . an important characteristic of aneurysm in bd is predilection for the recurrence , which can occur in different locations . matrix metalloproteinase proteins ( mmp ) which play an important role in tissue remodeling and destruction of extracellular matrix components are thought to have significant role in aneurysm formation . reported that serum level of mmp-9 and mmp-2 are important marker in vasculo - behcet . behcet 's disease is a condition without known - specific etiology and evidence that supports the immunologic basis it . therefore , cornerstone of medical management is manipulation of immune system and administering agents against some putative microorganisms and finally surgical intervention in case of vascular complications . managing vascular events in bd is debating and very important to all clinical disciplines including vascular rheumatology , cardiovascular medicine , vascular surgery , general internal medicine , general and interventional radiology , and also thrombologists . due to early diagnosis , aggressive treatments and new techniques of vascular surgery , the prognosis of large vessel involvement in bd is improving . treatment plan is generally based on the symptom(s ) , the organ involved , and severity of conditions that should be tailored individually . as mentioned earlier , bd is a multifeature condition . in the next part they are the pillar of management in any organ involvement in autoinflammatory and autoimmune disease ; therefore corticosteroids are administered for years in almost any organ involvement in bd with dramatic response in most cases . ( not traditional textbook based doses ) tailored for each individual patient could be rational in handling with corticosteroids . one of the agents most studied in randomized controlled trials and nonrandomized trials in bd is azathioprine [ 137139 ] and almost all of them reported good clinical response with negligible adverse effects . our anecdotal reports from iran do not match with this evidence . despite eular recommendations conducted by hatemi et al . denoting use of azathioprine in neurologic involvement in bd , we hardly use this agent in major neurologic manifestations in our center ( and also in iran ) due to uncertainty or lack of clinical benefit and cytopenia in tolerable doses in our practice . the eular recommended this agent in both arterial ( aneurysm ) and venous ( thrombosis ) involvement in central or peripheral vascular bed . its side effects of leukopenia , hair loss , and anorexia are reported to be nonsignificant . the mechanism of action for colchicine is not fully understood , but it is shown that the drug accumulates mostly in neutrophils and interferes with microtubule assembly . colchicine is believed to not only prevent and treat oral and genital aphthi and uveitis but also is used to prevent recurring intracardiac thrombus in bd . similarly it is used for some other autoinflammatory conditions like fmf , pyoderma gangrenosum , and other pmn - mediated conditions like sweet 's syndrome . therefore colchicine should be considered as a safe systemic disease modifying antirheumatic drug ( dmard ) in any patient with potentially active bd . alternatively , considering infiltration of neutrophils in affected vessel wall , doxycycline , a matrix metaloproteinase inhibitor was used in a 2 weeks period after surgery of abdominal aortic aneurysm with better results than placebo . this agent with inhibition of il-2 and il-17 is a powerful drug in controlling bd . thalidomide , an old drug which discovered in 1950s , revisited in recent years as a good antiinflammatory in conditions like aphthous ulcerations , hiv - associated cachexia , inflammatory bowel diseases , and mucocutaneous and intestinal bd with mechanism of inhibitions of angiogenesis and tnf - alpha [ 142 , 143 ] . as all clinicians know , standard of care in deep vein thrombosis is anticoagulation in almost all cases . even there are some indications to thrombolytic therapy in some selected cases of dvts . the challenging issue is holding anticoagulation in setting of bd complicated with dvt due to lack of risk of embolic events as supposed by caspary . the authors of this review believe that inflammation is an additional feature of bd complicated with thrombosis . presence of several thrombophilia states in bd [ 25 , 58 , 145 , 146 ] may offset in part the theory of adhering clot to highly inflammatory veins with no risk of embolisation . in our interdisciplinary discussion , we argued about cases in whom repeated attacks of dvt may precede months or years before full - picture bd and despite the same story for pathophysiology of dvt in these cases , it seems not to prudent withholding anticoagulation in these cases . in addition , we believe any mentioned and well - accepted risk of major bleeding in routine anticoagulation therapies ( about 7% ) may be due to any underlying occult vascular pathologies such as hidden aneurysms or vascular disintegrities like subclinical bd . showed that anticoagulation is not only an effective but also a safe treatment in cerebral venous thrombosis . it seems that thrombophilia in bd is a sophisticated process of intervening inherent or hereditary condition , endothelial hyperactivity , and low - grade inflammation . therefore , it is better to just recommend management of dvt in bd to be accomplished by highly expert physicians and not to discourage anticoagulation [ 147 , 148 ] even in pediatric bd except for pulmonary arterial involvement . some authors limit control of inflammation for the purpose of prevention of thrombosis rather than treatment of it and handling complicated cases of cerebral sinus , vein and pulmonary vein thrombosis is based on per - case decision made by a multidisciplinary therapeutic team sometimes necessitating more aggressive treatments such as local thrombolysis or surgical interventions . regarding intracardiac thrombus , some authors experienced successful thrombolytic therapy in the case of recurrent right ventricular thrombosis [ 150 , 151 ] . khammar and some other authors suggested colchicine , anticoagulant therapy , and corticosteroids for intracardiac thrombosis in bd [ 84 , 152 ] . in the case of neurovascular bd treatment , ( in contrary to eye , joint and mucocutaneous studies with level ib evidence ) there is no high - quality controlled trials and almost all studies are case - control or comparative ( level iii ) . benign vascular headache in bd is managed conservatively as usual migraine . in cvt , some authors use anticoagulants along with short course glucocorticoids and the others manage it as other parenchymal brain disease in bd . in the newest recommendation , eular did not address separately to cvt regarding anticoagulation usage but recommended corticosteroids in dural sinus thrombosis . it has also been emphasized that in cns bd , cyclosporine a should be withheld except in patients with concomitant eye involvement . surgical repair is the standard care in cases of organ threatening aneurysmal dilatation , stenosis , or obstruction of clinically significant arteries . in some cases , ligation of affected vessel ( mostly peripheral ones ) is mandatory even in the cases of pulmonary artery [ 154 , 155 ] . endovascular approach is a minimally invasive technique for aneurysm repair and is growingly advised by some authors [ 125 , 134 , 156 ] . in bd , this technique has less complications and better results comparing to atherosclerotic lesions . endarterectomy was performed in rare incidence of bilateral common carotid artery occlusion and chronic thromboembolic pulmonary hypertension secondary to bd [ 158 , 159 ] . other approach to some kinds of aneurysms is percutaneous embolization of individual aneurysm using substances like n - butyl cyanoacrylate . this is important to mention that systemic ( and/or local ) inflammation should be controlled before any elective surgical intervention . biologic agents are emerged in recent decades and are targeted drugs against specific mediators of inflammations . in general , biologic agents are the second choice in cases of bd refractory to conventional immunosuppression . in one study , baki et al . used infliximab de novo without preceding glucocorticoids and cyclophosphamide in a man with paa and reported complete remission in two years . the most popular biologic agents are agents against tnf - alpha ( infliximab , etanercept and adalimumab ) , b - cell depleting agents ( rituximab ) , and specific monoclonal antibodies to interleukin ( il)-1 ( anakinra ) , il-6 . most of studies on infliximab deal with inflammatory eye disease in bd . considering the role of tnf - alpha in triggering prothrombotic adhesion molecules , using tnf blocking agents could be theoretically of some interest in vasculo - bd . infliximab was used in two cases of paa ( with intracardiac thrombosis ) , one case with femoral profunda artery aneurysm and two cases with magic syndrome with more than a year of complete remission [ 166168 ] . employing tnf ( and probably other biological agents ) in thrombosis of bd was disappointing with especial regard to potential prothrombotic activities and predisposition to frequent autoantibody formation ( notably anticardiolipin ) of these agents . infliximab was used in several studies as an agent to improve endothelia dysfunction and suppress low - grade inflammation in the major cases of cardiovascular burden in most autoimmune and autoinflammatory conditions [ 36 , 170 , 171 ] . adalimumab is another tnf blocking agents which was used in a 43 years old man with paa . neither etanercept nor rituximab is reported to be useful in aneurysmal type bd so far . in recent eular recommendations little information is provided regarding these agents in cardiovascular bd . in a recent systematic review our search results showed only two studies ( both from iran ) addressing the use of rituximab in retinal vasculitis in bd and both had favorable response [ 172 , 173 ] and several trials mostly as case reports with etanercept uveoretinal involvement even in juvenile bd [ 174 , 175 ] . the reason for the far less studies on biological agents in vascular bd could be due to rather less frequent clinical vascular events and limited time for multiple drug trials with newly lunched drugs due to emergent nature of vasculo - behcet . dyslipidemia , a known risk factor for cardiovascular events in both autoimmune conditions and general population , is accelerated with most biologic agents except for adalimumab , rituximab , and etanercept . interferon alpha , a natural glycoproteins that has antiproliferative and antiviral properties , is used in retinal vasculitis . in a study it was shown that granulocytapheresis is more beneficial in 14 cases with long duration of bd with uveoretinitis . in addition , hematopoetic stem cell transplantation was used in several cases with necrotizing vasculitis including three cases with bd with inconsistent clinical results . as optimum management in bd is not achieved yet , future direction in treating bd could be focused on better understanding of common pathways in pathophysiology of vascular and nonvascular bd . no doubt is about the future role of biologic agents and targeted agents against proinflammatory cytokines such as il-15 , 17 , 21 , and adhesion molecules in controlling bd . using biological agents targeting tnf - alpha may improve endothelial dysfunction responsible for more features of bd . addressing many disease flare - ups after major or even minor physical or emotional stressors , we should also emphasize on potential therapeutic role of hpa axis support to prevent and control bd . taking into consideration the important role of activated platelets and unique thrombosis - inflammation relationship in bd , more clinical application of antiplatelet ( aspirin , clopidegrel ) solely or in combination with colchicine as a pmn inhibitor is justified . role of statins as lipid lowering and antiinflammatory agents and improving endothelial dysfunction in bd could be matter of future studies for clinical use . detection of possible putative triggering microorganism(s ) could direct treatment to some specific antibiotics and even categorize bd as a potentially infectious disease .

behcet 's disease ( bd ) is the only systemic vasculitis involving both arteries and vein in any sizes . it frequently encounters in rheumatology clinics . it has some major morbidities and even fatal outcomes in some cases . the aim of this paper is to analyze the main concepts on pathophysiology and treatment options in bd , focusing on cardiovascular aspects , thrombosis , and potential future treatment .

1. Introduction 2. Cardiovascular Etiopathogenesis and Pathology 3. Cardiac Manifestations 4. Thrombosis 5. Aneurysm 6. Treatment

it clinically diagnoses with recurrent oral and/or genital aphthosis , mucocutaneous lesions , central nervous system manifestation , and ocular , vascular , articular , gastrointestinal , and inflammatory eye lesions . these conditions share in some clinical aspects including uveitis , fever , arthritis , and mucocutanous lesions as seen in hyperimmunoglobulinemia d syndrome , periodic fever syndrome ( hids ) , familial mediterranean fever ( fmf ) , and tumor necrosis factor receptor - associated fever syndrome ( traps ) . the highest prevalence was reported in turkey ( 40370 per 100000 ) , while this was 80 per 100000 in iran and 7 to 8.5 per 100000 in japan . by contrast , the disease is less common in north america , northern europe , usa , uk , and germany ( 0.12 to 0.64 ) . despite lower sensitivity of this manifestation in bd , its higher specificity with respect to eye and skin involvement merits special consideration for behcetologists . most authors believed that venous manifestation is more common in bd . according to hamza classification the leading cause of sudden death in patients with bd is usually ruptured of a large aortic or arterial aneurysm . we performed a comprehensive search through medline , web of science , scopus , and google scholar for recently published articles , using the following keywords : behcet 's disease , behcet 's syndrome , cardiovascular , thrombosis , aneurysm , pseudoaneurysm , deep vein thrombosis , embolism , heart , vessel , biological therapy , thrombophilia , infliximab , etanercept , rituximab , atherosclerosis , platelet , and aspirin . the aim of this study is to review the main new concepts on pathophysiology , clinical manifestation , and current and potential future treatment in cardiovascular bd . hla - b51expression is believed to be associated with more severe disease in several studies and is related to familial bd . in various studies from iran , tunisia , turkey , israel , france , and canada , it was shown that ( tumor necrosis factor ) tnf - alpha-1031c allele and polymorphism in il-21 , 10 , and 8 genes are related to pathogenesis of bd [ 1117 ] . the role of il-23/il-17 axis is recently introduced in inducing bd [ 18 , 19 ] . delayed - type hypersensitivity to infections such as streptococcus species ( especially sanguis ) are supposed in part by some authors to be an important factor in inducing bd . these bacterial antigens along with heat shock protein ( hsp ) 60/65 kda activate t cells to promote inflammatory response in bd [ 9 , 21 ] . besides from antiinflammatory activities of antibiotics , favorable response of joint , and mucucutaneous manifestations of bd to benzathine penicillin may potentiate the hypothesis of some role for infection [ 2224 ] . coagulation abnormalities and platelet overactivity may have some additional role on endothelial damage , inflammation and vascular pathologies in bd ( figure 1 ) [ 2527 ] . in addition , hypovitaminosis d was shown to be related to immune aberrancy leading to bd . it has been postulated that platelet - neutrophils complexes ( pnc ) can play an important role in thrombosis - inflammation which is seen in some critical vascular events including bd [ 3035 ] . also it has been shown that inhibition of pnc formation led to marked reduction in pulmonary injuries and improved pulmonary function . elevation in some markers of endothelial dysfunction ( thrombomodulin and adhesion molecules ) and low - grade inflammation ( proinflammatory cytokines , c - reactive protein , and serum amyloid a ) disclose their important role in pathophysiology of most immune - mediated cardiovascular events in bd . impaired hypothalamus - pituitary - adrenal ( hpa ) axis which is an important background in most autoimmune disease may be involved in etiopathogenesis of bd , while this issue has been less addressed in the literatures . the main reason for this is due to the fact that frequently symptoms of patients will appear after a major or even minor emotional stresses . in addition , similar to the other disorders with increased risk of thrombosis formation , there is endothelial cell injury and hypercoagulable state in bd . e - selectin is expressed by activated endothelial cells and p - selectin is synthesized and stored in platelets and endothelial cells . increased p and e - selectin level in bd in addition , it has been reported that factor v leiden [ 4548 ] may be a factor in the development of thrombotic events in bd . also other factors which may play some role in the hypercoagulability are protein c or protein s deficiency [ 49 , 50 ] , impaired fibrinolytic activity , increased homocysteine levels [ 51 , 52 ] , decreased thrombomodulin , elevated lipoprotein ( a ) [ 54 , 55 ] , platelet hyperactivity , high von willebrand factor antigen , and the presence of antiphospholipid antibodies [ 5658 ] . besides inherent factors inducing thrombophilia in bd ; vasculitis can lead to the enhancement of platelet aggregation , and an impairment of fibrinolysis resulting in thrombosis . following endothelial injury , the first step in thrombosis , formation is the platelet adherence , aggregation and activation . increased mean platelet volume ( mpv ) , a marker of platelet function , is associated with thrombosis in bd [ 60 , 61 ] . necrotizing leukocytoclastic or polymorphonuclear ( pmn ) , obliterative perivasculitis and lymphoplasma cell infiltration of capillaries , veins , and arteries , and vasa vasorum ( and not vasa nervorum ) are the main pathologic features in affected tissues . in pseudoaneurysm all layers of intima , media , and adventitia are not involved as seen in aneurysmal lesion ( figure 2 ) . although accelerated atherosclerosis is a major contributor to cardiovascular events in most autoimmune disorders like systemic lupus erythematous and rheumatoid arthritis , several studies showed that atherosclerosis is characteristically not increased in bd [ 64 , 65 ] . despite nonincreased incidence of overt atherosclerosis in bd , intima / media thickness ( imt ) is shown to be increased in some studies that may be due to nonatherosclerotic causes of endothelial hyperactivity . they include pericarditis , coronary artery stenosis or aneurysm , myocarditis , cardiomyopathy , congestive heart failure , valvular pathology , endocarditis , intracardiac thrombosis , and aneurysm of aorta or its branches . the incidence and nature of the cardiovascular involvement in bd are not yet clearly documented . acute pericarditis , tamponade , constrictive pericarditis , and even only pericardial effusion without any symptom have been reported [ 7275 ] . cad can lead to clinical manifestation such as myocardial infarction , silent ischemia , and stable or unstable angina . aneurysm , stenosis , occlusion , and arteritis are the most common etiologies for cad in bd . coronary aneurysms are more frequent than the stenosis and can present as acute coronary syndrome and myocardial infarction , but sometimes are symptomatic . in general population , acute myocardial infarction ( ami ) is always associated with atherosclerotic coronary artery disease . in young adults with myocardial infarction , nonatherosclerotic etiologies such as embolization , trauma , arteritis , spasm , dissection , and congenital abnormalities should be considered . however , in bd , coronary arteritis has been supposed as an independent pathophysiological mechanism for ami . coronary arteritis may lead to myocardial infarction , but in some of patients with mi , coronary artery is normal . it was shown that occlusion is developed as a result of thrombosis formation in cad , consequently producing ami . understanding the etiology of ami in bd may be important for determination of best treatment strategy . silent myocardial infarction ( smi ) has also been reported in bd [ 81 , 82 ] . based on autopsy findings , it looks that endomyocardial fibrosis plays a role in the intracardiac thrombus development in some patients , but it is not clear whether they are secondary to underlying endocarditis or endomyocardial fibrosis , because in some case reports , a normal underlying myocardium was described . due to high specificity of right heart thrombus in bd , in any patient with this finding , diagnosis of bd should be considered [ 86 , 88 , 89 ] . intracardiac thrombus is the major differential diagnosis when a young patient presents with an intracardiac mass . endocardial involvement may present with mitral and aortic valve prolapse , mitral or aortic insufficiency , aneurysm of sinus valsalva , and endocarditis mimicking bacterial endocarditis [ 67 , 97 , 98 ] . the fibrosis secondary to endocardial involvement in bd may predispose to intracardiac thrombus formation . thrombosis is one of the most common and important features in bd with prevalence ranging 1030% in different studies [ 102107 ] . deep vein thrombosis of the lower extremities is the most frequent site for thrombosis in bd ( 6080% of vascular lesion ) [ 103 , 109 ] . this thrombosis has been described at any sites such as cerebral sinuses , intracardiac , renal , ulnar , subclavian , hepatic ( budd - chiari syndrome ) or portal veins , and vena cava . classic vessel - related lesions in central nervous system ( cns ) are related to thrombosis , aneurysms , and frank vasculitis . both arteries ( ischemic and embolic stroke - like picture ) and veins ( dural sinus thrombosis ) are vulnerable in bd . in addition , cerebral venous thrombosis ( cvt ) is a serious manifestation of bd . however , cvt might be the presenting feature in up to 20% of patients and could be subacute or chronic . persistent headache , papilledema , nausea , vomiting , and focal deficits are the main complaint among the patients . association between major vessel disorders and cvt has also been described . although it has been reported that venous involvement is the most common vasculo - behcet complication but jamoussi reported that arterial involvement is more frequently seen than the venous complication . abdominal aorta is the common site of aneurysm and approximately 60% of reported arterial lesions associated with bd is aneurysms . in thoracoabdominal aorta unusual aneurysms and pseudoaneurysms like ulnar , celiac , subclavian , left anterior descending , tibioperoneal , iliac , and superior mesenteric artery aneurysm were reported as rare manifestations of bd [ 124129 ] . pulmonary artery aneurysm ( paa ) with an associated rate of 1%10% of patients in bd is the most common pulmonary lesion . yekeler and goueffic believed that bd is the only vasculitis which could be associated with paa . the patients usually present with cough , dyspnea , fever , chest pain , and hemoptysis . the occurrence of intracranial aneurysm in bd is very rare ( e.g. middle cerebral artery is the most common affected vessel and subarachnoid hemorrhage is the most presenting manifestation secondary to aneurysm rupture . most of the time , aortic aneurysms show rapid progression to rupture , and as a result , huge retroperitoneal hematoma or hemoperitoneum is developed as initial manifestations . angiography is the best method in diagnosis of aneurysm and pseudoanurysm , but in bd the risk of new aneurysm formation is high . inflammatory obliteration of the vasa vasorum and consequent transmural necrosis may develop a pseudoanurysm or rupture an arterial wall . an important characteristic of aneurysm in bd is predilection for the recurrence , which can occur in different locations . reported that serum level of mmp-9 and mmp-2 are important marker in vasculo - behcet . behcet 's disease is a condition without known - specific etiology and evidence that supports the immunologic basis it . managing vascular events in bd is debating and very important to all clinical disciplines including vascular rheumatology , cardiovascular medicine , vascular surgery , general internal medicine , general and interventional radiology , and also thrombologists . due to early diagnosis , aggressive treatments and new techniques of vascular surgery , the prognosis of large vessel involvement in bd is improving . treatment plan is generally based on the symptom(s ) , the organ involved , and severity of conditions that should be tailored individually . in the next part they are the pillar of management in any organ involvement in autoinflammatory and autoimmune disease ; therefore corticosteroids are administered for years in almost any organ involvement in bd with dramatic response in most cases . ( not traditional textbook based doses ) tailored for each individual patient could be rational in handling with corticosteroids . one of the agents most studied in randomized controlled trials and nonrandomized trials in bd is azathioprine [ 137139 ] and almost all of them reported good clinical response with negligible adverse effects . denoting use of azathioprine in neurologic involvement in bd , we hardly use this agent in major neurologic manifestations in our center ( and also in iran ) due to uncertainty or lack of clinical benefit and cytopenia in tolerable doses in our practice . its side effects of leukopenia , hair loss , and anorexia are reported to be nonsignificant . colchicine is believed to not only prevent and treat oral and genital aphthi and uveitis but also is used to prevent recurring intracardiac thrombus in bd . similarly it is used for some other autoinflammatory conditions like fmf , pyoderma gangrenosum , and other pmn - mediated conditions like sweet 's syndrome . therefore colchicine should be considered as a safe systemic disease modifying antirheumatic drug ( dmard ) in any patient with potentially active bd . thalidomide , an old drug which discovered in 1950s , revisited in recent years as a good antiinflammatory in conditions like aphthous ulcerations , hiv - associated cachexia , inflammatory bowel diseases , and mucocutaneous and intestinal bd with mechanism of inhibitions of angiogenesis and tnf - alpha [ 142 , 143 ] . even there are some indications to thrombolytic therapy in some selected cases of dvts . the challenging issue is holding anticoagulation in setting of bd complicated with dvt due to lack of risk of embolic events as supposed by caspary . the authors of this review believe that inflammation is an additional feature of bd complicated with thrombosis . presence of several thrombophilia states in bd [ 25 , 58 , 145 , 146 ] may offset in part the theory of adhering clot to highly inflammatory veins with no risk of embolisation . it seems that thrombophilia in bd is a sophisticated process of intervening inherent or hereditary condition , endothelial hyperactivity , and low - grade inflammation . therefore , it is better to just recommend management of dvt in bd to be accomplished by highly expert physicians and not to discourage anticoagulation [ 147 , 148 ] even in pediatric bd except for pulmonary arterial involvement . khammar and some other authors suggested colchicine , anticoagulant therapy , and corticosteroids for intracardiac thrombosis in bd [ 84 , 152 ] . benign vascular headache in bd is managed conservatively as usual migraine . in cvt , some authors use anticoagulants along with short course glucocorticoids and the others manage it as other parenchymal brain disease in bd . in the newest recommendation , eular did not address separately to cvt regarding anticoagulation usage but recommended corticosteroids in dural sinus thrombosis . it has also been emphasized that in cns bd , cyclosporine a should be withheld except in patients with concomitant eye involvement . surgical repair is the standard care in cases of organ threatening aneurysmal dilatation , stenosis , or obstruction of clinically significant arteries . in some cases , ligation of affected vessel ( mostly peripheral ones ) is mandatory even in the cases of pulmonary artery [ 154 , 155 ] . in bd , this technique has less complications and better results comparing to atherosclerotic lesions . the most popular biologic agents are agents against tnf - alpha ( infliximab , etanercept and adalimumab ) , b - cell depleting agents ( rituximab ) , and specific monoclonal antibodies to interleukin ( il)-1 ( anakinra ) , il-6 . most of studies on infliximab deal with inflammatory eye disease in bd . in a recent systematic review our search results showed only two studies ( both from iran ) addressing the use of rituximab in retinal vasculitis in bd and both had favorable response [ 172 , 173 ] and several trials mostly as case reports with etanercept uveoretinal involvement even in juvenile bd [ 174 , 175 ] . dyslipidemia , a known risk factor for cardiovascular events in both autoimmune conditions and general population , is accelerated with most biologic agents except for adalimumab , rituximab , and etanercept . as optimum management in bd is not achieved yet , future direction in treating bd could be focused on better understanding of common pathways in pathophysiology of vascular and nonvascular bd . no doubt is about the future role of biologic agents and targeted agents against proinflammatory cytokines such as il-15 , 17 , 21 , and adhesion molecules in controlling bd . taking into consideration the important role of activated platelets and unique thrombosis - inflammation relationship in bd , more clinical application of antiplatelet ( aspirin , clopidegrel ) solely or in combination with colchicine as a pmn inhibitor is justified . role of statins as lipid lowering and antiinflammatory agents and improving endothelial dysfunction in bd could be matter of future studies for clinical use . detection of possible putative triggering microorganism(s ) could direct treatment to some specific antibiotics and even categorize bd as a potentially infectious disease .

the online version of this article ( doi:10.1007/s10334 - 014 - 0457 - 0 ) contains supplementary material , which is available to authorized users . intervertebral disc degeneration ( ivdd ) is a common clinical problem whose pathogenesis is still not very well understood . however , it is known that ivdd causes biochemical and morphological changes in the structure of the disc , leading to the deterioration of the biomechanical function of the joint and low back pain [ 14 ] . disc degeneration may result from mechanical trauma [ 3 , 5 ] , patient age , familial predisposition [ 5 , 6 ] , and an imbalance between anabolic and catabolic processes . a healthy disc consists of three elements : the nucleus pulposus ( np ) , the annulus fibrosus ( af ) , and endplates separating the nucleus from the adjacent vertebral bone . the central region of the nucleus pulposus is highly hydrated and acts as a hydraulic cushion to withstand the forces of compression and torsion . the annulus fibrosus prevents the nucleus pulposus from herniating or leaking out of the disc by hydraulically sealing the nucleus and evenly distributing any pressure and force imposed on the intervertebral disc . radiography , discography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) are used to diagnose disc degeneration [ 813 ] . interpreting the results of imaging is difficult and complicated because discs are composed of several subtissues ( nucleus , annulus , endplate ) and there is poor correlation between morphological findings , spinal biomechanics , and patient symptoms [ 3 , 1417 ] . high resolution magic angle spinning ( hr mas ) h nmr spectroscopy is a nondestructive technique that has been applied to characterize the composition of various intact human cancer tissues , such as breast [ 18 , 19 ] , brain [ 20 , 21 ] , prostate [ 2224 ] , lung , and colon cancers . [ 13 ] observed changes in the chemical composition of discs , especially increased levels of unbound hydroxyproline and glycine , associated with collagen breakdown . they noticed a decrease in the concentration of proteoglycans correlated with degeneration and suggested that lactate , collagen , and proteoglycan may serve as metabolic markers in discogenic back pain . a synergic combination of nmr spectroscopy and chemometric techniques offers a useful tool for the identification of markers of degenerative disc disease . the purpose of this study is the determination of disc metabolites in order to evaluate their applicability in the diagnosis of intervertebral disc degeneration by means of h hr mas nmr . we hope that in the near future hr mas nmr ex vivo studies of metabolic profiles combined with in vivo studies using mri scanners ( mrs ) may become part of a new diagnostic protocol . analysis was performed for 81 disc tissue samples ( control samples n = 21 , degenerated disc tissue samples tissue samples were harvested from 60 patients who underwent intervertebral disc surgery at the wam university hospital , central veteran hospital of the medical university of d , poland . the reference samples originated from non - degenerated discs after mechanical trauma ( n = 20 ) and from a post - mortem section ( n = 1 ) . after standard preparation of the operation field , intervertebral disc exposure was performed using microsurgery . the annulus fibrosus was fenestrated under the operating microscope and a 3 mm 3 mm specimen was taken . afterwards , tissue was harvested from the deeper layers of the intervertebral disc and denoted as nucleus . for each patient , annulus fibrosus and nucleus pulposus samples were analyzed by means of h hr mas nmr . analysis was performed on the following lumbar ( l ) and cervical ( c ) intervertebral discs : l1/l2 ( n = 2 ) , l2/l3 ( n = 2 ) , l3/l4 ( n = 3 ) , l4/l5 ( n = 20 ) , l5/s1 ( n = 30 ) , c2/c3 ( n = 2 ) , c5/c6 ( n = 12 ) , and c6/c7 ( n = 10 ) . all tissue samples were placed on ice after surgery for 15 min and then stored at 80 c until hr mas analysis within 1 week . no changes in h nmr spectra were observed after 7 days in storage at 80 c temperature . prior to hr mas analysis , all samples were cut to fit the 4 mm zirconium hr mas rotor ( a total sample volume of 50 l ) . the mean age of the patients was 46.5 13.7 ( sd ) years ( range 1678 years ) . all spectra were acquired using a bruker avance ii plus 16.4 t spectrometer ( brukerbiospin , germany ) operating atan h frequency ( 700.08 mhz ) . the instrument was equipped with a 4 mm h / c hr mas probe with the gradient aligned along the magic angle axis . samples were spun at 6 khz to keep rotation sidebands out of the acquisition window . all experiments were conducted at nominally 25 c for 20 min , to avoid the tca cycle metabolites such as succinate , citrate , and oxaloacetate formation . changes of the concentration of the other metabolites were not observed during this short time . at lower temperatures ( 5 and 15 c ) the resolution of the h spectra phosphate - buffered saline ( pbs 0.1 m , ph 7.4 , 30 l ) made with deuterium oxide and containing 3.8 mm tsp ( sodium-3-trimethylsilylpropionate-2,2,3,3-d4 ) was added to the each sample . gill ( cpmgpr , bruker ) spin - echo sequence was applied with a delay of 1 ms , repeated 140 times . spectra were recorded with 1.5 s water presaturation during the relaxation delay , and a calibrated 90 pulse was applied for 128 scans , collecting 64 k data points over a spectral width of 14,097 hz . the repetition time of 10.32 s , including a relaxation delay of 8 s , was calculated as 7t1 , which had the longest relaxation time to ensure complete magnetization recovery . an exponential line broadening of 0.30 hz was applied to raw data prior to fourier transformation . the tsp peak at 0 ppm was used as a chemical shift standard and a linear baseline correction was applied . homonuclear correlated spectra ( cosy ) and total correlation spectroscopy ( tocsy ) experiments were performed on selected samples to aid peak identification . spectra were recorded with acquisition of 16 transients for each of the 512 increments with 2 k data points . in spectra recorded for 20 min , only correlations for highly concentrated metabolites were observed . quantitative analysis was performed for the spectral regions from 0.8 to 4.65 , from 5.0 to 8.2 , and the region corresponding to tsp ( from 0.1 to 0.1 ppm ) . molar metabolite concentrations were calculated from the equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{\text{met } } ] = \frac{{a_{\text{met } } } } { { a_{\text{tsp } } } } \times \frac{{h_{\text{tsp } } } } { { h_{\text{met } } } } \times \frac{{n_{\text{tsp } } } } { { m_{\text{sample } } } } , $ $ \end{document}[met]=ametatsphtsphmetntspmsample , where amet and atsp are areas of metabolite and tsp signals , respectively ; hmet and htsp are the numbers of protons per metabolite and tsp signals ; ntsp is the number of moles the tsp signal represents , and msample is the weight of the sample in the mas rotor . the variation of the data was explored by principal component analysis ( pca ) , with np spectra being more suitable for this type of analysis . the spectral region from 3.29 to 4.05 of spin - echo h nmr spectra was chosen as input data for pca analysis . linear combinations of metabolites that explain most of the overall variance in the data set were calculated by means of pca . baseline offset was corrected , and the selected spectral region was mean - normalized to arrive at the total area for each sample . analysis was performed for 81 disc tissue samples ( control samples n = 21 , degenerated disc tissue samples tissue samples were harvested from 60 patients who underwent intervertebral disc surgery at the wam university hospital , central veteran hospital of the medical university of d , poland . the reference samples originated from non - degenerated discs after mechanical trauma ( n = 20 ) and from a post - mortem section ( n = 1 ) . after standard preparation of the operation field , intervertebral disc exposure was performed using microsurgery . the annulus fibrosus was fenestrated under the operating microscope and a 3 mm 3 mm specimen was taken . afterwards , tissue was harvested from the deeper layers of the intervertebral disc and denoted as nucleus . for each patient , annulus fibrosus and nucleus pulposus samples were analyzed by means of h hr mas nmr . analysis was performed on the following lumbar ( l ) and cervical ( c ) intervertebral discs : l1/l2 ( n = 2 ) , l2/l3 ( n = 2 ) , l3/l4 ( n = 3 ) , l4/l5 ( n = 20 ) , l5/s1 ( n = 30 ) , c2/c3 ( n = 2 ) , c5/c6 ( n = 12 ) , and c6/c7 ( n = 10 ) . all tissue samples were placed on ice after surgery for 15 min and then stored at 80 c until hr mas analysis within 1 week . no changes in h nmr spectra were observed after 7 days in storage at 80 c temperature . prior to hr mas analysis , all samples were cut to fit the 4 mm zirconium hr mas rotor ( a total sample volume of 50 l ) . the mean age of the patients was 46.5 13.7 ( sd ) years ( range 1678 years ) . all spectra were acquired using a bruker avance ii plus 16.4 t spectrometer ( brukerbiospin , germany ) operating atan h frequency ( 700.08 mhz ) . the instrument was equipped with a 4 mm h / c hr mas probe with the gradient aligned along the magic angle axis . samples were spun at 6 khz to keep rotation sidebands out of the acquisition window . all experiments were conducted at nominally 25 c for 20 min , to avoid the tca cycle metabolites such as succinate , citrate , and oxaloacetate formation . changes of the concentration of the other metabolites were not observed during this short time . at lower temperatures ( 5 and 15 c ) the resolution of the h spectra phosphate - buffered saline ( pbs 0.1 m , ph 7.4 , 30 l ) made with deuterium oxide and containing 3.8 mm tsp ( sodium-3-trimethylsilylpropionate-2,2,3,3-d4 ) was added to the each sample . gill ( cpmgpr , bruker ) spin - echo sequence was applied with a delay of 1 ms , repeated 140 times . spectra were recorded with 1.5 s water presaturation during the relaxation delay , and a calibrated 90 pulse was applied for 128 scans , collecting 64 k data points over a spectral width of 14,097 hz . the repetition time of 10.32 s , including a relaxation delay of 8 s , was calculated as 7t1 , which had the longest relaxation time to ensure complete magnetization recovery . an exponential line broadening of 0.30 hz was applied to raw data prior to fourier transformation . the tsp peak at 0 ppm was used as a chemical shift standard and a linear baseline correction was applied . homonuclear correlated spectra ( cosy ) and total correlation spectroscopy ( tocsy ) experiments were performed on selected samples to aid peak identification . spectra were recorded with acquisition of 16 transients for each of the 512 increments with 2 k data points . in spectra recorded for 20 min , only correlations for highly concentrated metabolites were observed . quantitative analysis was performed for the spectral regions from 0.8 to 4.65 , from 5.0 to 8.2 , and the region corresponding to tsp ( from 0.1 to 0.1 ppm ) . molar metabolite concentrations were calculated from the equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{\text{met } } ] = \frac{{a_{\text{met } } } } { { a_{\text{tsp } } } } \times \frac{{h_{\text{tsp } } } } { { h_{\text{met } } } } \times \frac{{n_{\text{tsp } } } } { { m_{\text{sample } } } } , $ $ \end{document}[met]=ametatsphtsphmetntspmsample , where amet and atsp are areas of metabolite and tsp signals , respectively ; hmet and htsp are the numbers of protons per metabolite and tsp signals ; ntsp is the number of moles the tsp signal represents , and msample is the weight of the sample in the mas rotor . the variation of the data was explored by principal component analysis ( pca ) , with np spectra being more suitable for this type of analysis . the spectral region from 3.29 to 4.05 of spin - echo h nmr spectra was chosen as input data for pca analysis . linear combinations of metabolites that explain most of the overall variance in the data set were calculated by means of pca . baseline offset was corrected , and the selected spectral region was mean - normalized to arrive at the total area for each sample . hr mas nmr spectroscopy makes it possible to determine metabolite concentrations in intact tissues . sample preparation for hr mas is simpler than analysis of extracts , which requires sample homogenization and standardization for each metabolite . representative ex vivo h hr masnmr spectra of healthy and degenerated disc tissues are shown in fig . 1 . assignment of metabolite resonances was based on analysis of one dimensional h , two dimensional correlation spectroscopy ( cosy ) , and total correlation spectroscopy ( tocsy ) nmr spectra . the collected data were compared with those from the literature and with the spectra of metabolites recorded on a bruker avance ii plus700 mhz spectrometer . theh hr mas nmr spectra for nucleus pulposus and annulus fibrosus specimens were very similar ( see figure sm1 in supplementary material ) . in the hr mas spectra of disc tissues , signals of five groups of compounds were observed . the main metabolites characterizing disc tissues are labeled in fig . 1 and the pool of metabolites the largest group consisted of 12 amino acids , which were derived from protein cores and were also formed by collagen breakdown . the second group of compounds was comprised of carboxylic acids and their derivatives , mainly lactic acid , citric acid , formate , acetate , and acetone . moreover , some carbohydrates ( mainly - and -glucose and chondroitin sulfate ) and pyrimidine derivatives ( e.g. uracil ) were found in the spectra of disc tissues . alcohols , such as myo - inositol , scyllo - inositol , and 2-propanol , were also identified . in the case of 2-propanol , the possibility of its exogenous origin was checked at every step from surgery to hr mas measurements , but it was deemed possible for 2-propanol to have been introduced into disc tissue samples as a result of its antiseptic use . the patients were being administered pain relief and non - steroidal anti - inflammatory drugs , but 2-propanol was not found to be a component or metabolite of those drugs . resonances of 2-propanol was observed due to good resolution of h nmr spectra and confirmed by cosy / tocsy experiments ( see figure sm3sm4 in supplementary materials ) . the unassigned signal at 1.2 ppm reported earlier probably corresponds to 2-propanol , but was not recognized in poorly resolved spectra.fig . 1representative ex vivo h hr mas nmr spectra of a healthy disc ( a ) , and of a degenerated disc for the aliphatic ( b ) and aromatic ( c ) regions of the spectrumtable 1metabolites assigned in hr mas spectra of disc tissues , their diagnostic h signals , chemical shifts ( h ) and multiplicitiesassignment numbermetaboliteassignment h multiplicity h , ppm1acetatech3 s1.932acetonech3 s2.233alaninech3 d1.48chq3.784aspartate-ch2 dd , dd2.67 , 2.81-chdd3.915chondroitin sulfate n - acetyls2.056citric acidch2 d2.54ch2 d2.667creatinech3 s3.03ch2 s3.938fatty acidsch3 t0.90(ch2)n m1.29ch2ch2coq1.559formates8.4510glycine-ch2 s3.5511-glucosec4h3.40c2hdd3.54c3hdd3.71c6h3.83c5h3.85c1hd5.2312-glucosec2hdd3.24c4h3.41c5hdd3.46c3ht3.49c6ht3.76c6hdd3.90c1hd4.6413histidinec2h , rings7.05c4h , rings7.7514hydroxyproline-chm2.13-chm2.36-chdt3.35-chdd3.42-chdd4.26-cht4.6715isoleucine-ch3 t0.94-ch3 d1.01-ch2 m1.271.47-chm1.98-chd3.6716lactatech3 d1.33chq4.1117leucine-ch3 d0.96-ch3 d0.97-chm1.70-ch2 m1.72-chm3.7418lysine-ch2 m1.41-ch2 m1.67-ch2 m1.70-ch2 t3.02-cht3.7719 myo - inositolc5ht3.27c1h , c3hdd3.53c4h , c6ht3.62c2ht4.0620phenylalanine-chdd3.21-chdd3.97c2h , c6h , ringm7.33c4h , ringm7.38c3h , c5h , ringm7.43212-propanolch3 d1.17chsp4.0222 scyllo - inositolchs3.3523taurines - ch2 t3.29n - ch2 t3.4324tyrosine-chdd3.20-chdd3.05-chdd3.94c3h , c5h , ringd6.91c2h , c6h , ringd7.1925uracil5-ch , ringd5.806-ch , ringd7.5326valine-ch3 d1.00-ch3 d1.04-chm2.27-chd3.61 notation : s singlet , d dublet , dd dublet of dublets , t triplet , dt dublet of triplets , sp septet , q quartet , m multiplet representative ex vivo h hr mas nmr spectra of a healthy disc ( a ) , and of a degenerated disc for the aliphatic ( b ) and aromatic ( c ) regions of the spectrum metabolites assigned in hr mas spectra of disc tissues , their diagnostic h signals , chemical shifts ( h ) and multiplicities notation : s singlet , d dublet , dd dublet of dublets , t triplet , dt dublet of triplets , sp septet , q quartet , m multiplet the presented study revealed that in degenerated disc samples , the concentrations of 2-propanol and lactate in the nucleus pulposus and the annulus fibrosus increase significantly with age , whereas the concentrations of other metabolites do not change significantly with age . the concentrations of these compounds are elevated in degenerated discs , particularly in patients above the age of 45 . linear curve fitting ( 045 and 4590 ) was performed to reveal the age effect on concentrations of 2-propanol and lactate . for annulus fibrosous the slope of the curve increased from 0.095 to 0.31 for 2-propanol , and from 0.093 to 0.18 for lactate . for the nucleus pulposus analogue correlation showed an increase of the curve slope from 0.019 to 0.27 for 2-propanol , and from 0.089 to 0.18 for lactate . 2relationship between patient age and the concentrations of 2-propanol , lactate , and alanine in the annulus fibrosus of degenerated discsfig . 3relationship between age and the concentrations of 2-propanol , lactate , and alanine in the nucleus pulposus of degenerated discs relationship between patient age and the concentrations of 2-propanol , lactate , and alanine in the annulus fibrosus of degenerated discs relationship between age and the concentrations of 2-propanol , lactate , and alanine in the nucleus pulposus of degenerated discs metabolic changes in degenerative disc disease were also observed based on analysis of the main components of intervertebral discs : proteoglycans and glycosaminoglycans . the degradation of proteoglycans was observed in h hr mas spectra as an increase in amino acid levels , mainly glycine , hydroxyproline , isoleucine , leucine , and valine . 4 . due to the decomposition of glycosaminoglycans ( mainly chondroitin sulfate ) , the intensity of the n - acetyl peak in the proton spectra of the nucleus pulposus and the annulus fibrosus decreased with patient age.fig . other metabolites are reported as supplementary material in tables sm1sm3 and figure sm2 concentrations of metabolites in degenerated discs and the control group . other metabolites are reported as supplementary material in tables sm1sm3 and figure sm2 pca explains the variance in structure of a set of variables through linear combinations of the variables ( principal components , pcs ) . the obtained pca score plot distinguished between degenerated disc tissue samples and healthy tissues , based on selected signals of protons detected by h hr mas nmr . analysis of the scores of pc1 versus pc2 versus pc3 ( describing 96.06 % of total variation ) led to two distinct groups . one consists of degenerated disc samples , while the other one is healthy disc samples . the metabolic profile of the healthy samples is characterized by the absence of 2-propanol and the presence of scyllo - inositol and taurine at the 3.294.05 ppm level . moreover , a higher level of glycine and lower concentrations of myo - inositol , creatine , and glucose were observed in degenerated discs than in healthy tissues . a pca score plot based on the spin - echo spectra of 60 samples . 5a score plot of pc1 versus pc2 versus pc3 and a loading plot of pc1 from pca of spin - echo spectra from patients diagnosed with intervertebral disc degeneration ( circles ) and from healthy samples ( squares ) a score plot of pc1 versus pc2 versus pc3 and a loading plot of pc1 from pca of spin - echo spectra from patients diagnosed with intervertebral disc degeneration ( circles ) and from healthy samples ( squares ) pca explains the variance in structure of a set of variables through linear combinations of the variables ( principal components , pcs ) . the obtained pca score plot distinguished between degenerated disc tissue samples and healthy tissues , based on selected signals of protons detected by h hr mas nmr . analysis of the scores of pc1 versus pc2 versus pc3 ( describing 96.06 % of total variation ) led to two distinct groups . one consists of degenerated disc samples , while the other one is healthy disc samples . the metabolic profile of the healthy samples is characterized by the absence of 2-propanol and the presence of scyllo - inositol and taurine at the 3.294.05 ppm level . moreover , a higher level of glycine and lower concentrations of myo - inositol , creatine , and glucose were observed in degenerated discs than in healthy tissues . a pca score plot based on the spin - echo spectra of 60 samples . 5a score plot of pc1 versus pc2 versus pc3 and a loading plot of pc1 from pca of spin - echo spectra from patients diagnosed with intervertebral disc degeneration ( circles ) and from healthy samples ( squares ) a score plot of pc1 versus pc2 versus pc3 and a loading plot of pc1 from pca of spin - echo spectra from patients diagnosed with intervertebral disc degeneration ( circles ) and from healthy samples ( squares ) intervertebral disc cells play an integral and vital role in maintaining disc health and function . the composition and degradation of ivd tissue is controlled by the cells , because they synthesize the extracellular matrix of the discs as well as matrix metalloproteinases ( mmps ) , which are responsible for matrix breakdown . nutrient and oxygen diffusion across the ivd matrix , soluble regulators of cell function , genetic influences , ageing , and mechanical load are known to be major factors affecting disc function . nmr spectra of non - degenerated and degenerated disc tissues revealed that they had different metabolic profiles . in particular , taurine and scyllo - inositol were observed only in healthy disc tissues , whereas myo - inositol was found only in small amounts in degenerated discs . myo - inositol and scyllo - inositol are important osmolytes responsible for the regulation of long term hypoosmotic and hyperosmotic stress . changes in the concentrations of these three metabolites may indicate an imbalance in the osmolyte function of discs in degenerative diseases . myo - inositol is crucial for the optimal functioning of neurons , and alteration of its concentration leads to disturbances in the physiological properties of nerves . it also serves as a neurotransmitter in the brain , an antioxidant , and a facilitator in the transport of ions such as sodium , calcium , potassium , and magnesium . in addition , a lower concentration of creatine and elevated concentrations of glycine and hydroxyproline were observed in degenerated discs . increased concentrations of glycine and hydroxyproline in degenerated discs have been observed previously , and have been associated with collagen breakdown [ 13 ] . this amino acid acts as an atp shuttle , carrying atp to the sites where it is utilized . the observed elevated 2-propanol level may originate from a deficiency of disc nutrition . during anaerobic glycolysis , disc cells obtain their energy in the form of adenosine triphosphate ( atp ) , which is produced during quantitative conversion of glucose to lactic acid . an accumulation of lactic acid in the tissues causes acidification ( ph < 6.4 ) , which may lead to cell death . furthermore , low glucose concentrations ( below 0.5 mm ) persisting for more than approximately 3 days may also result in cell death [ 41 , 42 ] . under slightly less acidic conditions ( ph < 6.8 ) , the rate of matrix production decreases while the rate of matrix degradation remains unchanged , causing an imbalance favoring matrix breakdown . the lower ph resulting from increased lactic acid levels enhances the enzymatic activity of metalloproteinases , which in turn causes the degradation of collagen into its amino acid constituents . the intervertebral disc is the largest avascular tissue in the body , and thus nutritional supply to disc tissue may occur either by diffusion of small molecules such as glucose or by convection of larger molecules [ 4547 ] . dehydration and the related disc degeneration cause changes in disc structure and may constrain the transport of nutrients . a lack of sufficient cellular nutrition may lead to new ways of glucose delivery , for instance , by fatty acid metabolism . the main product of this metabolism is acetyl - coa , which is precipitated under certain conditions such as starvation , chronic alcoholism , or high fat intake . acetoacetate is formed by coupling two molecules of acetyl - coa , which then undergo spontaneous decarboxylation to acetone . acetone in the presence of elevated nadh / nad ratios in reactions catalyzed by alcohol dehydrogenase is reduced to 2-propanol . the presence of 2-propanol has been reported previously in diabetic ketoacidosis , hypothermia , starvation , dehydration , chronic ethanol use , breast cancer , and in exhaled breath from smokers . pca of ex vivo h hr mas nmr data distinguished between two groups : healthy and degenerated disc tissues . our results indicate that the metabolic profile of degenerated discs is characterized by the presence of 2-propanol and the absence of scyllo - inositol and taurine . moreover , a decrease in creatine and myo - inositol concentrations was observed in degenerated discs . supplementary material 1 ( docx 29 kb ) supplementary material 2 ( tiff 1384 kb ) supplementary material 3 ( tiff 91 kb ) supplementary material 4 ( docx 154 kb ) supplementary material 5 ( docx 15 kb ) supplementary material 6 ( docx 17 kb ) supplementary material 7 ( docx 15 kb ) this project has been approved by the ethico - legal committee of the medical university of lodz , approval no . all patients were informed about the surgical procedure and their informed consent was obtained prior to surgery .

objectthe objective of this study is the identification of metabolites by means of 1h high resolution magic angle spinning nuclear magnetic resonance ( 1h hr mas nmr ) spectroscopy and the evaluation of their applicability in distinguishing between healthy and degenerated disc tissues . materials and methodsdifferences between the metabolic profiles of healthy and degenerated disc tissues were studied by means of 1h hr mas nmr . analysis was performed for 81 disc tissue samples ( control samples n = 21 , degenerated disc tissue samples n = 60 ) . twenty six metabolites ( amino acids , carbohydrates , and alcohols ) were identified and quantified.resultsthe results indicate that the metabolic profile of degenerated discs is characterized by the presence of 2-propanol and the absence of scyllo - inositol and taurine . the concentrations of 2-propanol and lactate increase with age.conclusionpca analysis of ex vivo 1h hr mas nmr data revealed the occurrence of two groups : healthy and degenerative disc tissues . the effects of insufficient nutrient supply of discs , leading to their degeneration and back pain , are discussed.electronic supplementary materialthe online version of this article ( doi:10.1007/s10334 - 014 - 0457 - 0 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Materials and methods Disc tissue samples HR MAS experiments Quantification of metabolites in disc samples Statistical analysis Results Principal component analysis Discussion Conclusion Electronic supplementary material Conflict of interest Ethical standard

the online version of this article ( doi:10.1007/s10334 - 014 - 0457 - 0 ) contains supplementary material , which is available to authorized users . however , it is known that ivdd causes biochemical and morphological changes in the structure of the disc , leading to the deterioration of the biomechanical function of the joint and low back pain [ 14 ] . radiography , discography , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) are used to diagnose disc degeneration [ 813 ] . high resolution magic angle spinning ( hr mas ) h nmr spectroscopy is a nondestructive technique that has been applied to characterize the composition of various intact human cancer tissues , such as breast [ 18 , 19 ] , brain [ 20 , 21 ] , prostate [ 2224 ] , lung , and colon cancers . [ 13 ] observed changes in the chemical composition of discs , especially increased levels of unbound hydroxyproline and glycine , associated with collagen breakdown . they noticed a decrease in the concentration of proteoglycans correlated with degeneration and suggested that lactate , collagen , and proteoglycan may serve as metabolic markers in discogenic back pain . a synergic combination of nmr spectroscopy and chemometric techniques offers a useful tool for the identification of markers of degenerative disc disease . the purpose of this study is the determination of disc metabolites in order to evaluate their applicability in the diagnosis of intervertebral disc degeneration by means of h hr mas nmr . we hope that in the near future hr mas nmr ex vivo studies of metabolic profiles combined with in vivo studies using mri scanners ( mrs ) may become part of a new diagnostic protocol . analysis was performed for 81 disc tissue samples ( control samples n = 21 , degenerated disc tissue samples tissue samples were harvested from 60 patients who underwent intervertebral disc surgery at the wam university hospital , central veteran hospital of the medical university of d , poland . the reference samples originated from non - degenerated discs after mechanical trauma ( n = 20 ) and from a post - mortem section ( n = 1 ) . for each patient , annulus fibrosus and nucleus pulposus samples were analyzed by means of h hr mas nmr . analysis was performed on the following lumbar ( l ) and cervical ( c ) intervertebral discs : l1/l2 ( n = 2 ) , l2/l3 ( n = 2 ) , l3/l4 ( n = 3 ) , l4/l5 ( n = 20 ) , l5/s1 ( n = 30 ) , c2/c3 ( n = 2 ) , c5/c6 ( n = 12 ) , and c6/c7 ( n = 10 ) . all tissue samples were placed on ice after surgery for 15 min and then stored at 80 c until hr mas analysis within 1 week . the instrument was equipped with a 4 mm h / c hr mas probe with the gradient aligned along the magic angle axis . quantitative analysis was performed for the spectral regions from 0.8 to 4.65 , from 5.0 to 8.2 , and the region corresponding to tsp ( from 0.1 to 0.1 ppm ) . molar metabolite concentrations were calculated from the equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{\text{met } } ] = \frac{{a_{\text{met } } } } { { a_{\text{tsp } } } } \times \frac{{h_{\text{tsp } } } } { { h_{\text{met } } } } \times \frac{{n_{\text{tsp } } } } { { m_{\text{sample } } } } , $ $ \end{document}[met]=ametatsphtsphmetntspmsample , where amet and atsp are areas of metabolite and tsp signals , respectively ; hmet and htsp are the numbers of protons per metabolite and tsp signals ; ntsp is the number of moles the tsp signal represents , and msample is the weight of the sample in the mas rotor . linear combinations of metabolites that explain most of the overall variance in the data set were calculated by means of pca . baseline offset was corrected , and the selected spectral region was mean - normalized to arrive at the total area for each sample . analysis was performed for 81 disc tissue samples ( control samples n = 21 , degenerated disc tissue samples tissue samples were harvested from 60 patients who underwent intervertebral disc surgery at the wam university hospital , central veteran hospital of the medical university of d , poland . the reference samples originated from non - degenerated discs after mechanical trauma ( n = 20 ) and from a post - mortem section ( n = 1 ) . for each patient , annulus fibrosus and nucleus pulposus samples were analyzed by means of h hr mas nmr . analysis was performed on the following lumbar ( l ) and cervical ( c ) intervertebral discs : l1/l2 ( n = 2 ) , l2/l3 ( n = 2 ) , l3/l4 ( n = 3 ) , l4/l5 ( n = 20 ) , l5/s1 ( n = 30 ) , c2/c3 ( n = 2 ) , c5/c6 ( n = 12 ) , and c6/c7 ( n = 10 ) . all tissue samples were placed on ice after surgery for 15 min and then stored at 80 c until hr mas analysis within 1 week . the instrument was equipped with a 4 mm h / c hr mas probe with the gradient aligned along the magic angle axis . quantitative analysis was performed for the spectral regions from 0.8 to 4.65 , from 5.0 to 8.2 , and the region corresponding to tsp ( from 0.1 to 0.1 ppm ) . molar metabolite concentrations were calculated from the equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{\text{met } } ] = \frac{{a_{\text{met } } } } { { a_{\text{tsp } } } } \times \frac{{h_{\text{tsp } } } } { { h_{\text{met } } } } \times \frac{{n_{\text{tsp } } } } { { m_{\text{sample } } } } , $ $ \end{document}[met]=ametatsphtsphmetntspmsample , where amet and atsp are areas of metabolite and tsp signals , respectively ; hmet and htsp are the numbers of protons per metabolite and tsp signals ; ntsp is the number of moles the tsp signal represents , and msample is the weight of the sample in the mas rotor . linear combinations of metabolites that explain most of the overall variance in the data set were calculated by means of pca . baseline offset was corrected , and the selected spectral region was mean - normalized to arrive at the total area for each sample . hr mas nmr spectroscopy makes it possible to determine metabolite concentrations in intact tissues . sample preparation for hr mas is simpler than analysis of extracts , which requires sample homogenization and standardization for each metabolite . representative ex vivo h hr masnmr spectra of healthy and degenerated disc tissues are shown in fig . assignment of metabolite resonances was based on analysis of one dimensional h , two dimensional correlation spectroscopy ( cosy ) , and total correlation spectroscopy ( tocsy ) nmr spectra . theh hr mas nmr spectra for nucleus pulposus and annulus fibrosus specimens were very similar ( see figure sm1 in supplementary material ) . in the hr mas spectra of disc tissues , signals of five groups of compounds were observed . 1 and the pool of metabolites the largest group consisted of 12 amino acids , which were derived from protein cores and were also formed by collagen breakdown . uracil ) were found in the spectra of disc tissues . alcohols , such as myo - inositol , scyllo - inositol , and 2-propanol , were also identified . in the case of 2-propanol , the possibility of its exogenous origin was checked at every step from surgery to hr mas measurements , but it was deemed possible for 2-propanol to have been introduced into disc tissue samples as a result of its antiseptic use . 1representative ex vivo h hr mas nmr spectra of a healthy disc ( a ) , and of a degenerated disc for the aliphatic ( b ) and aromatic ( c ) regions of the spectrumtable 1metabolites assigned in hr mas spectra of disc tissues , their diagnostic h signals , chemical shifts ( h ) and multiplicitiesassignment numbermetaboliteassignment h multiplicity h , ppm1acetatech3 s1.932acetonech3 s2.233alaninech3 d1.48chq3.784aspartate-ch2 dd , dd2.67 , 2.81-chdd3.915chondroitin sulfate n - acetyls2.056citric acidch2 d2.54ch2 d2.667creatinech3 s3.03ch2 s3.938fatty acidsch3 t0.90(ch2)n m1.29ch2ch2coq1.559formates8.4510glycine-ch2 s3.5511-glucosec4h3.40c2hdd3.54c3hdd3.71c6h3.83c5h3.85c1hd5.2312-glucosec2hdd3.24c4h3.41c5hdd3.46c3ht3.49c6ht3.76c6hdd3.90c1hd4.6413histidinec2h , rings7.05c4h , rings7.7514hydroxyproline-chm2.13-chm2.36-chdt3.35-chdd3.42-chdd4.26-cht4.6715isoleucine-ch3 t0.94-ch3 d1.01-ch2 m1.271.47-chm1.98-chd3.6716lactatech3 d1.33chq4.1117leucine-ch3 d0.96-ch3 d0.97-chm1.70-ch2 m1.72-chm3.7418lysine-ch2 m1.41-ch2 m1.67-ch2 m1.70-ch2 t3.02-cht3.7719 myo - inositolc5ht3.27c1h , c3hdd3.53c4h , c6ht3.62c2ht4.0620phenylalanine-chdd3.21-chdd3.97c2h , c6h , ringm7.33c4h , ringm7.38c3h , c5h , ringm7.43212-propanolch3 d1.17chsp4.0222 scyllo - inositolchs3.3523taurines - ch2 t3.29n - ch2 t3.4324tyrosine-chdd3.20-chdd3.05-chdd3.94c3h , c5h , ringd6.91c2h , c6h , ringd7.1925uracil5-ch , ringd5.806-ch , ringd7.5326valine-ch3 d1.00-ch3 d1.04-chm2.27-chd3.61 notation : s singlet , d dublet , dd dublet of dublets , t triplet , dt dublet of triplets , sp septet , q quartet , m multiplet representative ex vivo h hr mas nmr spectra of a healthy disc ( a ) , and of a degenerated disc for the aliphatic ( b ) and aromatic ( c ) regions of the spectrum metabolites assigned in hr mas spectra of disc tissues , their diagnostic h signals , chemical shifts ( h ) and multiplicities notation : s singlet , d dublet , dd dublet of dublets , t triplet , dt dublet of triplets , sp septet , q quartet , m multiplet the presented study revealed that in degenerated disc samples , the concentrations of 2-propanol and lactate in the nucleus pulposus and the annulus fibrosus increase significantly with age , whereas the concentrations of other metabolites do not change significantly with age . the concentrations of these compounds are elevated in degenerated discs , particularly in patients above the age of 45 . linear curve fitting ( 045 and 4590 ) was performed to reveal the age effect on concentrations of 2-propanol and lactate . 2relationship between patient age and the concentrations of 2-propanol , lactate , and alanine in the annulus fibrosus of degenerated discsfig . 3relationship between age and the concentrations of 2-propanol , lactate , and alanine in the nucleus pulposus of degenerated discs relationship between patient age and the concentrations of 2-propanol , lactate , and alanine in the annulus fibrosus of degenerated discs relationship between age and the concentrations of 2-propanol , lactate , and alanine in the nucleus pulposus of degenerated discs metabolic changes in degenerative disc disease were also observed based on analysis of the main components of intervertebral discs : proteoglycans and glycosaminoglycans . the degradation of proteoglycans was observed in h hr mas spectra as an increase in amino acid levels , mainly glycine , hydroxyproline , isoleucine , leucine , and valine . other metabolites are reported as supplementary material in tables sm1sm3 and figure sm2 concentrations of metabolites in degenerated discs and the control group . the obtained pca score plot distinguished between degenerated disc tissue samples and healthy tissues , based on selected signals of protons detected by h hr mas nmr . one consists of degenerated disc samples , while the other one is healthy disc samples . the metabolic profile of the healthy samples is characterized by the absence of 2-propanol and the presence of scyllo - inositol and taurine at the 3.294.05 ppm level . moreover , a higher level of glycine and lower concentrations of myo - inositol , creatine , and glucose were observed in degenerated discs than in healthy tissues . the obtained pca score plot distinguished between degenerated disc tissue samples and healthy tissues , based on selected signals of protons detected by h hr mas nmr . one consists of degenerated disc samples , while the other one is healthy disc samples . the metabolic profile of the healthy samples is characterized by the absence of 2-propanol and the presence of scyllo - inositol and taurine at the 3.294.05 ppm level . moreover , a higher level of glycine and lower concentrations of myo - inositol , creatine , and glucose were observed in degenerated discs than in healthy tissues . the composition and degradation of ivd tissue is controlled by the cells , because they synthesize the extracellular matrix of the discs as well as matrix metalloproteinases ( mmps ) , which are responsible for matrix breakdown . nmr spectra of non - degenerated and degenerated disc tissues revealed that they had different metabolic profiles . in particular , taurine and scyllo - inositol were observed only in healthy disc tissues , whereas myo - inositol was found only in small amounts in degenerated discs . myo - inositol and scyllo - inositol are important osmolytes responsible for the regulation of long term hypoosmotic and hyperosmotic stress . changes in the concentrations of these three metabolites may indicate an imbalance in the osmolyte function of discs in degenerative diseases . myo - inositol is crucial for the optimal functioning of neurons , and alteration of its concentration leads to disturbances in the physiological properties of nerves . in addition , a lower concentration of creatine and elevated concentrations of glycine and hydroxyproline were observed in degenerated discs . increased concentrations of glycine and hydroxyproline in degenerated discs have been observed previously , and have been associated with collagen breakdown [ 13 ] . during anaerobic glycolysis , disc cells obtain their energy in the form of adenosine triphosphate ( atp ) , which is produced during quantitative conversion of glucose to lactic acid . the intervertebral disc is the largest avascular tissue in the body , and thus nutritional supply to disc tissue may occur either by diffusion of small molecules such as glucose or by convection of larger molecules [ 4547 ] . the main product of this metabolism is acetyl - coa , which is precipitated under certain conditions such as starvation , chronic alcoholism , or high fat intake . acetone in the presence of elevated nadh / nad ratios in reactions catalyzed by alcohol dehydrogenase is reduced to 2-propanol . the presence of 2-propanol has been reported previously in diabetic ketoacidosis , hypothermia , starvation , dehydration , chronic ethanol use , breast cancer , and in exhaled breath from smokers . pca of ex vivo h hr mas nmr data distinguished between two groups : healthy and degenerated disc tissues . our results indicate that the metabolic profile of degenerated discs is characterized by the presence of 2-propanol and the absence of scyllo - inositol and taurine . moreover , a decrease in creatine and myo - inositol concentrations was observed in degenerated discs . supplementary material 1 ( docx 29 kb ) supplementary material 2 ( tiff 1384 kb ) supplementary material 3 ( tiff 91 kb ) supplementary material 4 ( docx 154 kb ) supplementary material 5 ( docx 15 kb ) supplementary material 6 ( docx 17 kb ) supplementary material 7 ( docx 15 kb ) this project has been approved by the ethico - legal committee of the medical university of lodz , approval no .

periodontal disease is one of the most common chronic bacterial infections of the tooth supporting structures , which is predominantly associated with gram negative bacteria that exist in the subgingival biofilm . atherosclerosis , on the other hand , is a highly prevalent disease in humans with significant morbidity and mortality . previous data suggests that subjects with the chronic periodontitis are at a significantly increased risk for developing coronary artery disease ( cad ) , some of the studies could not substantiate the correlation in this regard . apart from the known risk factors , such as smoking , diabetes , high blood pressure and socio - economic factors , the chronic periodontal inflammation is said to be an important contributing factor for cardiovascular diseases . the important risk markers for cardiovascular disease , such as plasma fibrinogen level , c - reactive protein , total white blood corpuscles count have been reported to be elevated in patients with chronic periodontitis . animal and human studies have found an association between the periodontal inflammation and the incidence of coronary events . though the studies performed in the past have shown the presence of periodontal bacteria in coronary plaque , but most of these have shown conflicting results . moreover , very few studies have shown its association with the amount of periodontal destruction . hence to explore further , the present study was undertaken to evaluate the presence of the 16s rrna gene of red complex bacteria ( tf , pg , td ) , pg ( fima ) and aa in the subgingival and the atherosclerotic plaques of periodontitis patients with cad undergoing coronary artery bypass grafting ( cabg ) . the periodontal status also compared with the presence of pathogenic bacteria in coronary plaque . one hundred and fifty patients with cad , scheduled to undergo cabg in the institute of cardiovascular diseases , madras medical mission , chennai , india were recruited consecutively for the study , out of which fifty one patients ( 40 males and 11 females ) in the age group of 40 - 80 years ( age : 61 8.75 years ) with chronic generalized periodontitis were selected based on following specific criteria . they were diagnosed with chronic generalized periodontitis with atleast 30% of sites with clinical attachment loss ( cal ) and alveolar bone exceeding 1/3 of the root in atleast 30% of the entire dentition . exclusion criteria included smokers , hypertensive patients , those with respiratory diseases , diabetes or any other major systemic diseases that could affect the periodontium . subjects with antibiotic intake or a history of periodontal treatment carried out in the previous 6 months were also excluded from the study . this was done to rule out the confounding factors in atherogenesis other than periodontal diseases . the periodontal examination of all the subjects included plaque index , gingival index , clinical attachment level and pocket depth index and oral hygiene index . the two deepest periodontal sites with periodontal depth 5 mm were selected for microbial sampling . after removal of the supragingival plaque , the subgingival plaque samples were obtained with the help of a curette and were pooled for analysis . the surgeon excised one or two small bits of atherosclerotic plaque ( 0.5 - 1 mm ) from the edge of the coronary arteriotomy performed for anatomizing the graft . to eliminate the blood contamination , the plaque samples were placed in sterilized phosphate - buffered saline and mixed gently and was transferred to fresh vials containing the transporting media . the samples were then homogenized by the tissue homogenizer as described by saiki et al . both the subgingival plaque and the coronary atherosclerotic plaque samples were centrifuged for 10 min at 10,000 rpm ( remi centrifuge , mumbai , india ) . the supernatant was discarded and the resulting pellet was resuspended in 200 l of lysis solution ( 100 mm tris , 1.0 mm ethylenediamine tetra - acetic acid , 1.0% triton x-100 , ph 7.8 ) . the samples were kept in a boiling water bath for 10 min , allowed to cool and again centrifuged for 5 min at 10,000 rpm . pcr primers used in the study were designed as per the protocol of larsen et al . the upstream and downstream sequence primers were then verified for their species specificity by comparing the sequences with all the available 16s rrna sequences in the rdp database . briefly , 10 l of dna template of the sample was added to 40 l of working stock reaction mixture containing 5 l of 10 pcr buffer , 1.25 units of taq dna polymerase ( 0.4 l ) , 0.2 mm ( 1 l ) of each deoxyribonucleotides ( dntp 's ) , primers ( 1 l ) forward and ( 1 l ) reverse of the specific microorganisms and 31.6 l of milli q water . the pcr reaction was carried out using a pcr thermocycler ( applied biosystems , ca , usa ) . the temperature profile of the bacteria tf , pg and td was 95c ( 2 min ) , 36 cycles of 95c ( 30 s ) , 60c ( 1 min ) , 72c ( 1 min ) and 72c ( 2 min ) . the temperature profile for aa was 95 c ( 2 min ) , 36 cycles of 94c ( 30 s ) , 30 cycles , annealing at 55c ( 1 min ) , 72c ( 2 min ) and final extension at 72c ( 10 min ) . pg ( fima gene ) had a temperature profile of 94c ( 5 min ) , 35 cycles of denaturation 94c ( 1 min ) , 50c ( 1 min ) , 72c ( 1.5 min ) and final 72c ( 7 min ) . after amplification , 10 l aliquot of the amplified pcr product was subjected to electrophoresis is in a 0.75% agarose gel containing 0.5 g / ml ethidium bromide in 1x tae buffer . the gel was photographed under a 300-nm ultraviolet light trans - illuminator . a 100 bp dna ladder ( bangalore genei pvt . limited ) served as a molecular weight marker ( bio - rad laboratories , ca , usa ) . the gel images in the figures represented the presence of bacterial dna of tf , pg , pg ( fima ) gene and td in the subgingival plaque and atherosclerotic plaque of the same patient [ figure 1 ] . 16s ribosomal ribonucleic acid - based polymerase chain reaction detection of tannerella forsythia ( a ) , porphyromonas gingivali ( b ) , p. gingivalis ( fima ) gene ( 1c ) and treponema denticola ( d ) . ( a ) lane 1 deoxyribonucleic acid ( dna ) ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( b ) lane 1 dna ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( c ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control . ( d ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control the pcr - amplified products were sequenced in an automated sequencer ( genetic analyzer ; applied biosystems , ca , u.s.a . ) . the sequencer data was blasted with available data in genbank and compared for possible homologies . the association between the periodontal parameters and presence of pg ( fima ) , aa and the red complex ( td , tf and pg ) in the atherosclerotic plaque samples , was analyzed by the student 's independent t - test . kappa analysis was applied to access the association between the presence of the bacterial species in the subgingival biofilm and atherosclerotic plaques . one hundred and fifty patients with cad , scheduled to undergo cabg in the institute of cardiovascular diseases , madras medical mission , chennai , india were recruited consecutively for the study , out of which fifty one patients ( 40 males and 11 females ) in the age group of 40 - 80 years ( age : 61 8.75 years ) with chronic generalized periodontitis were selected based on following specific criteria . they were diagnosed with chronic generalized periodontitis with atleast 30% of sites with clinical attachment loss ( cal ) and alveolar bone exceeding 1/3 of the root in atleast 30% of the entire dentition . exclusion criteria included smokers , hypertensive patients , those with respiratory diseases , diabetes or any other major systemic diseases that could affect the periodontium . subjects with antibiotic intake or a history of periodontal treatment carried out in the previous 6 months were also excluded from the study . this was done to rule out the confounding factors in atherogenesis other than periodontal diseases . the periodontal examination of all the subjects included plaque index , gingival index , clinical attachment level and pocket depth index and oral hygiene index . the two deepest periodontal sites with periodontal depth 5 mm were selected for microbial sampling . after removal of the supragingival plaque , the subgingival plaque samples were obtained with the help of a curette and were pooled for analysis . the surgeon excised one or two small bits of atherosclerotic plaque ( 0.5 - 1 mm ) from the edge of the coronary arteriotomy performed for anatomizing the graft . to eliminate the blood contamination , the plaque samples were placed in sterilized phosphate - buffered saline and mixed gently and was transferred to fresh vials containing the transporting media . the samples were then homogenized by the tissue homogenizer as described by saiki et al . both the subgingival plaque and the coronary atherosclerotic plaque samples were centrifuged for 10 min at 10,000 rpm ( remi centrifuge , mumbai , india ) . the supernatant was discarded and the resulting pellet was resuspended in 200 l of lysis solution ( 100 mm tris , 1.0 mm ethylenediamine tetra - acetic acid , 1.0% triton x-100 , ph 7.8 ) . the samples were kept in a boiling water bath for 10 min , allowed to cool and again centrifuged for 5 min at 10,000 rpm . pcr primers used in the study were designed as per the protocol of larsen et al . the upstream and downstream sequence primers were then verified for their species specificity by comparing the sequences with all the available 16s rrna sequences in the rdp database . briefly , 10 l of dna template of the sample was added to 40 l of working stock reaction mixture containing 5 l of 10 pcr buffer , 1.25 units of taq dna polymerase ( 0.4 l ) , 0.2 mm ( 1 l ) of each deoxyribonucleotides ( dntp 's ) , primers ( 1 l ) forward and ( 1 l ) reverse of the specific microorganisms and 31.6 l of milli q water . the pcr reaction was carried out using a pcr thermocycler ( applied biosystems , ca , usa ) . the temperature profile of the bacteria tf , pg and td was 95c ( 2 min ) , 36 cycles of 95c ( 30 s ) , 60c ( 1 min ) , 72c ( 1 min ) and 72c ( 2 min ) . the temperature profile for aa was 95 c ( 2 min ) , 36 cycles of 94c ( 30 s ) , 30 cycles , annealing at 55c ( 1 min ) , 72c ( 2 min ) and final extension at 72c ( 10 min ) . pg ( fima gene ) had a temperature profile of 94c ( 5 min ) , 35 cycles of denaturation 94c ( 1 min ) , 50c ( 1 min ) , 72c ( 1.5 min ) and final 72c ( 7 min ) . after amplification , 10 l aliquot of the amplified pcr product was subjected to electrophoresis is in a 0.75% agarose gel containing 0.5 g / ml ethidium bromide in 1x tae buffer . the gel was photographed under a 300-nm ultraviolet light trans - illuminator . a 100 bp dna ladder ( bangalore genei pvt . limited ) served as a molecular weight marker ( bio - rad laboratories , ca , usa ) . the gel images in the figures represented the presence of bacterial dna of tf , pg , pg ( fima ) gene and td in the subgingival plaque and atherosclerotic plaque of the same patient [ figure 1 ] . 16s ribosomal ribonucleic acid - based polymerase chain reaction detection of tannerella forsythia ( a ) , porphyromonas gingivali ( b ) , p. gingivalis ( fima ) gene ( 1c ) and treponema denticola ( d ) . ( a ) lane 1 deoxyribonucleic acid ( dna ) ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( b ) lane 1 dna ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( c ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control . ( d ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control the pcr - amplified products were sequenced in an automated sequencer ( genetic analyzer ; applied biosystems , ca , u.s.a . ) . the sequencer data was blasted with available data in genbank and compared for possible homologies . the association between the periodontal parameters and presence of pg ( fima ) , aa and the red complex ( td , tf and pg ) in the atherosclerotic plaque samples , was analyzed by the student 's independent t - test . kappa analysis was applied to access the association between the presence of the bacterial species in the subgingival biofilm and atherosclerotic plaques . the study showed the presence of bacteria , in all the subgingival and atherosclerotic plaque samples . all the samples were positive for bacterial dna ( 100% ) . in the pcr analysis , aa sequence of the 16srrna gene was detected only in one subgingival plaque sample and could not be detected in any of the atherosclerotic plaque samples . pg ( fima ) , td , tf and pg were detected in 62.7% , 66.7% , 43.1% and 64.71% of subgingival plaque samples and in 39.2% , 51% , 31.4% and 45.10% of atherosclerotic plaque samples , respectively . in both subgingival plaque and atherosclerotic plaque samples , pg ( fima ) was detected in 33.3% , td was detected in 35.3% , tf in 19.6% and pg in 39.22% [ figure 2 , bar diagram ] . we found a significant association of pg ( p - 0.003 ) and pg ( fima ) ( p - 0.008 ) in subgingival plaque and coronary plaque samples [ table 2 ] . the incidence of periodontal pathogens in subgingival plaque , atherosclerotic plaque and both sites kappa analysis for the presence of the microorganisms in subgingival and atherosclerotic plaque samples the present study further showed a statistically significant relationship between the presence of tf in the atherosclerotic plaque with gingival index ( p - 0.03 ) and probing pocket depth ( p - 0.01 ) [ table 3 ] . however , all the periodontal parameters analyzed were non - significant [ table 3 ] with the presence of td , pg and pg ( fima ) , in the atherosclerotic plaque samples . periodontal diseases are one of the most common bacterial infections characterized by inflammation and the subsequent destruction of the tooth supporting tissues . the disease is associated with a widely diverse and complex subgingival microorganisms encompassing gram - positive and gram - negative bacteria , facultative and anaerobic organisms . the past evidence states that dental plaque is the primarily etiological factor for the progression of periodontal diseases . several microorganisms including chlamydia pneumoniae and cytomegalovirus have been implicated in the infectious etiology of atherosclerosis . likewise , the chronic periodontal infections have also been reported to increase the risk for cad . it has been postulated that chronic infections can contribute to the development of atheromas by direct ( platelet aggregation , invasion of endothelial cells and endothelial injury ) and indirect ( induction of intracellular adhesion molecules , production of antibodies to lipopolysaccharide as well as cytokines and dysfunction of the immune system ) mechanisms . in recent years , many researchers have focused their attention on the ability of periodontal pathogens to colonize in the atheromatous plaque in order to substantiate the potential role of the periodontal bacteria in the progression of atherosclerosis . nevertheless , a clear correlation between the detection rates of periodontopathic bacterial dna in atheromas and periodontal pockets has not been established . in view of the above , the present study was undertaken to assess the presence of the bacterial dna of putative periodontal pathogens in the subgingival and coronary plaque of patients with chronic periodontitis and to compare the periodontal status with the presence of periodontal bacteria in the coronary plaque . the study revealed the presence of the red complex bacteria ( td , tf and pg ) in 51% , 31.4% and 45.10% of atherosclerotic plaque samples , respectively . pg was detected in 24.4% of subgingival plaque and 45.10% of atherosclerotic plaque , showing a significant correlation of the microorganism between the two plaque samples thus indicating its potential role toward the progression of coronary events . zhong et al . , gotsman et al . and zhang et al . also identified pg in 7.4% , 52% , 38.7% , 86% , 33% from atherosclerotic plaque samples , respectively . haraszthy et al . examined 50 carotid endarterectomy samples using pcr and found the presence of pg in 26% of the samples . we detected pg ( fima ) in 33.3% of both the subgingival and coronary plaque samples . the presence of pg ( fima ) was found to be significantly associated among both the plaque samples [ table 2 ] . our finding supports the animal study conducted by chou et al . , who demonstrated that pg fimbria - mediated invasion upregulates inflammatory gene expression in ( human aortic endothelial cells ) and in aortic tissues . pg is one of the major causative factor in periodontal disease and its fimbriae ( fima ) , a filamentous component located on the cell surface , plays a vital role in the colonization and invasion of the periodontal pathogen in the aortic tissues . we also detected td in 51% of the atherosclerotic plaque samples ; however , it remained non - significant [ table 2 ] . nakano et al . and gotsman et al . detected t. denticola in 44.4% and 85% of the atherosclerotic plaque samples respectively . in addition to the motility of td , it is possible that protease activity may also play a role in their penetration into the periodontal tissues by infiltrating between the gingival cells and the blood stream . the results of our study were in contrast to the findings of padilla et al . who could not detect td in the coronary plaque samples . in the present study showed the detection of tf in 43.1% of the subgingival plaques and 31.4% of atherosclerotic plaques . this was in contrast with padilla et al . and okuda et al . , who could not identify tf in the coronary plaque samples . in our study , only one patient was positive for aa in the subgingival plaque sample whereas 16s rrna gene for the microorganism could not be detected in the atherosclerotic plaque . , padilla et al . , ishihara et al . , marques da silva et al . , and cairo et al . , who detected aa in 33.3% , 58% , 29% , 7.1% and 5% of the subgingival plaque samples , respectively . however , the present study was in concurrence with the study done by zhong et al . , zhang et al . , and okuda et al . aa has been suggested to contribute to the pathogenesis of coronary heart disease and antibody levels against this pathogen in serum have been viewed as indicators of the risk for this disease . aa probably invades endothelial cells through a mechanism dependent upon the engagement of the platelet - activating factor receptor by bacterial phosphorylcholine . the bacterium possesses a number of putative virulence factors including a leucotoxin ( ltxa ) that target and destroy the host immune cells , such as monocytes and neutrophils . the reason could be that aa being more prevalent in the younger age group , was absent in our samples as the age of the selected subjects was between 40 and 80 years in this study . in the present study , the mean of the selected periodontal parameters namely gingival index , oral hygiene index , mean probing pocket depth , clinical attachment level , number of teeth present and the presence of the periodontal pathogens in the atherosclerotic plaque of the cardiac patients was analyzed [ table 3 ] . tf , showed a significant association with the gingival index suggesting that with an increase in the gingival inflammation , the above microorganism is more liable to be prevalent in the coronary plaque . tf has been reported to express a number of enzymatic and proteolytic activities that could contribute to its ability to compete effectively in the complex biofilm of the subgingival pocket . the previous studies have also shown the significant association between the periodontal parameters and the presence of the microorganisms . investigated whether poor periodontal condition was associated with an acute episode of myocardial infarction ( ami ) among chronic coronary heart disease patients . the proportion of patients with bleeding on probing and probing pocket depth 4 mm was found to be significantly associated with ami . similarly , gotsman et al . determined the relationship between periodontal disease measures and cad . patients with severe cad had significantly more periodontal destruction than those with mild cad . logistic regression analysis showed that percentage of teeth with cal more than 5 mm was significantly associated with cad severity . the correlation of td , pg and fima gene with periodontal parameters in the present study was not found to be significant . in addition , aa , could not show any significant association with the periodontal parameters . overall , the present study indicated that a good agreement existed between the percentage prevalence of the periodontal pathogens , in particular pg and pg fima in the subgingival and atherosclerotic plaque samples suggesting that the presence of these microorganisms in the subgingival plaque will have significant chances of their presence in atherosclerotic plaque as well contributing to the progression of atherosclerosis and vice - versa . it is suggested that the bacteria growing in the biofilm become resistant to the phagocytosis and host immune system . the ulcerated pocket epithelium becomes the portal of entry of the periodontal pathogens and their products to the general circulation in small and major blood vessels . on the other hand , the significant virulence factors of these bacteria are able to evade the host clearance mechanisms , participate in the tissue destruction and enter the general circulation in small and major blood vessels thus contributing to the coronary events . however , at this point of time , it is difficult to pin point whether it is the stand - alone role of individual periodontal pathogen or the total periodontal burden as a whole , which contribute toward atherosclerosis . further molecular studies are required to elucidate the role of each periodontal pathogen in a complex form for a better understanding of this association . in future , large cross sectional and longitudinal studies are required to elicit the role of aa , the microorganisms of red complex and their virulence factors in the pathogenesis of atherosclerosis .

aim : the objective of the present study was to detect the presence of specific periodontopathogenic bacteria in the coronary plaque of patients with coronary artery disease and to find out the significant association between the periodontal status and the presence of pathogenic bacteria in the coronary plaque.materials and methods : the study population consisted of 51 patients with chronic generalized periodontitis undergoing coronary artery bypass grafting . periodontal parameters were recorded and deoxyribonucleic acid was extracted from the subgingival plaque and coronary atherosclerotic plaque samples of the same patients . polymerase chain reaction was used to amplify the part of 16s ribosomal ribonucleic acid ( rrna ) gene to detect the presence of aggregatibacter actinomycetemcomitans ( aa ) , tannerella forsythia ( tf ) , porphyromonas gingivali ( pg ) , porphyromonas gingivalis ( fima ) gene and treponema denticola ( td).results : aa , tf , pg , pg ( fima ) gene and td were detected in 0% , 31.4% , 45.1% 39.2% and 51% of atherosclerotic plaque samples , respectively . tf was detected in 19.6% , pg in 39.2% , pg ( fima ) gene in 33.3% and td in 35.3% of both , subgingival plaque and atherosclerotic plaque samples . periodontal parameters correlated with the presence of bacteria in coronary plaque . aa could not be detected in coronary plaque samples.conclusions:the study confirmed the detection of red complex bacteria in coronary plaque samples and these bacteria correlated with the severity of periodontal destruction .

INTRODUCTION MATERIALS AND METHODS Patients selection Subgingival plaque collection Collection of atherosclerotic plaque Deoxyribonucleic acid (DNA) extraction Polymerase chain reaction (PCR) amplification Sequencing of the PCR products Statistical analysis RESULTS DISCUSSION CONCLUSION

periodontal disease is one of the most common chronic bacterial infections of the tooth supporting structures , which is predominantly associated with gram negative bacteria that exist in the subgingival biofilm . previous data suggests that subjects with the chronic periodontitis are at a significantly increased risk for developing coronary artery disease ( cad ) , some of the studies could not substantiate the correlation in this regard . apart from the known risk factors , such as smoking , diabetes , high blood pressure and socio - economic factors , the chronic periodontal inflammation is said to be an important contributing factor for cardiovascular diseases . the important risk markers for cardiovascular disease , such as plasma fibrinogen level , c - reactive protein , total white blood corpuscles count have been reported to be elevated in patients with chronic periodontitis . animal and human studies have found an association between the periodontal inflammation and the incidence of coronary events . though the studies performed in the past have shown the presence of periodontal bacteria in coronary plaque , but most of these have shown conflicting results . moreover , very few studies have shown its association with the amount of periodontal destruction . hence to explore further , the present study was undertaken to evaluate the presence of the 16s rrna gene of red complex bacteria ( tf , pg , td ) , pg ( fima ) and aa in the subgingival and the atherosclerotic plaques of periodontitis patients with cad undergoing coronary artery bypass grafting ( cabg ) . the periodontal status also compared with the presence of pathogenic bacteria in coronary plaque . one hundred and fifty patients with cad , scheduled to undergo cabg in the institute of cardiovascular diseases , madras medical mission , chennai , india were recruited consecutively for the study , out of which fifty one patients ( 40 males and 11 females ) in the age group of 40 - 80 years ( age : 61 8.75 years ) with chronic generalized periodontitis were selected based on following specific criteria . they were diagnosed with chronic generalized periodontitis with atleast 30% of sites with clinical attachment loss ( cal ) and alveolar bone exceeding 1/3 of the root in atleast 30% of the entire dentition . subjects with antibiotic intake or a history of periodontal treatment carried out in the previous 6 months were also excluded from the study . this was done to rule out the confounding factors in atherogenesis other than periodontal diseases . after removal of the supragingival plaque , the subgingival plaque samples were obtained with the help of a curette and were pooled for analysis . the surgeon excised one or two small bits of atherosclerotic plaque ( 0.5 - 1 mm ) from the edge of the coronary arteriotomy performed for anatomizing the graft . to eliminate the blood contamination , the plaque samples were placed in sterilized phosphate - buffered saline and mixed gently and was transferred to fresh vials containing the transporting media . both the subgingival plaque and the coronary atherosclerotic plaque samples were centrifuged for 10 min at 10,000 rpm ( remi centrifuge , mumbai , india ) . the supernatant was discarded and the resulting pellet was resuspended in 200 l of lysis solution ( 100 mm tris , 1.0 mm ethylenediamine tetra - acetic acid , 1.0% triton x-100 , ph 7.8 ) . pcr primers used in the study were designed as per the protocol of larsen et al . briefly , 10 l of dna template of the sample was added to 40 l of working stock reaction mixture containing 5 l of 10 pcr buffer , 1.25 units of taq dna polymerase ( 0.4 l ) , 0.2 mm ( 1 l ) of each deoxyribonucleotides ( dntp 's ) , primers ( 1 l ) forward and ( 1 l ) reverse of the specific microorganisms and 31.6 l of milli q water . the pcr reaction was carried out using a pcr thermocycler ( applied biosystems , ca , usa ) . the temperature profile of the bacteria tf , pg and td was 95c ( 2 min ) , 36 cycles of 95c ( 30 s ) , 60c ( 1 min ) , 72c ( 1 min ) and 72c ( 2 min ) . pg ( fima gene ) had a temperature profile of 94c ( 5 min ) , 35 cycles of denaturation 94c ( 1 min ) , 50c ( 1 min ) , 72c ( 1.5 min ) and final 72c ( 7 min ) . after amplification , 10 l aliquot of the amplified pcr product was subjected to electrophoresis is in a 0.75% agarose gel containing 0.5 g / ml ethidium bromide in 1x tae buffer . the gel images in the figures represented the presence of bacterial dna of tf , pg , pg ( fima ) gene and td in the subgingival plaque and atherosclerotic plaque of the same patient [ figure 1 ] . 16s ribosomal ribonucleic acid - based polymerase chain reaction detection of tannerella forsythia ( a ) , porphyromonas gingivali ( b ) , p. gingivalis ( fima ) gene ( 1c ) and treponema denticola ( d ) . ( a ) lane 1 deoxyribonucleic acid ( dna ) ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( b ) lane 1 dna ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( c ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control . ( d ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control the pcr - amplified products were sequenced in an automated sequencer ( genetic analyzer ; applied biosystems , ca , u.s.a . ) the association between the periodontal parameters and presence of pg ( fima ) , aa and the red complex ( td , tf and pg ) in the atherosclerotic plaque samples , was analyzed by the student 's independent t - test . kappa analysis was applied to access the association between the presence of the bacterial species in the subgingival biofilm and atherosclerotic plaques . one hundred and fifty patients with cad , scheduled to undergo cabg in the institute of cardiovascular diseases , madras medical mission , chennai , india were recruited consecutively for the study , out of which fifty one patients ( 40 males and 11 females ) in the age group of 40 - 80 years ( age : 61 8.75 years ) with chronic generalized periodontitis were selected based on following specific criteria . they were diagnosed with chronic generalized periodontitis with atleast 30% of sites with clinical attachment loss ( cal ) and alveolar bone exceeding 1/3 of the root in atleast 30% of the entire dentition . subjects with antibiotic intake or a history of periodontal treatment carried out in the previous 6 months were also excluded from the study . this was done to rule out the confounding factors in atherogenesis other than periodontal diseases . after removal of the supragingival plaque , the subgingival plaque samples were obtained with the help of a curette and were pooled for analysis . the surgeon excised one or two small bits of atherosclerotic plaque ( 0.5 - 1 mm ) from the edge of the coronary arteriotomy performed for anatomizing the graft . to eliminate the blood contamination , the plaque samples were placed in sterilized phosphate - buffered saline and mixed gently and was transferred to fresh vials containing the transporting media . both the subgingival plaque and the coronary atherosclerotic plaque samples were centrifuged for 10 min at 10,000 rpm ( remi centrifuge , mumbai , india ) . the supernatant was discarded and the resulting pellet was resuspended in 200 l of lysis solution ( 100 mm tris , 1.0 mm ethylenediamine tetra - acetic acid , 1.0% triton x-100 , ph 7.8 ) . pcr primers used in the study were designed as per the protocol of larsen et al . briefly , 10 l of dna template of the sample was added to 40 l of working stock reaction mixture containing 5 l of 10 pcr buffer , 1.25 units of taq dna polymerase ( 0.4 l ) , 0.2 mm ( 1 l ) of each deoxyribonucleotides ( dntp 's ) , primers ( 1 l ) forward and ( 1 l ) reverse of the specific microorganisms and 31.6 l of milli q water . the pcr reaction was carried out using a pcr thermocycler ( applied biosystems , ca , usa ) . the temperature profile of the bacteria tf , pg and td was 95c ( 2 min ) , 36 cycles of 95c ( 30 s ) , 60c ( 1 min ) , 72c ( 1 min ) and 72c ( 2 min ) . pg ( fima gene ) had a temperature profile of 94c ( 5 min ) , 35 cycles of denaturation 94c ( 1 min ) , 50c ( 1 min ) , 72c ( 1.5 min ) and final 72c ( 7 min ) . the gel images in the figures represented the presence of bacterial dna of tf , pg , pg ( fima ) gene and td in the subgingival plaque and atherosclerotic plaque of the same patient [ figure 1 ] . 16s ribosomal ribonucleic acid - based polymerase chain reaction detection of tannerella forsythia ( a ) , porphyromonas gingivali ( b ) , p. gingivalis ( fima ) gene ( 1c ) and treponema denticola ( d ) . ( a ) lane 1 deoxyribonucleic acid ( dna ) ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( b ) lane 1 dna ladder 100 bp , lane 2 and 4 subgingival plaque samples , lane 3 and 5 coronary artery plaque samples , lane 6 negative control . ( c ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control . ( d ) lane 1 dna ladder 100 bp , lane 2 , 4 and 6 subgingival plaque samples , lane 3 , 5 and 7 coronary artery plaque samples , lane 8 negative control the pcr - amplified products were sequenced in an automated sequencer ( genetic analyzer ; applied biosystems , ca , u.s.a . ) the association between the periodontal parameters and presence of pg ( fima ) , aa and the red complex ( td , tf and pg ) in the atherosclerotic plaque samples , was analyzed by the student 's independent t - test . kappa analysis was applied to access the association between the presence of the bacterial species in the subgingival biofilm and atherosclerotic plaques . the study showed the presence of bacteria , in all the subgingival and atherosclerotic plaque samples . in the pcr analysis , aa sequence of the 16srrna gene was detected only in one subgingival plaque sample and could not be detected in any of the atherosclerotic plaque samples . pg ( fima ) , td , tf and pg were detected in 62.7% , 66.7% , 43.1% and 64.71% of subgingival plaque samples and in 39.2% , 51% , 31.4% and 45.10% of atherosclerotic plaque samples , respectively . in both subgingival plaque and atherosclerotic plaque samples , pg ( fima ) was detected in 33.3% , td was detected in 35.3% , tf in 19.6% and pg in 39.22% [ figure 2 , bar diagram ] . we found a significant association of pg ( p - 0.003 ) and pg ( fima ) ( p - 0.008 ) in subgingival plaque and coronary plaque samples [ table 2 ] . the incidence of periodontal pathogens in subgingival plaque , atherosclerotic plaque and both sites kappa analysis for the presence of the microorganisms in subgingival and atherosclerotic plaque samples the present study further showed a statistically significant relationship between the presence of tf in the atherosclerotic plaque with gingival index ( p - 0.03 ) and probing pocket depth ( p - 0.01 ) [ table 3 ] . however , all the periodontal parameters analyzed were non - significant [ table 3 ] with the presence of td , pg and pg ( fima ) , in the atherosclerotic plaque samples . periodontal diseases are one of the most common bacterial infections characterized by inflammation and the subsequent destruction of the tooth supporting tissues . the past evidence states that dental plaque is the primarily etiological factor for the progression of periodontal diseases . in recent years , many researchers have focused their attention on the ability of periodontal pathogens to colonize in the atheromatous plaque in order to substantiate the potential role of the periodontal bacteria in the progression of atherosclerosis . nevertheless , a clear correlation between the detection rates of periodontopathic bacterial dna in atheromas and periodontal pockets has not been established . in view of the above , the present study was undertaken to assess the presence of the bacterial dna of putative periodontal pathogens in the subgingival and coronary plaque of patients with chronic periodontitis and to compare the periodontal status with the presence of periodontal bacteria in the coronary plaque . the study revealed the presence of the red complex bacteria ( td , tf and pg ) in 51% , 31.4% and 45.10% of atherosclerotic plaque samples , respectively . pg was detected in 24.4% of subgingival plaque and 45.10% of atherosclerotic plaque , showing a significant correlation of the microorganism between the two plaque samples thus indicating its potential role toward the progression of coronary events . also identified pg in 7.4% , 52% , 38.7% , 86% , 33% from atherosclerotic plaque samples , respectively . examined 50 carotid endarterectomy samples using pcr and found the presence of pg in 26% of the samples . we detected pg ( fima ) in 33.3% of both the subgingival and coronary plaque samples . the presence of pg ( fima ) was found to be significantly associated among both the plaque samples [ table 2 ] . pg is one of the major causative factor in periodontal disease and its fimbriae ( fima ) , a filamentous component located on the cell surface , plays a vital role in the colonization and invasion of the periodontal pathogen in the aortic tissues . we also detected td in 51% of the atherosclerotic plaque samples ; however , it remained non - significant [ table 2 ] . detected t. denticola in 44.4% and 85% of the atherosclerotic plaque samples respectively . in addition to the motility of td , it is possible that protease activity may also play a role in their penetration into the periodontal tissues by infiltrating between the gingival cells and the blood stream . who could not detect td in the coronary plaque samples . in the present study showed the detection of tf in 43.1% of the subgingival plaques and 31.4% of atherosclerotic plaques . , who could not identify tf in the coronary plaque samples . in our study , only one patient was positive for aa in the subgingival plaque sample whereas 16s rrna gene for the microorganism could not be detected in the atherosclerotic plaque . , who detected aa in 33.3% , 58% , 29% , 7.1% and 5% of the subgingival plaque samples , respectively . however , the present study was in concurrence with the study done by zhong et al . aa has been suggested to contribute to the pathogenesis of coronary heart disease and antibody levels against this pathogen in serum have been viewed as indicators of the risk for this disease . the reason could be that aa being more prevalent in the younger age group , was absent in our samples as the age of the selected subjects was between 40 and 80 years in this study . in the present study , the mean of the selected periodontal parameters namely gingival index , oral hygiene index , mean probing pocket depth , clinical attachment level , number of teeth present and the presence of the periodontal pathogens in the atherosclerotic plaque of the cardiac patients was analyzed [ table 3 ] . tf , showed a significant association with the gingival index suggesting that with an increase in the gingival inflammation , the above microorganism is more liable to be prevalent in the coronary plaque . tf has been reported to express a number of enzymatic and proteolytic activities that could contribute to its ability to compete effectively in the complex biofilm of the subgingival pocket . the previous studies have also shown the significant association between the periodontal parameters and the presence of the microorganisms . the proportion of patients with bleeding on probing and probing pocket depth 4 mm was found to be significantly associated with ami . patients with severe cad had significantly more periodontal destruction than those with mild cad . the correlation of td , pg and fima gene with periodontal parameters in the present study was not found to be significant . in addition , aa , could not show any significant association with the periodontal parameters . overall , the present study indicated that a good agreement existed between the percentage prevalence of the periodontal pathogens , in particular pg and pg fima in the subgingival and atherosclerotic plaque samples suggesting that the presence of these microorganisms in the subgingival plaque will have significant chances of their presence in atherosclerotic plaque as well contributing to the progression of atherosclerosis and vice - versa . it is suggested that the bacteria growing in the biofilm become resistant to the phagocytosis and host immune system . the ulcerated pocket epithelium becomes the portal of entry of the periodontal pathogens and their products to the general circulation in small and major blood vessels . on the other hand , the significant virulence factors of these bacteria are able to evade the host clearance mechanisms , participate in the tissue destruction and enter the general circulation in small and major blood vessels thus contributing to the coronary events . in future , large cross sectional and longitudinal studies are required to elicit the role of aa , the microorganisms of red complex and their virulence factors in the pathogenesis of atherosclerosis .

coronary heart disease is the leading cause of death worldwide , responsible for over 7 million deaths annually . indian asians ( people originating from india , pakistan , bangladesh and sri lanka ) comprise one quarter of the globe 's population and are at high risk of developing chd . recent estimates from the global burden of disease ( gbd ) 2010 study indicate that chd deaths are highest in south asia , increasing by 87.8% between 1990 and 2010 , second only to east asia ( table 1 ) . chd prevalence is presently twice as high in urban compared to rural india ( 1012% vs. 45% ) . with chd rates increasing more quickly in urban vs. rural areas ( figure 1 ) , and compounded by the current rapid rate of urbanization in india , this may result in an underestimation of chd mortality projections . india experiences amongst the highest number of potentially productive life years lost due to cardiovascular disease ; 9.2 million years in 2000 , expected to double to 17.9 million years by 2030 . the world health organisation estimated that india lost 8.7 billion us dollars in national income due to combined mortality from chd , stroke and diabetes in 2005 . high chd mortality rates have also been reported in migrant indian asian populations in england and wales , the united states , canada , singapore , mauritius , south africa , and trinidad ( table 2 ) . in the uk overall chd mortality rates have fallen in most minority ethnic groups residing in england and wales since 1980 , including those born on the indian subcontinent . importantly , increasing mortality rate ratio figures for indian asians compared to europeans suggest that the overall observed decline in chd mortality is slower amongst uk indian asian populations ( table 3 ) . conventional chd risk factors are known to have a pivotal role in chd in all populations , including indian asians ( table 4 ) . interheart , a case - control study of 15,152 first myocardial infarction cases and 14,820 controls recruited from 52 countries ( including 2,171 indian asian cases and 2,573 controls ) , compared hazard ratios and population - attributable risk fractions ( par ) for major chd risk factors ( smoking , hypertension , diabetes , central obesity , dyslipidaemia , physical activity level , psychosocial factors , alcohol , fruit and vegetable consumption ) . in the interheart study , these nine modifiable risk factors collectively account for 88% of chd in indian asians , similar to other populations ( table 5 ) . interheart was not designed to address reasons underlying differences in chd risk between population groups . previous studies showed that the differences in prevalence of smoking , hypertension , and dyslipidaemia were not consistently pointing to higher risk amongst uk indian asian sub - groups compared to europeans . these studies argued that conventional risk factors did not explain the 40% excess chd mortality among indian asians . in contrast , type-2 diabetes ( t2d ) , insulin resistance and related metabolic disturbances ( i.e. high triglycerides , low high - density lipoprotein cholesterol , central obesity ) were found to be more prevalent amongst indian asians than europeans ; in multivariate regression models , insulin resistance accounted for 70% of the excess risk of major q waves ( representing chd mortality risk ) in indian asians compared to europeans . most previous studies amongst indian asians have been either cross - sectional or case - control in design and hence the contribution of risk factors to incident chd has not been possible to assess in this population . thus reasons underlying the excess risk of chd amongst indian asians compared to europeans , and other population groups , remain poorly understood . the southall and brent revisited ( sabre ) study was a prospective comparison of chd mortality among uk indian asian and european men ( n = 1,420 and 1,787 respectively ) . sabre confirmed a 2-fold excess chd risk amongst indian asian men , which persisted after adjustment for chd risk factors including obesity , diabetes , insulin resistance , blood lipids , blood pressure and smoking ( table 6 ) . these prospective data do not support the widely held view that the excess chd risk amongst indian asians compared to europeans is largely attributable to central adiposity , insulin resistance or diabetes . however , sabre did not investigate the role of diet , physical activity , or genetic risk factors and did not study women . the lolipop study was established to investigate the reasons underlying higher susceptibility among indian asians to diabetes and cardiovascular disease , compared to europeans . participants were recruited from the lists of local general practitioners , thus providing a representative sample of the population living in west london . participants have had detailed clinical cardiovascular ( including 12-lead electrocardiogram ) and biochemical characterisation , with assessment of diet and physical activity . subsets of participants have genome - wide scans ( n20,000 ) , whole genome ( n400 ) or whole exome sequencing ( n2,000 ) , metabolome - wide ( n15,000 ) and epigenome - wide scans ( n3,300 ) . a representative lolipop sample of indian asians ( n1,000 ) and europeans ( n1,000 ) have also had echocardiography , 24hr blood pressure , 24hr holter monitor , cardiac computed tomography ( calcium scores ) , carotid intima media thickness ( with plaque characterisation ) , to examine the role of subclinical risk factors to high rates of chd in indian asians . indian asians have a 2-fold higher prevalence of chd compared to europeans which is evident at all age - groups ( figure 2 ) . in multivariate regression analysis , 1.862.40 , p < 0.001 ) amongst indian asians compared to europeans after adjustment for age , sex and conventional chd risk factors ( table 8) . after additional correction for insulin resistance , triglycerides and hdl cholesterol , a nearly 2-fold excess risk of chd remained amongst indian asians compared to europeans ( or 1.81 , 95% c.i . results therefore show that the higher risk of chd amongst uk indian asians is not explained by conventional risk factors , insulin resistance and related metabolic disturbances . lolipop participants are completing 10 year follow - up for cardiovascular events as well as other major health outcomes . ascertainment of events is being carried out through a multi - faceted approach including events occurring in primary care , hospital events , face - to - face follow - up , and national mortality records . this effort is expected to yield in excess of 2,000 cardiovascular events , making it possible to conduct the first well - powered investigation into the role of established , emerging and novel risk factors to incident cardiovascular events amongst indian asians , and help identify the reasons underlying their excess of chd risk compared to europeans . genome - wide association studies ( gwas ) amongst indian asians and europeans together identify a total of 51 single nucleotide polymorphisms ( snps , from 45 loci ) associated with chd . these individual variants each have a modest effect size , with odds ratios of 1.051.30 per risk allele copy , and in aggregate account for only a small proportion of the total predicted genetic variance . we have found no significant differences in either allele frequencies or odds ratios for these known chd snps ( discovered in europeans ) between indian asians and europeans ( figure 3 ) . our results therefore imply that reported genetic variants thus far do not account for the increased risk of chd amongst indian asians compared to europeans . however , these findings do not exclude a role for genetic factors since the gwas approach is not well designed to identify either low frequency or rare variants , which could make an important contribution to the missing heritability . furthermore , the available gwas arrays are based on hapmap data from predominantly european populations and thus do not capture indian asian - specific genetic variation contributing to chd . previous studies provide evidence for population - specific genetic variation increasing susceptibility to cardiovascular disease in indian asians . a 25-base pair deletion in the gene encoding cardiac myosin binding protein c ( mybpc3 ) , found in 4% of the population of indian asian ancestry , but absent amongst europeans , the lack of a systematic catalogue of indian asian - specific genetic variation is a major limitation to the identification of the genetic factors contributing to chd in this population , and underlies the strategy of whole genome ( wgs ) and whole exome sequencing ( wes ) amongst indian asians participating in the lolipop study . the 1000 genomes project is actively extending the catalogue of known human variants down to a frequency of 1% through wgs of target populations . lolipop has carried out wgs ( n400 ) and wes ( n2,000 ) in order to catalogue indian asian - specific genetic variation . results of this effort will help in the design of future studies assessing the contribution of indian asian - specific variants ( including low frequency and rare variants ) to the excess risk of chd amongst indian asians compared to europeans . epigenetic factors which are known to alter gene function , but are not the result of changes in the dna sequence , are increasingly considered to have a role in susceptibility to cardiovascular disease . epigenetic regulation of gene function is determined by reversible molecular processes such as histone binding of dna and methylation at cytosine residues , both of which influence transcription factor binding and accessibility of dna to the polymerase enzymes . more recent evidence suggests that epigenetic patterns of gene regulation may also occur in response to environmental insults , and underlie susceptibility to future disease . in the context of cardiovascular disease , several studies suggest that in utero or early life exposure to a poor nutritional environment may predispose to the development of cardiovascular disease in adult life . more than 96% of low birth - weight occurs in the developing world , with highest incidence in indian asians ( 7.1% of live births ) . birth cohort studies show that maternal undernutrition , low birth - weight , and rapid postnatal child growth are all associated with increased risk of chd in the off - spring . these risks appear to be trans - generational raising the possibility that epigenetic mechanisms may underlie part of the increased risk of cardiovascular disease amongst indian asians . technological advances have enabled nuclear magnetic resonance ( nmr ) spectrometry to detect a wide variety of metabolites from dietary , gut microbial , and host metabolism sources in one analytical sweep . bioinformatic approaches now allow combined analysis of metabolic phenotypes with genomic , epigenomic , and transcriptomic markers providing greater insight into potential mechanisms underlying cardiovascular disorders . the incorporation of these omic markers ( genomics , metabolomics and epigenomics ) into the prospective lolipop study design provides an exciting opportunity for discovery of novel risk factors and disease mechanisms underlying the excess risk of chd amongst the indian asian population . reasons underlying the 2-fold increased chd risk amongst indian asians compared to europeans remain poorly understood . priorities should include optimal management of indian asians with established chd , treatment of those with cardiovascular risk factors , and of first degree relatives of indian asians with premature chd . measures to improve education and management of cardiovascular risk factors in the population are also required . healthcare organisations and professionals need awareness of the higher risk of chd and adverse outcomes faced by indian asians . comprehensive screening for cardiovascular risk factors such as diabetes , hypertension and dyslipidaemia needs to be targeted in this population . of the available risk stratification tools , the framingham risk score ( frs ) has been shown to underestimate chd risk amongst indian asians thus limiting its usage . modifications to improve frs discrimination amongst indian asians have been suggested , such as multiplying the frs by a factor of 1.4 ( for males ) , or adding 10 years to the patients age . qrisk2 is a contemporary cardiovascular risk prediction tool developed and independently validated in uk populations . the qrisk2 algorithm takes ethnicity and deprivation into account , and is continuously updated , but the study population is mainly caucasian ( 1% indian asian ) . qrisk2 has not been externally validated in indian asians . despite chd being a major threat and huge public health challenge , only 1.1% of the gross domestic product was spent on healthcare in india ( 2008 - 9 ) , lower than most other emerging economies . major inequities based on gender , caste , geography , and social status persist . government funding and policymakers need to establish preventative strategies to reduce the burden of chd and its risk factors . additionally , major investment is required into research aimed at understanding increased susceptibility to chd amongst indian asians . high chd mortality rates have also been reported in migrant indian asian populations in england and wales , the united states , canada , singapore , mauritius , south africa , and trinidad ( table 2 ) . in the uk overall chd mortality rates have fallen in most minority ethnic groups residing in england and wales since 1980 , including those born on the indian subcontinent . importantly , increasing mortality rate ratio figures for indian asians compared to europeans suggest that the overall observed decline in chd mortality is slower amongst uk indian asian populations ( table 3 ) . conventional chd risk factors are known to have a pivotal role in chd in all populations , including indian asians ( table 4 ) . interheart , a case - control study of 15,152 first myocardial infarction cases and 14,820 controls recruited from 52 countries ( including 2,171 indian asian cases and 2,573 controls ) , compared hazard ratios and population - attributable risk fractions ( par ) for major chd risk factors ( smoking , hypertension , diabetes , central obesity , dyslipidaemia , physical activity level , psychosocial factors , alcohol , fruit and vegetable consumption ) . in the interheart study , these nine modifiable risk factors collectively account for 88% of chd in indian asians , similar to other populations ( table 5 ) . interheart was not designed to address reasons underlying differences in chd risk between population groups . previous studies showed that the differences in prevalence of smoking , hypertension , and dyslipidaemia were not consistently pointing to higher risk amongst uk indian asian sub - groups compared to europeans . these studies argued that conventional risk factors did not explain the 40% excess chd mortality among indian asians . in contrast , type-2 diabetes ( t2d ) , insulin resistance and related metabolic disturbances ( i.e. high triglycerides , low high - density lipoprotein cholesterol , central obesity ) were found to be more prevalent amongst indian asians than europeans ; in multivariate regression models , insulin resistance accounted for 70% of the excess risk of major q waves ( representing chd mortality risk ) in indian asians compared to europeans . most previous studies amongst indian asians have been either cross - sectional or case - control in design and hence the contribution of risk factors to incident chd has not been possible to assess in this population . thus reasons underlying the excess risk of chd amongst indian asians compared to europeans , and other population groups , remain poorly understood . the southall and brent revisited ( sabre ) study was a prospective comparison of chd mortality among uk indian asian and european men ( n = 1,420 and 1,787 respectively ) . sabre confirmed a 2-fold excess chd risk amongst indian asian men , which persisted after adjustment for chd risk factors including obesity , diabetes , insulin resistance , blood lipids , blood pressure and smoking ( table 6 ) . these prospective data do not support the widely held view that the excess chd risk amongst indian asians compared to europeans is largely attributable to central adiposity , insulin resistance or diabetes . however , sabre did not investigate the role of diet , physical activity , or genetic risk factors and did not study women . the lolipop study was established to investigate the reasons underlying higher susceptibility among indian asians to diabetes and cardiovascular disease , compared to europeans . participants were recruited from the lists of local general practitioners , thus providing a representative sample of the population living in west london . participants have had detailed clinical cardiovascular ( including 12-lead electrocardiogram ) and biochemical characterisation , with assessment of diet and physical activity . subsets of participants have genome - wide scans ( n20,000 ) , whole genome ( n400 ) or whole exome sequencing ( n2,000 ) , metabolome - wide ( n15,000 ) and epigenome - wide scans ( n3,300 ) . a representative lolipop sample of indian asians ( n1,000 ) and europeans ( n1,000 ) have also had echocardiography , 24hr blood pressure , 24hr holter monitor , cardiac computed tomography ( calcium scores ) , carotid intima media thickness ( with plaque characterisation ) , to examine the role of subclinical risk factors to high rates of chd in indian asians . indian asians have a 2-fold higher prevalence of chd compared to europeans which is evident at all age - groups ( figure 2 ) . in multivariate regression analysis , 1.862.40 , p < 0.001 ) amongst indian asians compared to europeans after adjustment for age , sex and conventional chd risk factors ( table 8) . after additional correction for insulin resistance , triglycerides and hdl cholesterol , a nearly 2-fold excess risk of chd remained amongst indian asians compared to europeans ( or 1.81 , 95% c.i . results therefore show that the higher risk of chd amongst uk indian asians is not explained by conventional risk factors , insulin resistance and related metabolic disturbances . lolipop participants are completing 10 year follow - up for cardiovascular events as well as other major health outcomes . ascertainment of events is being carried out through a multi - faceted approach including events occurring in primary care , hospital events , face - to - face follow - up , and national mortality records . this effort is expected to yield in excess of 2,000 cardiovascular events , making it possible to conduct the first well - powered investigation into the role of established , emerging and novel risk factors to incident cardiovascular events amongst indian asians , and help identify the reasons underlying their excess of chd risk compared to europeans . genome - wide association studies ( gwas ) amongst indian asians and europeans together identify a total of 51 single nucleotide polymorphisms ( snps , from 45 loci ) associated with chd . these individual variants each have a modest effect size , with odds ratios of 1.051.30 per risk allele copy , and in aggregate account for only a small proportion of the total predicted genetic variance . we have found no significant differences in either allele frequencies or odds ratios for these known chd snps ( discovered in europeans ) between indian asians and europeans ( figure 3 ) . our results therefore imply that reported genetic variants thus far do not account for the increased risk of chd amongst indian asians compared to europeans . however , these findings do not exclude a role for genetic factors since the gwas approach is not well designed to identify either low frequency or rare variants , which could make an important contribution to the missing heritability . furthermore , the available gwas arrays are based on hapmap data from predominantly european populations and thus do not capture indian asian - specific genetic variation contributing to chd . previous studies provide evidence for population - specific genetic variation increasing susceptibility to cardiovascular disease in indian asians . a 25-base pair deletion in the gene encoding cardiac myosin binding protein c ( mybpc3 ) , found in 4% of the population of indian asian ancestry , but absent amongst europeans , is associated with an 7-fold increased risk of heart failure in indian populations . the lack of a systematic catalogue of indian asian - specific genetic variation is a major limitation to the identification of the genetic factors contributing to chd in this population , and underlies the strategy of whole genome ( wgs ) and whole exome sequencing ( wes ) amongst indian asians participating in the lolipop study . the 1000 genomes project is actively extending the catalogue of known human variants down to a frequency of 1% through wgs of target populations . lolipop has carried out wgs ( n400 ) and wes ( n2,000 ) in order to catalogue indian asian - specific genetic variation . results of this effort will help in the design of future studies assessing the contribution of indian asian - specific variants ( including low frequency and rare variants ) to the excess risk of chd amongst indian asians compared to europeans . epigenetic factors which are known to alter gene function , but are not the result of changes in the dna sequence , are increasingly considered to have a role in susceptibility to cardiovascular disease . epigenetic regulation of gene function is determined by reversible molecular processes such as histone binding of dna and methylation at cytosine residues , both of which influence transcription factor binding and accessibility of dna to the polymerase enzymes . more recent evidence suggests that epigenetic patterns of gene regulation may also occur in response to environmental insults , and underlie susceptibility to future disease . in the context of cardiovascular disease , several studies suggest that in utero or early life exposure to a poor nutritional environment may predispose to the development of cardiovascular disease in adult life . more than 96% of low birth - weight occurs in the developing world , with highest incidence in indian asians ( 7.1% of live births ) . birth cohort studies show that maternal undernutrition , low birth - weight , and rapid postnatal child growth are all associated with increased risk of chd in the off - spring . these risks appear to be trans - generational raising the possibility that epigenetic mechanisms may underlie part of the increased risk of cardiovascular disease amongst indian asians . technological advances have enabled nuclear magnetic resonance ( nmr ) spectrometry to detect a wide variety of metabolites from dietary , gut microbial , and host metabolism sources in one analytical sweep . bioinformatic approaches now allow combined analysis of metabolic phenotypes with genomic , epigenomic , and transcriptomic markers providing greater insight into potential mechanisms underlying cardiovascular disorders . the incorporation of these omic markers ( genomics , metabolomics and epigenomics ) into the prospective lolipop study design provides an exciting opportunity for discovery of novel risk factors and disease mechanisms underlying the excess risk of chd amongst the indian asian population . reasons underlying the 2-fold increased chd risk amongst indian asians compared to europeans remain poorly understood . priorities should include optimal management of indian asians with established chd , treatment of those with cardiovascular risk factors , and of first degree relatives of indian asians with premature chd . measures to improve education and management of cardiovascular risk factors in the population are also required . healthcare organisations and professionals need awareness of the higher risk of chd and adverse outcomes faced by indian asians . comprehensive screening for cardiovascular risk factors such as diabetes , hypertension and dyslipidaemia needs to be targeted in this population . of the available risk stratification tools , the framingham risk score ( frs ) has been shown to underestimate chd risk amongst indian asians thus limiting its usage . modifications to improve frs discrimination amongst indian asians have been suggested , such as multiplying the frs by a factor of 1.4 ( for males ) , or adding 10 years to the patients age . qrisk2 is a contemporary cardiovascular risk prediction tool developed and independently validated in uk populations . the qrisk2 algorithm takes ethnicity and deprivation into account , and is continuously updated , but the study population is mainly caucasian ( 1% indian asian ) . qrisk2 has not been externally validated in indian asians . despite chd being a major threat and huge public health challenge , only 1.1% of the gross domestic product was spent on healthcare in india ( 2008 - 9 ) , lower than most other emerging economies . major inequities based on gender , caste , geography , and social status persist . government funding and policymakers need to establish preventative strategies to reduce the burden of chd and its risk factors . additionally , major investment is required into research aimed at understanding increased susceptibility to chd amongst indian asians .

the indian asian population accounts for a fifth of all global deaths from coronary heart disease ( chd ) . chd deaths on the indian subcontinent have doubled since 1990 , and are predicted to rise a further 50% by 2030 . reasons underlying the increased chd mortality among indian asians remain unknown . although conventional cardiovascular risk factors contribute to chd in indian asians as in other populations , these do not account for their increased risk . type-2 diabetes , insulin resistance and related metabolic disturbances are more prevalent amongst indian asians than europeans , and have been proposed as major determinants of higher chd risk among indian asians . however , this view is not supported by prospective data . genome - wide association studies have not identified differences in allele frequencies or effect sizes in known loci to explain the increased chd risk in indian asians . limited knowledge of mechanisms underlying higher chd risk amongst indian asians presents a major obstacle to reducing the burden of chd in this population . systems biology approaches such as genomics , epigenomics , metabolomics and transcriptomics , provide a non - biased approach for discovery of novel biomarkers and disease pathways underlying chd . incorporation of these omic approaches in prospective indian asian cohorts such as the london life sciences population study ( lolipop ) provide an exciting opportunity for the identification of new risk factors underlying chd in this high risk population .

Introduction Epidemiology of CHD in Indian Asians Genomic, epigenomic and metabolomic approaches to identify novel CHD risk factors among Indian Asians Strategies to reduce CHD amongst Indian Asians

indian asians ( people originating from india , pakistan , bangladesh and sri lanka ) comprise one quarter of the globe 's population and are at high risk of developing chd . recent estimates from the global burden of disease ( gbd ) 2010 study indicate that chd deaths are highest in south asia , increasing by 87.8% between 1990 and 2010 , second only to east asia ( table 1 ) . with chd rates increasing more quickly in urban vs. rural areas ( figure 1 ) , and compounded by the current rapid rate of urbanization in india , this may result in an underestimation of chd mortality projections . high chd mortality rates have also been reported in migrant indian asian populations in england and wales , the united states , canada , singapore , mauritius , south africa , and trinidad ( table 2 ) . in the uk overall chd mortality rates have fallen in most minority ethnic groups residing in england and wales since 1980 , including those born on the indian subcontinent . importantly , increasing mortality rate ratio figures for indian asians compared to europeans suggest that the overall observed decline in chd mortality is slower amongst uk indian asian populations ( table 3 ) . conventional chd risk factors are known to have a pivotal role in chd in all populations , including indian asians ( table 4 ) . interheart , a case - control study of 15,152 first myocardial infarction cases and 14,820 controls recruited from 52 countries ( including 2,171 indian asian cases and 2,573 controls ) , compared hazard ratios and population - attributable risk fractions ( par ) for major chd risk factors ( smoking , hypertension , diabetes , central obesity , dyslipidaemia , physical activity level , psychosocial factors , alcohol , fruit and vegetable consumption ) . in the interheart study , these nine modifiable risk factors collectively account for 88% of chd in indian asians , similar to other populations ( table 5 ) . interheart was not designed to address reasons underlying differences in chd risk between population groups . previous studies showed that the differences in prevalence of smoking , hypertension , and dyslipidaemia were not consistently pointing to higher risk amongst uk indian asian sub - groups compared to europeans . these studies argued that conventional risk factors did not explain the 40% excess chd mortality among indian asians . in contrast , type-2 diabetes ( t2d ) , insulin resistance and related metabolic disturbances ( i.e. high triglycerides , low high - density lipoprotein cholesterol , central obesity ) were found to be more prevalent amongst indian asians than europeans ; in multivariate regression models , insulin resistance accounted for 70% of the excess risk of major q waves ( representing chd mortality risk ) in indian asians compared to europeans . most previous studies amongst indian asians have been either cross - sectional or case - control in design and hence the contribution of risk factors to incident chd has not been possible to assess in this population . thus reasons underlying the excess risk of chd amongst indian asians compared to europeans , and other population groups , remain poorly understood . the southall and brent revisited ( sabre ) study was a prospective comparison of chd mortality among uk indian asian and european men ( n = 1,420 and 1,787 respectively ) . sabre confirmed a 2-fold excess chd risk amongst indian asian men , which persisted after adjustment for chd risk factors including obesity , diabetes , insulin resistance , blood lipids , blood pressure and smoking ( table 6 ) . these prospective data do not support the widely held view that the excess chd risk amongst indian asians compared to europeans is largely attributable to central adiposity , insulin resistance or diabetes . however , sabre did not investigate the role of diet , physical activity , or genetic risk factors and did not study women . the lolipop study was established to investigate the reasons underlying higher susceptibility among indian asians to diabetes and cardiovascular disease , compared to europeans . subsets of participants have genome - wide scans ( n20,000 ) , whole genome ( n400 ) or whole exome sequencing ( n2,000 ) , metabolome - wide ( n15,000 ) and epigenome - wide scans ( n3,300 ) . a representative lolipop sample of indian asians ( n1,000 ) and europeans ( n1,000 ) have also had echocardiography , 24hr blood pressure , 24hr holter monitor , cardiac computed tomography ( calcium scores ) , carotid intima media thickness ( with plaque characterisation ) , to examine the role of subclinical risk factors to high rates of chd in indian asians . indian asians have a 2-fold higher prevalence of chd compared to europeans which is evident at all age - groups ( figure 2 ) . in multivariate regression analysis , 1.862.40 , p < 0.001 ) amongst indian asians compared to europeans after adjustment for age , sex and conventional chd risk factors ( table 8) . after additional correction for insulin resistance , triglycerides and hdl cholesterol , a nearly 2-fold excess risk of chd remained amongst indian asians compared to europeans ( or 1.81 , 95% c.i . results therefore show that the higher risk of chd amongst uk indian asians is not explained by conventional risk factors , insulin resistance and related metabolic disturbances . this effort is expected to yield in excess of 2,000 cardiovascular events , making it possible to conduct the first well - powered investigation into the role of established , emerging and novel risk factors to incident cardiovascular events amongst indian asians , and help identify the reasons underlying their excess of chd risk compared to europeans . genome - wide association studies ( gwas ) amongst indian asians and europeans together identify a total of 51 single nucleotide polymorphisms ( snps , from 45 loci ) associated with chd . these individual variants each have a modest effect size , with odds ratios of 1.051.30 per risk allele copy , and in aggregate account for only a small proportion of the total predicted genetic variance . we have found no significant differences in either allele frequencies or odds ratios for these known chd snps ( discovered in europeans ) between indian asians and europeans ( figure 3 ) . our results therefore imply that reported genetic variants thus far do not account for the increased risk of chd amongst indian asians compared to europeans . however , these findings do not exclude a role for genetic factors since the gwas approach is not well designed to identify either low frequency or rare variants , which could make an important contribution to the missing heritability . furthermore , the available gwas arrays are based on hapmap data from predominantly european populations and thus do not capture indian asian - specific genetic variation contributing to chd . a 25-base pair deletion in the gene encoding cardiac myosin binding protein c ( mybpc3 ) , found in 4% of the population of indian asian ancestry , but absent amongst europeans , the lack of a systematic catalogue of indian asian - specific genetic variation is a major limitation to the identification of the genetic factors contributing to chd in this population , and underlies the strategy of whole genome ( wgs ) and whole exome sequencing ( wes ) amongst indian asians participating in the lolipop study . results of this effort will help in the design of future studies assessing the contribution of indian asian - specific variants ( including low frequency and rare variants ) to the excess risk of chd amongst indian asians compared to europeans . more than 96% of low birth - weight occurs in the developing world , with highest incidence in indian asians ( 7.1% of live births ) . birth cohort studies show that maternal undernutrition , low birth - weight , and rapid postnatal child growth are all associated with increased risk of chd in the off - spring . these risks appear to be trans - generational raising the possibility that epigenetic mechanisms may underlie part of the increased risk of cardiovascular disease amongst indian asians . bioinformatic approaches now allow combined analysis of metabolic phenotypes with genomic , epigenomic , and transcriptomic markers providing greater insight into potential mechanisms underlying cardiovascular disorders . the incorporation of these omic markers ( genomics , metabolomics and epigenomics ) into the prospective lolipop study design provides an exciting opportunity for discovery of novel risk factors and disease mechanisms underlying the excess risk of chd amongst the indian asian population . reasons underlying the 2-fold increased chd risk amongst indian asians compared to europeans remain poorly understood . priorities should include optimal management of indian asians with established chd , treatment of those with cardiovascular risk factors , and of first degree relatives of indian asians with premature chd . measures to improve education and management of cardiovascular risk factors in the population are also required . healthcare organisations and professionals need awareness of the higher risk of chd and adverse outcomes faced by indian asians . comprehensive screening for cardiovascular risk factors such as diabetes , hypertension and dyslipidaemia needs to be targeted in this population . of the available risk stratification tools , the framingham risk score ( frs ) has been shown to underestimate chd risk amongst indian asians thus limiting its usage . modifications to improve frs discrimination amongst indian asians have been suggested , such as multiplying the frs by a factor of 1.4 ( for males ) , or adding 10 years to the patients age . qrisk2 has not been externally validated in indian asians . government funding and policymakers need to establish preventative strategies to reduce the burden of chd and its risk factors . additionally , major investment is required into research aimed at understanding increased susceptibility to chd amongst indian asians . high chd mortality rates have also been reported in migrant indian asian populations in england and wales , the united states , canada , singapore , mauritius , south africa , and trinidad ( table 2 ) . in the uk overall chd mortality rates have fallen in most minority ethnic groups residing in england and wales since 1980 , including those born on the indian subcontinent . importantly , increasing mortality rate ratio figures for indian asians compared to europeans suggest that the overall observed decline in chd mortality is slower amongst uk indian asian populations ( table 3 ) . conventional chd risk factors are known to have a pivotal role in chd in all populations , including indian asians ( table 4 ) . interheart , a case - control study of 15,152 first myocardial infarction cases and 14,820 controls recruited from 52 countries ( including 2,171 indian asian cases and 2,573 controls ) , compared hazard ratios and population - attributable risk fractions ( par ) for major chd risk factors ( smoking , hypertension , diabetes , central obesity , dyslipidaemia , physical activity level , psychosocial factors , alcohol , fruit and vegetable consumption ) . in the interheart study , these nine modifiable risk factors collectively account for 88% of chd in indian asians , similar to other populations ( table 5 ) . interheart was not designed to address reasons underlying differences in chd risk between population groups . previous studies showed that the differences in prevalence of smoking , hypertension , and dyslipidaemia were not consistently pointing to higher risk amongst uk indian asian sub - groups compared to europeans . these studies argued that conventional risk factors did not explain the 40% excess chd mortality among indian asians . in contrast , type-2 diabetes ( t2d ) , insulin resistance and related metabolic disturbances ( i.e. high triglycerides , low high - density lipoprotein cholesterol , central obesity ) were found to be more prevalent amongst indian asians than europeans ; in multivariate regression models , insulin resistance accounted for 70% of the excess risk of major q waves ( representing chd mortality risk ) in indian asians compared to europeans . most previous studies amongst indian asians have been either cross - sectional or case - control in design and hence the contribution of risk factors to incident chd has not been possible to assess in this population . thus reasons underlying the excess risk of chd amongst indian asians compared to europeans , and other population groups , remain poorly understood . the southall and brent revisited ( sabre ) study was a prospective comparison of chd mortality among uk indian asian and european men ( n = 1,420 and 1,787 respectively ) . sabre confirmed a 2-fold excess chd risk amongst indian asian men , which persisted after adjustment for chd risk factors including obesity , diabetes , insulin resistance , blood lipids , blood pressure and smoking ( table 6 ) . these prospective data do not support the widely held view that the excess chd risk amongst indian asians compared to europeans is largely attributable to central adiposity , insulin resistance or diabetes . the lolipop study was established to investigate the reasons underlying higher susceptibility among indian asians to diabetes and cardiovascular disease , compared to europeans . subsets of participants have genome - wide scans ( n20,000 ) , whole genome ( n400 ) or whole exome sequencing ( n2,000 ) , metabolome - wide ( n15,000 ) and epigenome - wide scans ( n3,300 ) . a representative lolipop sample of indian asians ( n1,000 ) and europeans ( n1,000 ) have also had echocardiography , 24hr blood pressure , 24hr holter monitor , cardiac computed tomography ( calcium scores ) , carotid intima media thickness ( with plaque characterisation ) , to examine the role of subclinical risk factors to high rates of chd in indian asians . in multivariate regression analysis , 1.862.40 , p < 0.001 ) amongst indian asians compared to europeans after adjustment for age , sex and conventional chd risk factors ( table 8) . after additional correction for insulin resistance , triglycerides and hdl cholesterol , a nearly 2-fold excess risk of chd remained amongst indian asians compared to europeans ( or 1.81 , 95% c.i . results therefore show that the higher risk of chd amongst uk indian asians is not explained by conventional risk factors , insulin resistance and related metabolic disturbances . this effort is expected to yield in excess of 2,000 cardiovascular events , making it possible to conduct the first well - powered investigation into the role of established , emerging and novel risk factors to incident cardiovascular events amongst indian asians , and help identify the reasons underlying their excess of chd risk compared to europeans . genome - wide association studies ( gwas ) amongst indian asians and europeans together identify a total of 51 single nucleotide polymorphisms ( snps , from 45 loci ) associated with chd . these individual variants each have a modest effect size , with odds ratios of 1.051.30 per risk allele copy , and in aggregate account for only a small proportion of the total predicted genetic variance . we have found no significant differences in either allele frequencies or odds ratios for these known chd snps ( discovered in europeans ) between indian asians and europeans ( figure 3 ) . our results therefore imply that reported genetic variants thus far do not account for the increased risk of chd amongst indian asians compared to europeans . however , these findings do not exclude a role for genetic factors since the gwas approach is not well designed to identify either low frequency or rare variants , which could make an important contribution to the missing heritability . furthermore , the available gwas arrays are based on hapmap data from predominantly european populations and thus do not capture indian asian - specific genetic variation contributing to chd . previous studies provide evidence for population - specific genetic variation increasing susceptibility to cardiovascular disease in indian asians . a 25-base pair deletion in the gene encoding cardiac myosin binding protein c ( mybpc3 ) , found in 4% of the population of indian asian ancestry , but absent amongst europeans , is associated with an 7-fold increased risk of heart failure in indian populations . the lack of a systematic catalogue of indian asian - specific genetic variation is a major limitation to the identification of the genetic factors contributing to chd in this population , and underlies the strategy of whole genome ( wgs ) and whole exome sequencing ( wes ) amongst indian asians participating in the lolipop study . results of this effort will help in the design of future studies assessing the contribution of indian asian - specific variants ( including low frequency and rare variants ) to the excess risk of chd amongst indian asians compared to europeans . more than 96% of low birth - weight occurs in the developing world , with highest incidence in indian asians ( 7.1% of live births ) . birth cohort studies show that maternal undernutrition , low birth - weight , and rapid postnatal child growth are all associated with increased risk of chd in the off - spring . these risks appear to be trans - generational raising the possibility that epigenetic mechanisms may underlie part of the increased risk of cardiovascular disease amongst indian asians . the incorporation of these omic markers ( genomics , metabolomics and epigenomics ) into the prospective lolipop study design provides an exciting opportunity for discovery of novel risk factors and disease mechanisms underlying the excess risk of chd amongst the indian asian population . reasons underlying the 2-fold increased chd risk amongst indian asians compared to europeans remain poorly understood . priorities should include optimal management of indian asians with established chd , treatment of those with cardiovascular risk factors , and of first degree relatives of indian asians with premature chd . comprehensive screening for cardiovascular risk factors such as diabetes , hypertension and dyslipidaemia needs to be targeted in this population . of the available risk stratification tools , the framingham risk score ( frs ) has been shown to underestimate chd risk amongst indian asians thus limiting its usage . modifications to improve frs discrimination amongst indian asians have been suggested , such as multiplying the frs by a factor of 1.4 ( for males ) , or adding 10 years to the patients age . the qrisk2 algorithm takes ethnicity and deprivation into account , and is continuously updated , but the study population is mainly caucasian ( 1% indian asian ) . qrisk2 has not been externally validated in indian asians . government funding and policymakers need to establish preventative strategies to reduce the burden of chd and its risk factors . additionally , major investment is required into research aimed at understanding increased susceptibility to chd amongst indian asians .

obesity and related disorders are on the rise throughout the globe at an alarming rate , the causes of which are not yet completely understood . on the one hand , individuals differ substantially in their tendency to accumulate fat , and there are strong familial tendencies suggesting that genetic predisposition may play a major role . on the other hand , it is obvious that no alleles can increase in frequency in a population with a rate matching the rate of spread of the obesity epidemic , indicating that genetics alone does not explain the rise in obesity . the most common perception , therefore , is that of a interplay between genes and environment . according to the current paradigm , a gene or a set of genes predisposing to obesity presumably evolved owing to a selective advantage in ancestral feast and famine environment and remained in polymorphic state in the population which is turning pathological in the modern urban environment selectively affecting individuals with the gene(s ) . this line of thinking was first stated explicitly by neel who suggested that a thrifty gene helped storage of fat under conditions of better availability of nutrients and allowed reutilization under starvation . the thrifty gene was under positive selection pressure in ancestral life when seasonal and climatic conditions resulted into fluctuating food availability . this concept soon became almost an axiom , although no such thrifty gene or set of genes have been convincingly demonstrated . later , the observation that individuals born small for gestational age had a greater probability to become obese and type 2 diabetic in later life led to the concept of fetal programming [ 24 ] . this hypothesis states that if a fetus faces inadequate nutrition in intrauterine life , the body is programmed to be one relates to an immediate gain in terms of survival during fetal and early infant life . both the concepts of thrifty gene and thrifty phenotype by fetal programming have recently faced serious criticism on several grounds [ 611 ] . the original suggestion of neel was that in individuals prone to obesity and type 2 diabetes ( t2d ) , a quick insulin trigger ensures rapid glucose uptake which is then converted into fat . in the early 1960s , it was well known that the levels of insulin are higher in prediabetics , and neel 's original proposal looked sound . however , insulin resistance was discovered soon , and it became clear that the quick insulin trigger is unlikely to work as believed by neel . fat cell - specific insulin receptor knock - outs fail to accumulate fat , raising the possibility that insulin resistance actually arrests lipogenesis . it is therefore not logical to call the diabetes - prone genotypes as thrifty . if thriftiness is due to lower metabolic rate conserving more energy which gets stored in fat tissue , a lower metabolic rate should be observed in people having a predisposition to obesity . consistent correlations between birth weight and metabolic rate are not found in empirical data to demonstrate the supposed thriftiness of low birth weight individuals [ 13 , 14 ] . studies using doubly labeled water have not consistently found lower metabolic rates in people with sedentary lifestyle in the modern urban societies [ 15 , 16 ] . impaired fat oxidation rather than lower metabolic rate appears to be the main contributor to obesity of developmentally stunted individuals [ 17 , 18 ] . if inability to reutilize stored fat is the major cause of obesity , the stored fat is unlikely to help under famine conditions , and this is a major blow to the thriftiness hypotheses . the doubly labeled water studies also suggest that obesity is more a product of hyperphagia than metabolic thriftiness [ 15 , 16 ] . the known genetic mechanisms of obesity also work by interfering with appetite control rather than through metabolic thriftiness . therefore , it is doubtful whether obesity actually offered sufficient advantage during famines to get selected in spite of the fact that obesity is associated with reduced fecundity . obesity and insulin resistance is associated with a number of changes in the different body systems and their functions as diverse as ovulation , spermatogenesis , innate immunity , wound healing , memory , and cognitive brain functions . the thriftiness hypotheses focus on energy homeostasis alone and offer no explanation as to why these diverse changes are associated with it . realizing the limitations , inadequacies , and flaws of the thrifty gene and fetal programming hypotheses , a number of alternatives have been suggested . ( a ) as alternatives to the thrifty gene hypothesis , ( i ) speakman postulated genetic drift rather than selection for obesity - related genes . an upper limit on obesity was set in hunter - gatherer life which was effectively lifted when humans became free of predation . argued that today 's obese , diabetic , and pcos - prone genotype was the ancestral one that had better fertility in famine conditions . in the modern era of food security since 1800 ad , an insulin - sensitive genotype that has better fertility under conditions of food abundance started spreading . . hypothesized that high plasma glucose lowers the freezing point of blood which prevents formation of ice crystals in cells through supercooling , and this has been suggested as an adaptation to the ice age . ( i ) hattersley and tooke argued that the association between low birth weight and insulin resistance arises out of a reverse causation , that is , babies with insulin resistance genotype are more likely to survive fetal undernourishment . ( ii ) wells [ 11 , 24 ] argued that there is maternal advantage in fetal programming in the form of optimizing maternal inputs per fetus or bet hedging , that is , distribution of risk among offspring . ( c ) there are hypotheses that account for genetic as well as intrauterine effects . ( i ) watve and yajnik argued that insulin resistance is a socioecological adaptation that mediates two phenotypic transitions , namely , ( i ) transition in reproductive strategy from r ( large number of offspring with little investment in each ) to k ( smaller number of offspring with more investment in each ) and ( ii ) transition from soldier to diplomat , that is , from a physically aggressive behavior to a socially manipulative one . according to this hypothesis , insulin resistance changes the differential budget allocation to tissues . since all tissues are not dependent on insulin for nutrient uptake , when insulin resistance develops , the uptake of insulin - dependent tissues reduces , and more nutrients become available for insulin independent tissues . muscles are insulin dependent , and brain is insulin independent , and , therefore , insulin resistance results in disinvestment from muscles and increased investment in brain . insulin resistance is likely to have evolved as a switch in reproductive and sustenance strategies rather than an adaptation to feast and famine . ( ii ) extending this logic further , rashidi et al . explained why pancreatic beta cells and those of females in particular are more susceptible to oxidative damage . under stress conditions , the release of stress hormones produces insulin resistance and , owing to reactive oxygen species ( ros ) preventing beta cells from secreting insulin at the level required to maintain homeostasis , diverts glucose to insulin - independent tissues , such as the brain and the fetus . they suggest that pancreatic beta cells lost part of their antioxidant defense in association with brain evolution , and lost even more in females when placental mammals evolved . the emergence of alternative hypotheses having different implications for the genetics of obesity has made it even more critical that the traditional hypothesis is examined analytically . the concept of thriftiness has been mostly discussed descriptively and qualitatively and almost never subject to quantitative methods commonly used by evolutionary geneticists to test or support any argument . we use a simple mathematical model here to examine the conditions under which thrifty gene or fetal programming is likely to gain a selective advantage and evaluate how likely it is for human ancestors to have evolved constitutive or programmable thriftiness . there are no empirical estimates of the actual fitness contributions of the hypothetical genes or phenotypes , and , therefore , a quantitatively predictive model can not be attempted at this stage . the limited objective of our model is to conceptually examine whether a thrifty gene or thrifty programming can evolve in principle , whether stable polymorphism in this character is possible , if yes , what are the necessary conditions for its evolutionary stability , and whether the thriftiness hypotheses adequately explain the current obesity epidemic . to model the possible evolution of thriftiness , we consider 3 hypothetical genotypes , namely , a nonthrifty wild type having no mechanism for thriftiness ( n ) , a thrifty genotype ( tg ) which is genetically programmed for thriftiness and a genotype with a capacity for fetal programming for thriftiness ( tp ) . taking a year as a natural time unit of seasonality , we assume a simple dichotomy of years with adequate food supply ( feast ) and those with inadequate food supply ( famine ) . fitness of an individual with nonthrifty genotype in feast conditions ( nf1 ) is assumed to be greater than that of individual with thrifty genotype ( tf1 ) because there is cost associated with thriftiness . when there is a feast , nonthrifty individuals will do better as they do not pay the cost for being thrifty ( nf1 > tf1 ) . we assume that thrifty individuals are fast to become obese in feast conditions , and there are multiple mechanisms by which obesity causes reduction in fecundity . on the one hand , obesity and insulin resistance are associated with ovulation disorders and are major risk factors for polycystic ovary syndrome ( pcos ) . on the other , reduced fecundity is also seen in obese women with regular cycles [ 20 , 27 ] . moreover , obesity also affects spermatogenesis in men , and the effects of obesity on male and female fertility become additive if a couple is obese . fitness of an individual with nonthrifty genotype in famine conditions ( nf0 ) is assumed to be less than that of individuals with thrifty genotype ( tf0 ) ( nf0 < tf0 ) . for individuals with nonthrifty genotype , ( 1)ln = pfnf0+(1pf)nf1 . for individuals with thrifty genotype , ( 2)ltg = pftf0+(1pf)tf1 . for individuals with thrifty phenotype or the capacity for irreversible fetal programming , assuming no correlation between birth and lifetime conditions , the total fitness is calculated as the sum of all years , with the assumption that in the birth year , the phenotype is best suited for given conditions . for the rest of the lifetime ( s ) therefore , ( 3)ltp=1s[pftf0+(1pf)nf1]+s1s {[pfltg]+[(1pf)ln]}. the first term in ( 3 ) denotes fitness contribution of birth year , and the second term sums the fitness contribution of the rest of the lifespan . solution : considering the nonthrifty and thrifty genotypes alone , it can be seen that , at large pf , thrifty gene has an advantage over nonthrifty gene , and , at small pf , nonthrifty gene can not be invaded by the thrifty one . the transition is at ln = ltg and this happens when ( 4)pf(1pf)=(tf1nf1)(nf0tf0 ) . it is important to note that the areas of advantage are decided not by the absolute fitness values but only by the ratios of the differences in fitness in feast and famine conditions . considering the nonthrifty and thrifty genotypes alone , it can be seen that , at large pf , thrifty gene has an advantage over nonthrifty gene , and , at small pf , nonthrifty gene can not be invaded by the thrifty one . the transition is at ln = ltg and this happens when ( 4)pf(1pf)=(tf1nf1)(nf0tf0 ) . it is important to note that the areas of advantage are decided not by the absolute fitness values but only by the ratios of the differences in fitness in feast and famine conditions . equation ( 4 ) implies that the evolution of thrifty gene needs a high frequency of famine . if surviving famines is the major selective force for thrifty gene , with pf = 0.01 , the advantage of thrifty over nonthrifty phenotype in famine conditions should be more than 100-times the relative loss suffered by thrifty gene in feast conditions . if the obesity - induced reduction in fecundity is sizable , the advantage of thriftiness in famines should be exceedingly large for thrifty genes to evolve . such a large advantage should be highly evident and easily measurable , but obese people have not been shown to survive famines significantly better than lean individuals . considering competition between nonthrifty and fetal programming genotypes , it can be seen that at higher pf and lower s , ltp > ln . the transition line resides at ln = ltp . for a given pf , this condition is met at ( 5)s=[pf(tf0ltg)]+[(1pf)(nf1ln)]pf(lnltg ) . figure 1(a ) shows the transition line demarcating the areas of selective advantage of ln and ltp when only these two genotypes compete . at very high pf , however , an all - time thrifty genotype might have an advantage over fetal programming . therefore , we need to examine the areas of advantage of ltp and ltg . taking a similar approach as above , ltg = ltp when ( 6)s=[pf(tf0ltg)]+[(1pf)(nf1ln)](1pf)(ltgln ) . a graph of pf versus s ( figure 1(b ) ) shows that , at greater probability of famine and greater longevity , thrifty gene(s ) can evolve . the two results together ( figure 2 ) imply that at low probabilities of famine , a nonthrifty gene has a net advantage , and at high pf , thrifty gene would evolve leaving a very narrow area of advantage for fetal programming . the area is narrower for long - lived species whereas for short - lived ones , the birth year advantage is large enough as compared to lifetime , and , therefore , fetal programming has a much larger width of advantage . the results so far are based on the assumption that seasonal or annual climatic variations are random with little or no predictability . if there exists a correlation r between uterine conditions and lifetime conditions , then it follows that the lifetime probability of famine would be higher if birth was in a famine year . we can , therefore , write the expected probability of facing a famine if the birth year was a famine year as ( 7)pfr0=(1pf)r+pf . the probability of facing a famine if the birth year was a feast year as ( 8)pfr1=pf(pfr ) , accordingly,(9)ltp=[pftf0+(1pf)nf1]s + { ( s1)[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}s.now , the new transition between areas of advantage of non thrifty and fetal programming ( when ln = ltp ) is obtained at ( 10)s=[pftf0+(1pf)nf1]ln{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1 } ] } {[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}ln{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}. similarly , the transition between areas of advantage of thrifty and fetal programming is obtained at ltg = ltp . this condition is satisfied when(11)s=[pftf0+(1pf)nf1]ltg{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1 } ] } {[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}ltg{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}.figure 3 shows that as the birth - time and lifetime correlation increases , the area of advantage of fetal programmer widens . however , in long - lived species , since the advantage to fetal programmer in the absence of correlation is very small , even a small negative correlation drives the fetal programmer to extinction ( figure 3(c ) ) . it is most important for the evolution of fetal programming that a positive correlation between birth - time and lifetime conditions exists . it can be seen that at r = 0.3 , fetal programmer has an advantage over a wide range of pf . climatic fluctuations from year to year are a complex phenomenon , and since prediction of important climatic features , such as rainfall , has important implications , there have been serious attempts to detect temporal patterns . however , temporal patterns are of little help in weather prediction since there are no consistent time lapse correlations in rainfall or other parameters . since india has the largest population of diabetics and monsoon is the most important determinant of food availability in this region , it would be enlightening to see the patterns in the indian monsoon . based on the public - domain data on indian monsoon by the indian institute of tropical meteorology ( ftp://www.tropmet.res.in/pub/data/rain/iitm-subdivrf.txt ) , we investigated whether birth year and subsequent year 's rainfall has any positive correlation in the short or long run for any of the 30 monsoon subdivisions in india . table 1 shows that rainfall in a given year is not correlated with that of the subsequent year , subsequent 10 years , or 40 years cumulative . over the 30 monsoon subdivisions , only 6 are statistically significant using individual level of 0.05 , out of which 4 are negative , contrary to the expectation . using bonferroni correction for significance level , applicable when a large number of tests are being done together , none of the correlations remain significant . as there is no detectable positive correlation between birth year and lifetime rainfall conditions , similarly no such correlational patterns in any other climatic variables are reported , fetal programming is unlikely to have evolved in anticipation of drought or famine . the model suggests that a thrifty gene or a set of genes can evolve only if the frequency of famines is very high and under such conditions the thrifty allele(s ) would not exist in a stable polymorphic state . selection for the ability to program the fetus is likely in a very narrow range of frequency of famines , and at lower or higher frequency of famines , fetal programming would not evolve . the parameter space for the evolution of fetal programming becomes very narrow and highly specific for long - lived species owing to which evolution of fetal programming for thriftiness is highly unlikely . the model results need to be interpreted carefully in the context of ancestral human ecology . one of the key questions is when in human history could selection for thriftiness , if any , have operated . ( a ) selection during hunting - gathering stage : paleoarcheological data suggests that chronic starvation was uncommon during hunter - gatherer stage . today 's hunter - gatherer societies do not seem to suffer starvation more frequently or more intensively than agricultural societies . therefore , the assumption that hunter - gatherer societies suffered frequent starvation is not well supported , but even if we assume hunter - gatherer societies to be prone to feast and famine selection , a number of other questions remain unanswered . since hominids were hunter - gatherers for the most part of human evolutionary history , selection would have been prolonged , and we would expect alleles to have reached equilibrium frequencies . the model implies that selection can not result in stable polymorphism of thrifty alleles . in the modern human society therefore , polymorphism with respect to genes predisposing to obesity and type 2 diabetes presumably exists . if there is no negative frequency dependence or heterozygote advantage , natural selection will be directional , resulting into the fixation of the advantageous genotype . in that case at no value of pf , the thrifty and nonthrifty genes can coexist stably . theoretically , if a heterozygote of thrifty and nonthrifty alleles gets a dual advantage by expressing the right allele in the right environment , the two alleles can coexist , and the population at any given time will consist of thrifty , nonthrifty , and all time fit individuals . however , there is no empirical evidence of heterozygote advantage so far . however , if fitness is decided by climatic conditions as assumed by the popular version of thrifty gene hypothesis , frequency dependence is unlikely . therefore , selection during hunter - gatherer stage does not explain the prevalent polymorphism in predisposition to obesity . ( b ) selection after the beginning of agriculture : chronic starvation due to famines became more serious and common with the beginning of agriculture [ 7 , 8 ] . signs of chronic starvation on teeth , such as linear enamel hypoplasia , are more common in early agricultural societies than in hunter - gatherer societies . this is owing to the fact that crops are highly seasonal in nature , and the failure of a single crop leads to long - term food scarcity . such long - term food shortages are much less probable in hunter - gatherer life , particularly in biodiversity rich areas . therefore , if selection for thriftiness started acting after the beginning of agriculture , there could be transient polymorphism . a testable prediction of the hypothesis would then be that ethnic groups that took to agriculture earlier should show higher tendencies to become obese and diabetic . this has not been rigorously tested with quantitative data . however , ethnic groups , such as the australian aborigines , remained hunter gatherers until recently , and the recently urbanized individuals of this community developed a high prevalence of diabetes and hypertension . it is difficult to argue , therefore , that thrifty genes evolved after the advent of agriculture . ( c ) selection in modern times : intensive agricultural and industrial societies are modern phenomena not more than a few hundred years old , and it is highly unlikely that this period could have brought about an evolutionary change . it can be seen from all the three possible scenarios that natural selection for the hypothetical thrifty gene(s ) is unable to explain the polymorphism observed today . since different geographic regions of the world differ in the climatic conditions , seasonality , and food availability , ethnic groups evolved in different areas should show differential predisposition to obesity and related disorders . people evolved in arid or drought - prone areas could have suffered more frequent famines and , therefore , should have a greater tendency to develop obesity . also , ethnic groups from harsh winter environments were unable to hunt , fish , or farm during the colder months and , thus , historically could have faced regular feast and famine conditions . although substantial cross - ethnic differences are observed , the trends are not as expected by the thriftiness hypotheses . people from caucasoid , eskimo , and some of the himalayan ethnic groups that have faced harsh winter environments have a considerably lower frequency of obesity and/or t2d [ 3438 ] whereas almost all ethnic groups of warmer habitats have a high frequency on adopting a western urban lifestyle [ 33 , 39 ] , contrary to the expectation . short - term survival advantages in fetal and early infant life and long - term predictive adaptive responses . our model incorporates both of the advantages separately . if the advantage is of a short duration , it is difficult to explain why a lifetime commitment to a particular metabolic state could have evolved . a number of developmental genes have age - specific expressions , and , therefore , any rigid lifelong programming for short - term advantage is a difficult proposition . if climatic fluctuations were the main selective force , it should evolve metabolic flexibility rather than lifelong rigid programming . metabolic programming of a lifelong duration based on intrauterine conditions is unlikely to offer a fitness advantage except in two sets of conditions . as the model suggests , if a species has a very short life span , fetal programming for adapting to the birth year conditions can be beneficial , since the birth year itself is a substantial part of the total life span . assuming one year to be the natural unit of seasonal cycles , species with a life span of less than 35 years can be expected to evolve lifelong fetal programming for thriftiness even though the adaptive advantage is of a short duration . for long - lived species , fetal programming is unlikely to evolve unless there is a significant positive correlation between birth conditions and lifelong conditions . since such correlations are not seen in climatological data , there are problems in explaining evolution of lifelong thrifty programming . since the thriftiness hypotheses are inadequate or weak on several grounds , there is a need to rethink the paradigm and consider alternative hypotheses seriously . a detailed comparative analysis of all the alternative hypotheses is beyond the scope of this paper . most of them are new , and all their implications have not yet come forward . we will only briefly evaluate the alternative hypotheses below to see whether they offer better explanations where the thriftiness hypotheses are weaker . ( 1 ) polymorphism : polymorphism in the predisposition to obesity can be potentially explained in three different ways . it is possible that the allelic composition in the population is close to equilibrium , and there is stable polymorphism . out of all alternative hypotheses , only the watve - yajnik hypothesis is able to predict stable polymorphism since there is negative frequency - dependent selection in a hawk and dove like game . an alternative view is that the human population today is not at a stable equilibrium proportion of alleles but is undergoing drift or selection . speakman also tries to quantify the drift dynamics ; however , his calculation is based on the assumption that human ancestors became free from predators about 1.8 million years ago , and this estimate is debatable . moreover , if the drift is an ongoing process , it would take a different direction in different populations and the cross - ethnic differences are expected to be random . instead , we see that almost all tropical ethnic groups have high tendency to develop obesity and t2d on urbanization , and such a generalizable pattern is not expected by the drift hypothesis . also , as we know now , a large number of genes are associated with obesity , and if drift has to operate independently on all the genes , it is highly unlikely that it will result in a directional change . it has not been critically questioned whether only about 200 years of selection is sufficient to generate the current levels of polymorphism . any data to test the dynamics of both the hypotheses using a predictive model are currently absent . the third possible explanation to the apparent polymorphism is that there is no real genetic polymorphism , but individuals are programmed differently depending upon environmental conditions faced in early life . apart from the thrifty phenotype hypothesis , only the watve and yajnik hypothesis accounts for lifetime programming in a way discussed below . the cold adaptation hypothesis of hattersley and tooke does not explain polymorphism at any of the above levels . if high blood glucose is adaptive in cold environments , then ethnic groups who evolved in cold climates should undergo directional selection leading to fixation . polymorphism in that case can only arise by cross - breeding between ethnic groups coming from warmer and colder environments . ( 2 ) low birth weight effects : there is globally consistent and strong evidence that a small birth weight is associated with altered metabolic and endocrine states in adult life [ 3 , 41 , 42 ] . different interpretations of the birth weight effects have been offered , and the birth weight association itself can not be considered a convincing evidence of fetal programming . however , rat experiments with maternal nutrient deficiencies have also given strong evidence in support of fetal programming . it can nevertheless be debated whether the programming is for thriftiness or for any other adaptation . speakman 's [ 7 , 8 ] drifty gene hypothesis , corbett et al . . wells and watve and yajnik have different interpretations of the effect of maternal nutrient limitation , but both of them adequately account for birth weight effects . wells ' hypothesis of maternal advantage does not have a predictive adaptation element and , therefore , does not explain lifetime rigidity in the programming . watve and yajnik argue that fetal undernourishment affects muscle mass more than brain mass and , therefore , a brain - dependent diplomat lifestyle marked by insulin resistance is adaptive for low birth weight individuals . since muscle cells do not replicate in adult life , this early developmental limitation persists throughout life , and , thus , lifetime programming could evolve . at the same time , low birth weight is not a necessary prerequisite for the watve and yajnik hypothesis to work . any hypothesis exclusively based on fetal programming suffers from another problem in that although low birth weight individuals have a greater probability of developing obesity and t2d , a large proportion of adult type 2 diabetics are not born with low weights . ( 3 ) birth time versus lifetime correlation : the model predicts that for lifetime fetal programming to evolve , a positive correlation between intrauterine and lifetime conditions is necessary . although climatic variations are unlikely to cause such correlations , there can be other causes of food deprivation apart from climatic factors . although population density may oscillate , the oscillations typically span over several generations so that an individual born at a high population density is expected to see high population density through most of his life . therefore , periodic food scarcity caused by population oscillations can result into positive correlations between birth year and lifetime nutritional conditions . in social species , an individual born smaller and weaker is more likely to face social subordination , and , therefore , an anticipatory fetal programming would be adaptive . these social causes can produce positive birth - lifetime correlations , and they are more likely candidates to select for fetal programming of lifelong duration . there exist a broad range of metabolic , endocrinological , behavioral , and cognitive adaptations that accompany social hierarchical positions . therefore , if fetal programming is in response to social hierarchies , we expect that it need not be restricted to diet and energy homeostasis , but it affects a large number of systems of the body along with brain and behavior . this indeed , is the case , and type 2 diabetes involves almost all systems of the body [ 10 , 45 ] . social subordination is an important predisposing factor for type 2 diabetes , and the link between the two is elaborately explained by belsare et al . . we suggest that on exposure to intrauterine malnourishment , there is fetal programming in anticipation of physical weakness and social subordination or anticipation of high population density rather than anticipation of famine . in primate societies , it is known that social ranks of juveniles and subadults are influenced by the ranks of their mothers [ 47 , 48 ] . if social rank influences preferential access to food resources , it is sufficient to produce the positive correlation between fetal and lifetime nutritional conditions needed for the evolution of fetal programming . it appears logical , therefore , that fetal programming could have evolved in anticipation of population- and social hierarchy - related factors than anticipation of famines or other climate - related factors . ( 4 ) multiple effects : obesity and t2d are not only about energy homeostasis but also about changes in innate immunity , sexual and reproductive function , vascular development and function , skin architecture , wound healing and tissue regeneration , memory , cognitive functions , behavior and mechanisms of decision making , social relations , and social signaling . therefore , any hypothesis about the origins of obesity needs to account for all the network of changes adequately . most hypotheses are too glucolipo - centric and , therefore , fall short of this criterion . the only possible exception is the watve - yajnik hypothesis which has an inherent expectation that a number of systems of the body would be simultaneously involved [ 10 , 40 ] . implications for genetics of obesity : a large body of research has now focused on the genetics of obesity . an increasing number of loci and mutants associated with obesity are being identified . however , there are certain internal paradoxes associated with these data . studies prior to the genomic era that were based on familial , twin - pair , and adoption studies typically predicted a large heritable component in obesity ( reviewed by ) . the genome - wide association studies , on the other hand , have identified a large number of associations , but together they explain a very small fraction of variance in obesity parameters [ 5054 ] , leaving a large gap between the pregenomic and emerging genomic picture . we are yet to understand the reasons for this discrepancy , but a most likely implication goes against all hypotheses that assume a gene or a set of genes for obesity . these hypotheses include neel 's thrifty gene , speakman 's drifty gene , corbett et al . 's reverse selection , and moalem et al . 's cold adaptation . on the other hand , fetal programming has a promise for filling the gap between the familial studies and genome - wide association studies . three of the above hypotheses involve fetal programming , namely , the classical thrifty phenotype hypothesis , maternal adaptation hypothesis by wells , and behavioural switch hypothesis by watve and yajnik . since our model has indicated the inadequacies of the thrifty phenotype hypothesis , the other two need to be considered more seriously . we suggest , here , that more than one type of metabolic programming may be involved in obesity , and they may be induced in various stages of life . there are strong data for fetal programming , but behavioral programming is also likely to affect metabolism , since associations between neuroendocrine mechanisms of behavior and metabolic states are known . the classical thriftiness family of hypotheses would expect genes associated with metabolic rates to be the obesity genes . the behavioural origins hypothesis , on the other hand , expects genes involved in sexual function , cognitive abilities , immunity , regulation of aggression , and other behavioral traits to be associated with obesity and related disorders . it would be useful to interpret the emerging data on genome - wide associations and , perhaps , near future studies on epigenetics of obesity in the light of the more promising hypotheses from the above . we have shown with a simple theoretical model that the classical concepts of thrifty gene as well as fetal programming for thriftiness face a number of problems and are inadequate to account for the observable trends in the modern epidemic of obesity and related disorders . it might be too early to discard any of the hypotheses right away , but a comparative analysis shows the strengths and weaknesses of each of them . since search for obesity - associated genes has so far given limited success in terms of explaining population variance in obesity parameters , the hypotheses involving phenotypic programming look more promising . in understanding obesity and related disorders , the need for a combined genomic - phenomic approach is increasingly being felt [ 55 , 56 ] , and such an integration can be best achieved on the platform of an evolutionary insight into the phenomenon . greater efforts are , therefore , needed to understand the evolutionary background of obesity and related pathophysiological conditions .

obesity and related disorders are thought to have their roots in metabolic thriftiness that evolved to combat periodic starvation . the association of low birth weight with obesity in later life caused a shift in the concept from thrifty gene to thrifty phenotype or anticipatory fetal programming . the assumption of thriftiness is implicit in obesity research . we examine here , with the help of a mathematical model , the conditions for evolution of thrifty genes or fetal programming for thriftiness . the model suggests that a thrifty gene can not exist in a stable polymorphic state in a population . the conditions for evolution of thrifty fetal programming are restricted if the correlation between intrauterine and lifetime conditions is poor . such a correlation is not observed in natural courses of famine . if there is fetal programming for thriftiness , it could have evolved in anticipation of social factors affecting nutrition that can result in a positive correlation .

1. Introduction 2. The Model 3. Discussion

obesity and related disorders are on the rise throughout the globe at an alarming rate , the causes of which are not yet completely understood . on the other hand , it is obvious that no alleles can increase in frequency in a population with a rate matching the rate of spread of the obesity epidemic , indicating that genetics alone does not explain the rise in obesity . the most common perception , therefore , is that of a interplay between genes and environment . according to the current paradigm , a gene or a set of genes predisposing to obesity presumably evolved owing to a selective advantage in ancestral feast and famine environment and remained in polymorphic state in the population which is turning pathological in the modern urban environment selectively affecting individuals with the gene(s ) . this line of thinking was first stated explicitly by neel who suggested that a thrifty gene helped storage of fat under conditions of better availability of nutrients and allowed reutilization under starvation . later , the observation that individuals born small for gestational age had a greater probability to become obese and type 2 diabetic in later life led to the concept of fetal programming [ 24 ] . both the concepts of thrifty gene and thrifty phenotype by fetal programming have recently faced serious criticism on several grounds [ 611 ] . the original suggestion of neel was that in individuals prone to obesity and type 2 diabetes ( t2d ) , a quick insulin trigger ensures rapid glucose uptake which is then converted into fat . in the early 1960s , it was well known that the levels of insulin are higher in prediabetics , and neel 's original proposal looked sound . if thriftiness is due to lower metabolic rate conserving more energy which gets stored in fat tissue , a lower metabolic rate should be observed in people having a predisposition to obesity . consistent correlations between birth weight and metabolic rate are not found in empirical data to demonstrate the supposed thriftiness of low birth weight individuals [ 13 , 14 ] . studies using doubly labeled water have not consistently found lower metabolic rates in people with sedentary lifestyle in the modern urban societies [ 15 , 16 ] . the known genetic mechanisms of obesity also work by interfering with appetite control rather than through metabolic thriftiness . therefore , it is doubtful whether obesity actually offered sufficient advantage during famines to get selected in spite of the fact that obesity is associated with reduced fecundity . obesity and insulin resistance is associated with a number of changes in the different body systems and their functions as diverse as ovulation , spermatogenesis , innate immunity , wound healing , memory , and cognitive brain functions . realizing the limitations , inadequacies , and flaws of the thrifty gene and fetal programming hypotheses , a number of alternatives have been suggested . ( i ) hattersley and tooke argued that the association between low birth weight and insulin resistance arises out of a reverse causation , that is , babies with insulin resistance genotype are more likely to survive fetal undernourishment . ( ii ) wells [ 11 , 24 ] argued that there is maternal advantage in fetal programming in the form of optimizing maternal inputs per fetus or bet hedging , that is , distribution of risk among offspring . insulin resistance is likely to have evolved as a switch in reproductive and sustenance strategies rather than an adaptation to feast and famine . the concept of thriftiness has been mostly discussed descriptively and qualitatively and almost never subject to quantitative methods commonly used by evolutionary geneticists to test or support any argument . we use a simple mathematical model here to examine the conditions under which thrifty gene or fetal programming is likely to gain a selective advantage and evaluate how likely it is for human ancestors to have evolved constitutive or programmable thriftiness . there are no empirical estimates of the actual fitness contributions of the hypothetical genes or phenotypes , and , therefore , a quantitatively predictive model can not be attempted at this stage . the limited objective of our model is to conceptually examine whether a thrifty gene or thrifty programming can evolve in principle , whether stable polymorphism in this character is possible , if yes , what are the necessary conditions for its evolutionary stability , and whether the thriftiness hypotheses adequately explain the current obesity epidemic . to model the possible evolution of thriftiness , we consider 3 hypothetical genotypes , namely , a nonthrifty wild type having no mechanism for thriftiness ( n ) , a thrifty genotype ( tg ) which is genetically programmed for thriftiness and a genotype with a capacity for fetal programming for thriftiness ( tp ) . fitness of an individual with nonthrifty genotype in feast conditions ( nf1 ) is assumed to be greater than that of individual with thrifty genotype ( tf1 ) because there is cost associated with thriftiness . when there is a feast , nonthrifty individuals will do better as they do not pay the cost for being thrifty ( nf1 > tf1 ) . for individuals with thrifty phenotype or the capacity for irreversible fetal programming , assuming no correlation between birth and lifetime conditions , the total fitness is calculated as the sum of all years , with the assumption that in the birth year , the phenotype is best suited for given conditions . solution : considering the nonthrifty and thrifty genotypes alone , it can be seen that , at large pf , thrifty gene has an advantage over nonthrifty gene , and , at small pf , nonthrifty gene can not be invaded by the thrifty one . considering the nonthrifty and thrifty genotypes alone , it can be seen that , at large pf , thrifty gene has an advantage over nonthrifty gene , and , at small pf , nonthrifty gene can not be invaded by the thrifty one . equation ( 4 ) implies that the evolution of thrifty gene needs a high frequency of famine . if surviving famines is the major selective force for thrifty gene , with pf = 0.01 , the advantage of thrifty over nonthrifty phenotype in famine conditions should be more than 100-times the relative loss suffered by thrifty gene in feast conditions . if the obesity - induced reduction in fecundity is sizable , the advantage of thriftiness in famines should be exceedingly large for thrifty genes to evolve . considering competition between nonthrifty and fetal programming genotypes , it can be seen that at higher pf and lower s , ltp > ln . at very high pf , however , an all - time thrifty genotype might have an advantage over fetal programming . a graph of pf versus s ( figure 1(b ) ) shows that , at greater probability of famine and greater longevity , thrifty gene(s ) can evolve . the two results together ( figure 2 ) imply that at low probabilities of famine , a nonthrifty gene has a net advantage , and at high pf , thrifty gene would evolve leaving a very narrow area of advantage for fetal programming . the area is narrower for long - lived species whereas for short - lived ones , the birth year advantage is large enough as compared to lifetime , and , therefore , fetal programming has a much larger width of advantage . if there exists a correlation r between uterine conditions and lifetime conditions , then it follows that the lifetime probability of famine would be higher if birth was in a famine year . we can , therefore , write the expected probability of facing a famine if the birth year was a famine year as ( 7)pfr0=(1pf)r+pf . the probability of facing a famine if the birth year was a feast year as ( 8)pfr1=pf(pfr ) , accordingly,(9)ltp=[pftf0+(1pf)nf1]s + { ( s1)[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}s.now , the new transition between areas of advantage of non thrifty and fetal programming ( when ln = ltp ) is obtained at ( 10)s=[pftf0+(1pf)nf1]ln{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1 } ] } {[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}ln{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}. similarly , the transition between areas of advantage of thrifty and fetal programming is obtained at ltg = ltp . this condition is satisfied when(11)s=[pftf0+(1pf)nf1]ltg{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1 } ] } {[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}ltg{[pf{(pfr0tf0)+(1pfr0)tf1}]+[(1pf){(pfr1nf0)+(1pfr1)nf1}]}.figure 3 shows that as the birth - time and lifetime correlation increases , the area of advantage of fetal programmer widens . however , in long - lived species , since the advantage to fetal programmer in the absence of correlation is very small , even a small negative correlation drives the fetal programmer to extinction ( figure 3(c ) ) . it is most important for the evolution of fetal programming that a positive correlation between birth - time and lifetime conditions exists . since india has the largest population of diabetics and monsoon is the most important determinant of food availability in this region , it would be enlightening to see the patterns in the indian monsoon . based on the public - domain data on indian monsoon by the indian institute of tropical meteorology ( ftp://www.tropmet.res.in/pub/data/rain/iitm-subdivrf.txt ) , we investigated whether birth year and subsequent year 's rainfall has any positive correlation in the short or long run for any of the 30 monsoon subdivisions in india . table 1 shows that rainfall in a given year is not correlated with that of the subsequent year , subsequent 10 years , or 40 years cumulative . as there is no detectable positive correlation between birth year and lifetime rainfall conditions , similarly no such correlational patterns in any other climatic variables are reported , fetal programming is unlikely to have evolved in anticipation of drought or famine . the model suggests that a thrifty gene or a set of genes can evolve only if the frequency of famines is very high and under such conditions the thrifty allele(s ) would not exist in a stable polymorphic state . selection for the ability to program the fetus is likely in a very narrow range of frequency of famines , and at lower or higher frequency of famines , fetal programming would not evolve . the parameter space for the evolution of fetal programming becomes very narrow and highly specific for long - lived species owing to which evolution of fetal programming for thriftiness is highly unlikely . the model results need to be interpreted carefully in the context of ancestral human ecology . one of the key questions is when in human history could selection for thriftiness , if any , have operated . ( a ) selection during hunting - gathering stage : paleoarcheological data suggests that chronic starvation was uncommon during hunter - gatherer stage . therefore , the assumption that hunter - gatherer societies suffered frequent starvation is not well supported , but even if we assume hunter - gatherer societies to be prone to feast and famine selection , a number of other questions remain unanswered . the model implies that selection can not result in stable polymorphism of thrifty alleles . in the modern human society therefore , polymorphism with respect to genes predisposing to obesity and type 2 diabetes presumably exists . if there is no negative frequency dependence or heterozygote advantage , natural selection will be directional , resulting into the fixation of the advantageous genotype . theoretically , if a heterozygote of thrifty and nonthrifty alleles gets a dual advantage by expressing the right allele in the right environment , the two alleles can coexist , and the population at any given time will consist of thrifty , nonthrifty , and all time fit individuals . however , there is no empirical evidence of heterozygote advantage so far . however , if fitness is decided by climatic conditions as assumed by the popular version of thrifty gene hypothesis , frequency dependence is unlikely . it is difficult to argue , therefore , that thrifty genes evolved after the advent of agriculture . since different geographic regions of the world differ in the climatic conditions , seasonality , and food availability , ethnic groups evolved in different areas should show differential predisposition to obesity and related disorders . people evolved in arid or drought - prone areas could have suffered more frequent famines and , therefore , should have a greater tendency to develop obesity . if the advantage is of a short duration , it is difficult to explain why a lifetime commitment to a particular metabolic state could have evolved . metabolic programming of a lifelong duration based on intrauterine conditions is unlikely to offer a fitness advantage except in two sets of conditions . as the model suggests , if a species has a very short life span , fetal programming for adapting to the birth year conditions can be beneficial , since the birth year itself is a substantial part of the total life span . assuming one year to be the natural unit of seasonal cycles , species with a life span of less than 35 years can be expected to evolve lifelong fetal programming for thriftiness even though the adaptive advantage is of a short duration . for long - lived species , fetal programming is unlikely to evolve unless there is a significant positive correlation between birth conditions and lifelong conditions . it is possible that the allelic composition in the population is close to equilibrium , and there is stable polymorphism . out of all alternative hypotheses , only the watve - yajnik hypothesis is able to predict stable polymorphism since there is negative frequency - dependent selection in a hawk and dove like game . an alternative view is that the human population today is not at a stable equilibrium proportion of alleles but is undergoing drift or selection . moreover , if the drift is an ongoing process , it would take a different direction in different populations and the cross - ethnic differences are expected to be random . instead , we see that almost all tropical ethnic groups have high tendency to develop obesity and t2d on urbanization , and such a generalizable pattern is not expected by the drift hypothesis . also , as we know now , a large number of genes are associated with obesity , and if drift has to operate independently on all the genes , it is highly unlikely that it will result in a directional change . apart from the thrifty phenotype hypothesis , only the watve and yajnik hypothesis accounts for lifetime programming in a way discussed below . if high blood glucose is adaptive in cold environments , then ethnic groups who evolved in cold climates should undergo directional selection leading to fixation . ( 2 ) low birth weight effects : there is globally consistent and strong evidence that a small birth weight is associated with altered metabolic and endocrine states in adult life [ 3 , 41 , 42 ] . different interpretations of the birth weight effects have been offered , and the birth weight association itself can not be considered a convincing evidence of fetal programming . wells and watve and yajnik have different interpretations of the effect of maternal nutrient limitation , but both of them adequately account for birth weight effects . watve and yajnik argue that fetal undernourishment affects muscle mass more than brain mass and , therefore , a brain - dependent diplomat lifestyle marked by insulin resistance is adaptive for low birth weight individuals . at the same time , low birth weight is not a necessary prerequisite for the watve and yajnik hypothesis to work . any hypothesis exclusively based on fetal programming suffers from another problem in that although low birth weight individuals have a greater probability of developing obesity and t2d , a large proportion of adult type 2 diabetics are not born with low weights . ( 3 ) birth time versus lifetime correlation : the model predicts that for lifetime fetal programming to evolve , a positive correlation between intrauterine and lifetime conditions is necessary . therefore , periodic food scarcity caused by population oscillations can result into positive correlations between birth year and lifetime nutritional conditions . in social species , an individual born smaller and weaker is more likely to face social subordination , and , therefore , an anticipatory fetal programming would be adaptive . therefore , if fetal programming is in response to social hierarchies , we expect that it need not be restricted to diet and energy homeostasis , but it affects a large number of systems of the body along with brain and behavior . we suggest that on exposure to intrauterine malnourishment , there is fetal programming in anticipation of physical weakness and social subordination or anticipation of high population density rather than anticipation of famine . if social rank influences preferential access to food resources , it is sufficient to produce the positive correlation between fetal and lifetime nutritional conditions needed for the evolution of fetal programming . it appears logical , therefore , that fetal programming could have evolved in anticipation of population- and social hierarchy - related factors than anticipation of famines or other climate - related factors . an increasing number of loci and mutants associated with obesity are being identified . three of the above hypotheses involve fetal programming , namely , the classical thrifty phenotype hypothesis , maternal adaptation hypothesis by wells , and behavioural switch hypothesis by watve and yajnik . since our model has indicated the inadequacies of the thrifty phenotype hypothesis , the other two need to be considered more seriously . we suggest , here , that more than one type of metabolic programming may be involved in obesity , and they may be induced in various stages of life . there are strong data for fetal programming , but behavioral programming is also likely to affect metabolism , since associations between neuroendocrine mechanisms of behavior and metabolic states are known . the behavioural origins hypothesis , on the other hand , expects genes involved in sexual function , cognitive abilities , immunity , regulation of aggression , and other behavioral traits to be associated with obesity and related disorders . it would be useful to interpret the emerging data on genome - wide associations and , perhaps , near future studies on epigenetics of obesity in the light of the more promising hypotheses from the above . we have shown with a simple theoretical model that the classical concepts of thrifty gene as well as fetal programming for thriftiness face a number of problems and are inadequate to account for the observable trends in the modern epidemic of obesity and related disorders . since search for obesity - associated genes has so far given limited success in terms of explaining population variance in obesity parameters , the hypotheses involving phenotypic programming look more promising . in understanding obesity and related disorders , the need for a combined genomic - phenomic approach is increasingly being felt [ 55 , 56 ] , and such an integration can be best achieved on the platform of an evolutionary insight into the phenomenon . greater efforts are , therefore , needed to understand the evolutionary background of obesity and related pathophysiological conditions .

the mediator complex is a multiprotein assembly comprising at least 30 subunits that functions as a regulator of gene transcription in multiple contexts including stem cell function , the immune response , inflammation , cell adhesion , the epithelial to mesenchymal transition and development . the mediator complex - associated kinase cdk8 and its paralog cdk19 are cyclin c - dependent enzymes that , along with med12 and med13 , form the kinase module of the mediator complex . cdk8 has been reported to regulate basal transcription by phosphorylation of rna polymerase ii and to phosphorylate e2f1 , thereby activating wnt signaling . interestingly , cdk8 gene expression correlates with activation of -catenin , a core transcriptional regulator of canonical wnt signaling , in colon and gastric cancers . cdk8 gene expression also correlates with increased mortality in colorectal , breast , and ovarian cancers ; furthermore cdk8 is overexpressed and essential for cell proliferation in melanoma . consistent with these reports , cdk8 is located in a region of chromosome 13 known to undergo copy number gain in 60% of colorectal cancers and inducible shrna - mediated knockdown of cdk8 protein reduces the growth of ht29 and colo205 colorectal cancer human tumor xenograft animal models harboring cdk8 gene amplification . notably , cdk8 expression transforms nih3t3 cells into a malignant phenotype whereas a kinase - dead mutant does not , thereby implicating the kinase function of cdk8 in oncogenesis . cdk19 has been reported to form mediator complexes independent of cdk8 ; however their context - dependent roles are the subject of ongoing study . previously reported small molecule ligands for cdk8 and its paralog cdk19 have been described in a recent comprehensive review . in brief , the steroidal natural product cortistatin a ( 1 ) was the first - reported high affinity and selective ligand for cdk8/19 ( chart 1 ) ; recent disclosures include a patent describing cortistatin a analogs and a report demonstrating potent in vitro and in vivo antileukemic activity of cortistatin a through dual cdk8/19 inhibition . the marketed kinase inhibitor sorafenib ( 2 ) has been cocrystallized with cdk8/cyclin c , and subsequently the same group reported a fragment - based approach to cdk8 ligands building from the urea moiety associated with the type ii binding mode of sorafenib . type ii kinase inhibitors linifanib ( 3 ) and ponatinib ( 4 ) have also been reported to bind both cdk8 and cdk19 . recently , a cell - based hts campaign seeking inhibitors of p21-activated transcription was reported ; this effort led to the discovery of aminoquinazoline - based cdk8/19 ligands , exemplified by senexin b ( 5 ) ( chart 1 ) . other series of small molecule cdk8 inhibitors have also been reported in the patent literature . we have previously reported the discovery of 6 ( cct251545 ) , a potent , orally bioavailable small molecule inhibitor of wnt signaling from a cell - based pathway screen ( chart 1 ) . we identified protein kinases cdk8 and cdk19 as the primary targets of this trisubstituted pyridine series and demonstrated a strong correlation between cdk8 and cdk19 binding affinities in this chemical series . here we describe the medicinal chemistry optimization of 6 to compound 109 , a potent , selective , and orally bioavailable inhibitor of cdk8 with equipotent affinity for cdk19 that demonstrates potent cell - based activity together with improved pharmacokinetic and pharmaceutical properties . we demonstrate inhibition of cdk8 function concomitant with reduced proliferation in a human tumor xenograft animal model of colorectal cancer . the general synthetic method for the preparation of 3-cl and 3-f substituted pyridine analogues involved initial snar displacement at the 4-position of 3-bromo-4,5-dichloropyridine ( 7 ) or 3-bromo-4-chloro-5-fluoropyridine ( 57 ) to give intermediates 8 , 2636 , 58 , 59 , and 96 , which were then subject to suzuki cross - coupling to give final compounds 1725 , 4056 , 6063 , and 100105 ( scheme 1 and tables 2 , 3 , 4 , and 7 ) . the corresponding cf3-substituted pyridines ( 70 , 71 , and 72 , table 4 ) were synthesized by two alternative routes . compound 71 was prepared by selective copper - mediated trifluoromethylation at the bromo - substituted carbon atom of 8 , followed by suzuki coupling at the pyridine chloro substituent . alternatively , compounds 70 and 72 were prepared by snar displacement at the 4-position of 3,5-diiodo-4-chloropyridine 64 , followed by copper - mediated trifluoromethylation at one iodo - substituted carbon followed by suzuki cross - coupling at the other . the asterisk ( ) indicates different solvent and/or slightly different conditions were used for compounds 17 , 18 , 42 , 47 , 50 , and 52 . 2-aminopyridines ( 88 , 9295 , 108 , and 111 , tables 5 and 8) were prepared by snar - mediated displacement of the 4-chloro substituent in pyridines 73 , 74 , and 75 followed by suzuki cross - coupling ( scheme 2 ) . in the case of 8587 , 8991 , 109 , and 110 , we found that protection of the primary amine with a pmb group improved both the snar displacement and the subsequent palladium - mediated cross - coupling reaction ; deprotection was achieved using trifluoroacetic acid at room temperature . the asterisk ( ) indicates that kf was added for the synthesis of compounds 76 and 82 . compound 6 is a high affinity ligand for cdk8 and cdk19 ( ic50 of 7.2 1.4 and 6.0 1.0 nm , respectively ) and demonstrates potent inhibition of wnt - dependent signaling using our previously described inducible luciferase reporter assay in human embryonic kidney cells ( hek293 ) that contains both estrogen receptor - dishevelled ( dvl2 ) and tcf - luciferase - ires - gfp constructs ( 7df3 ic50 = 5.0 2.0 nm , table 2 ) ; it also demonstrates potent activity in ls174 t human colorectal carcinoma cells that harbor a reporter - based readout measuring constitutive -catenin mutation - driven wnt pathway activity ( ic50 = 23 11 nm ) . compound 6 displays moderate in vivo clearance in both mouse and rat ; however , high predicted human clearance ( 76% liver blood flow ) prevented further progression ( table 1 ) . furthermore , the volumes of distribution ( vd ) across all species tested were low to medium and the aqueous kinetic and thermodynamic solubilities ( 94 m and 0.006 mg / ml , respectively ) were suboptimal . we therefore turned our attention to lowering the lipophilicity of compound 6 ( measured log d = 3.5 ) with the aim of decreasing the microsomal and in vivo clearance as well as improving aqueous solubility . to increase the vd , we also attempted the introduction of a weakly basic center . in the course of improving physicochemical , pharmaceutical , and pharmacokinetic properties , we monitored in vitro biochemical affinity for cdk8 and cdk19 which we found to consistently predict for cell - based activity in both the reporter - based 7df3 and ls174 t assays ; we also observed consistent sar in these two reporter - based cellular assays for compounds described in this manuscript ( figure s1 in supporting information ) . dose : 0.2 mg / kg ( iv ) , 0.5 mg / kg ( po ) . upon the basis of our work to identify the molecular targets of 6 , we knew that extended linear substituents were tolerated at c-5 of the pyridine ring ; therefore , we introduced a variety of polar groups on the c-5 phenylpyrazole anticipating that these would enhance metabolic stability and aqueous solubility ( table 2 , entries 25 ) . pleasingly , replacement of the pyrazole n1-methyl substituent by hydroxyethyl or 2-hydroxy-2-methylpropyl led to a significant improvement in microsomal stability in all species ; furthermore , aqueous solubility also increased ( table 2 , entries 2 and 3 ) while cdk8/19 biochemical affinity and cell - based 7df3 potency were maintained . introduction of a basic center resulted in a 4-fold drop in potency in both biochemical and cell - based assays , suggesting that permeability is not responsible for the observed decrease ( table 2 , entries 46 ) . however , we noticed that compounds such as 17 , 18 , and 21 ( table 2 , entries 2 , 3 , 6 ) had reduced microsomal clearance ( clint ) , which confirmed that the introduction of polarity could be fruitful . introduction of 5-substituted-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide ( henceforth abbreviated to sultam ) improved the microsomal stability of the molecule across all species tested ( table 2 , entry 7 ) . furthermore , the thermodynamic aqueous solubility of 22 was improved by 120-fold ; however a 23-fold drop in cell - based potency was also observed . in order to improve cell permeability by lowering h - bond donor count , the n - methyl sultam derivative 23 was prepared ( table 2 , entry 8) ; while this compound was potent in the 7df3 cell - based assay , both 23 and its regioisomer 24 exhibited significant metabolic instability ( table 2 , entries 8 and 9 ) . the aminoindazole 25 gave an acceptable in vitro clearance profile across all species and high aqueous solubility ( table 2 , entry 10 ) ; however , as for most of the derivatives in table 2 , we observed low oral bioavailability and no improvement of the in vivo mouse clearance compared to 6 . run in different conditions ( see reporter displacement assay in experimental section ) . in this case the chloro substituent is at c-5 according to nomenclature 8-(3-(3-amino-1h - indazol-6-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one . to understand whether the introduction of polarity influenced binding mode , we determined the crystal structure of aminoindazole derivative 25 in complex with the kinase domain of cdk8 and cyclin c ( figure 1 , panel a ) . as previously observed with 6 , analog 25 occupies the atp binding site ( figure 1 , panel b ) . all interactions were conserved including a cation interaction of the indazole phenyl ring with arg365 due to insertion of the c - terminal domain of cdk8 into the atp binding site . notably , binding to the hinge via the pyridine is conserved despite the introduction of a potential alternative 3-aminoindazole hinge - binding motif ; pleasingly , the only difference we observe between the binding modes of compounds 6 and 25 is the presence of a dmso molecule in a small pocket adjacent to 25 ( see figure 1 , panel a ) . crystal structure of 25 in cdk8/cyclin c ( panel a , pdb code 5fgk ) . overlay of 6 ( pink ) and 25 ( gray ) in cdk8/cyclin c ( panel b ) . although the n - methyl sultam group conferred metabolic instability ( table 2 , entry 8) , its beneficial effect on the potency was very attractive and we were keen to explore replacement of the spirolactam in combination either with the n - methyl sultam or with our hitherto preferred 4-(1-methyl-1h - pyrazol-4-yl)phenyl substituent at c-5 . consistent with our desire to replace the spirolactam with more polar heterocycles , mass spectrometry - mediated metabolite identification for 6 indicated that the spirolactam was the main site of oxidative metabolism ( data not shown ) . introduction of a carbamate ( table 3 , entry 3 ) resulted in > 100-fold decrease in cdk8 affinity , whereas isomer 41 had a potency profile similar to 6 suggesting that a hydrogen - bond - mediated interaction from the carbamate nh of compound 41 with asp173 is required for potency ( table 3 , entry 4 ) . compound 42 ( table 3 , entry 5 ) , which combines the optimal carbamate isomer and an n - methyl sultam , gave very potent cdk8/19 affinity with improved metabolic stability compared to 6 . addition of two methyl groups to block metabolically vulnerable sites in the spirocycle did not lead to improved metabolic stability , and a drop in potency was also observed ( table 3 , entry 6 ) . crystallization of compound 42 ( table 3 , entry 5 ) in cdk8/cyclin c confirmed the carbamate nh hydrogen bond interaction with asp173 and showed interaction of the carbamate carbonyl group with lys52 ( figure 2 ) . overlay of lactam 6 ( brown ) and carbamate 42 ( cyan ) in cdk8/cyclin c , pdb code 5hbe . cognizant of blocking oxidative metabolism on the spirolactam , we prepared additional polar derivatives where the lactam was replaced by imidazolidin-4-one , imidazolidine-2,4-dione , 2-methylimidazol-5-one , and piperazine-2,5-dione ( table 3 , entries 711 ) . pleasingly , all derivatives were significantly more stable in microsomal clearance assays ; however , translation to cell - based potency was eroded for the imidazolidine-2,4-dione , 2-methylimidazole-5-one , and piperazine-2,5-dione derivatives ( table 3 , entries 811 ) and no examples demonstrated significant improvement in in vivo mouse clearance ( table 3 , entries 711 ) . surprisingly , the 3-methylpyrazolone derivative 49 ( table 3 , entry 12 ) led to a 13-fold improvement in potency in the 7df3 assay compared to 23 ( table 3 , entry 2 ) but suffered from poor metabolic stability which was not overcome by exchanging the methyl for a trifluoromethyl group ( table 3 , entry 13 ) . given that the lower dynamic range of our in vitro biochemical assays for cdk8 and cdk19 is approximately 3 nm due to the concentration of enzyme ( 5 and 6.7 nm , respectively ) , it is possible that the exquisite cell - based potency for compound 49 is on - target . introduction of a basic center ( compound 51 , entry 14 ) demonstrated that polarity was tolerated at the piperidine c-4 position , but all analogs were metabolically unstable . unsurprisingly , spiropyrrolidine 52 ( table 3 , entry 15 ) led to a less active compound due to removal of the carbonyl group and loss of interaction with lys52 . as noted above , a dmso - filled pocket was observed in the x - ray crystal structure of 25 ( figure 1 , panel a ) , and we postulated that potency could be regained by targeting this region . addition of a linker on the pyrrolidine nitrogen ( table 3 , entries 1619 ) led to a 4-fold improvement in potency ; however , the metabolic stability of such compounds was poor and , disappointingly , cocrystallization of 54 in cdk8/cyclin c did not demonstrate electron density consistent with binding of the pendant methoxyethyl moiety in the dmso pocket ( figure 3 ) . despite its high in vivo clearance , the spirocarbamate 42 ( table 3 , entry 5 ) was attractive from its in vitro profile and we considered spirocarbamate as a potential replacement for the spirolactam in our further optimization . crystal structure of 54 in cdk8/cyclin c ( panel a , pdb code 5hbh ) . overlay of 25 ( gray ) with 54 ( green ) in cdk8/cyclin c ( panel b ) . next , we investigated variation of the pyridine c-3 position ( table 4 ) . we had previously demonstrated that small lipophilic residues were tolerated at this position consistent with the protein ligand crystal structure of 6 , 25 , and 42 in cdk8/cyclin c where the pyridine c3 substituent is proximal to the lipophilic gatekeeper residue phe97 . 3-fluoro derivatives ( table 4 , entries 14 ) led to a decrease in both biochemical and cell - based potency consistent with a weaker lipophilic interaction of fluorine compared to chlorine with phe97 . pleasingly , the combination of either spirolactam or spirocarbamate with the n - methyl sultam and c-3 fluoro group resulted in acceptable potency and good solubility ; however , both 61 and 63 showed high in vivo clearance in mouse pharmacokinetics ( table 4 , entries 2 and 4 ) . introduction of a c-3 trifluoromethyl group restored potency for spirolactams 70 and 71 ( table 4 , entries 56 ) ; however , they proved metabolically unstable . carbamate 72 , despite being surprisingly less potent than the spirolactam 70 , had an acceptable clint profile and in vivo mouse clearance similar to 6 . however , due to the consistently poor aqueous solubility of these cf3 derivatives , we did not progress them further . the protein ligand crystal structure of 25 in cdk8/cyclin c demonstrates that the c2-h of the pyridine is proximal to the backbone carbonyl of asp98 ( distance of 2.3 , figure 1 ) , an observation consistent with our previously reported crystal structure of 6 in cdk8/cyclin c. we hypothesized that introduction of a hydrogen bond donor at the 2 position of the pyridine could form a favorable interaction with the backbone carbonyl of asp98 . pleasingly , introduction of a 2-amino group ( 85 , table 5 , entry 1 ) maintained potency with acceptable metabolic stability and pharmacokinetics , albeit with poor aqueous solubility . at this stage , we decided to introduce the 2-amino functionality into selected optimal compounds from tables 2 , 3 , and 4 . similar to 85 , all additional 2-aminopyridines ( table 5 , entries 211 ) were significantly more metabolically stable than their corresponding pyridine matched pair . for example , the 2-aminopyridine 87 was at least 5-fold more stable in mouse clint experiments than 25 . the presence of the 2-amino functionality also led to lower mouse in vivo clearance ; for example , 92 ( table 5 , entry 8 , cl = 1.28 l / h / kg ) compared to its pyridine matched pair 61 ( table 4 , entry 2 , cl = 7.9 l / h / kg ) . however , we became concerned that reducing in vitro mouse metabolic clint to < 10 l / min / mg ( table 5 , entry 6 ) did not translate to improved mouse in vivo clearance . thus , in vitro clint did not appear to be a sufficient predictor of in vivo clearance for this series . predicted mouse blood clearance ( parallel tube model ) was estimated from the clint data to take into account the polarity of the compounds and their nonspecific protein binding in in vitro clearance assays ; however , this method also failed to explain the discrepancy between in vitro and in vivo data ( figure 4 ) . in vitro in vivo correlation of clearance for compounds from the pyridine and 2-aminopyridine series . x axis is calculated blood clearance from measured plasma clearance in mouse pk . we hypothesized that the increased in vivo clearance in comparison with the in vitro predicted clearance may be driven by active transport . caco-2 assay data confirmed that most compounds had a high efflux ratio ( er ) ( table s1 ) . however , it was unclear if the lack of correlation between predicted and observed blood clearance ( calculated from measured plasma clearance data ) was due to a p - glycoprotein ( p - gp ) transporter liability ( figure 4 ) . to clarify this matter , we conducted pharmacokinetic evaluation in p - gp knockout ( ko ) mice for a set of compounds ( table 6 ) . we observed lower in vivo clearance in p - gp ko mice for compounds 42 , 48 , and 90 , while clearance remained unchanged for both 6 and 89 that did not exhibit efflux in the caco-2 assay . furthermore , in p - gp ko mice we noted a lower percentage of parent compound cleared unchanged in the feces compared to wild - type mice for all compounds ( table 6 ) . taken together , these data are consistent with the notion that transporter - mediated hepatic uptake and elimination contribute to the overall clearance of some compounds in this series . however , we noted that even in the p - gp ko mice , compounds 48 and 90 , both of which have low clint , demonstrate moderate in vivo clearance and that compound 48 is still observed in the feces of p - gp ko mice , suggesting that additional nonhepatic elimination mechanisms are also involved . to understand the contribution of physicochemical parameters to efflux , we conducted a more detailed analysis of the caco-2 data . cognizant of the potential for variability in caco-2 assay data , we classified compounds into two large data sets , pyridines ( n = 165 ) and aminopyridines ( n = 39 ) , seeking trends to direct our medicinal chemistry design ( figure 5 ) . analysis of physicochemical property trends in the pyridine series showed that hbd 2 is necessary but not sufficient to give er < 3 . the probability of compounds in this series exhibiting a low efflux ratio could be further improved by limiting physicochemical properties within the range log p > 2.5 , tpsa < 80100 , hba 7 with no basic center . analysis of physicochemical properties versus efflux ratio in a madin darby canine kidney epithelial cell line ( mdck ) , transfected to express human p - gp ( mdr - mdck ) , led to broadly similar trends . analysis of caco-2 data in the 2-aminopyridine series indicated that physicochemical properties in the range hbd 3 and tpsa < 100 or hbd 3 and log p > 3 are necessary for low efflux in this chemical series , and a combination of hbd 3 , tpsa < 100 , and log p > 3 is more likely to lead to a low efflux ratio . these observations are consistent with previously published studies demonstrating that a fine balance of physicochemical properties is often required to achieve potency , metabolic stability , and low efflux in a single molecule . cognizant of these analyses , we set out to reduce efflux in both the pyridine and 2-aminopyridine series . in the pyridine series , compound 25 ( table 7 , entry 1 ) has an attractive solubility coupled with low clint and acceptable potency ; however , this compound harbors four hydrogen bond donors with an efflux ratio of > 1200 . alkylation of the 3-aminoindazole nitrogen in 25 significantly reduced caco-2 efflux but to the detriment of metabolic stability and potency ( table 7 , entries 24 ) . methylation of the hydantoin ( table 7 , entry 6 ) also led to a lower efflux ratio compared to the parent compound 45 , again to the detriment of microsomal stability . the potent , metabolically stable , and soluble compound 44 ( table 7 , entry 7 ) bears two hydrogen bond donors and exhibits an efflux ratio of 3.7 . methylation of the n-1 nitrogen of the imidazolidinone ring reduced the efflux ratio ( table 7 , entry 8) ; however , poor microsomal stability was again observed . replacement of the methyl group by a variety of substituents including trifluoroethyl did not improve the microsomal stability ( table 7 , entry 9 ) . as these attempts proved unsuccessful , we turned our attention to the aminopyridine series . in this case log p was calculated using percepta batch , version 2015 ( www.acdlabs.com ) . tpsa was calculated in the program moe ( www.chemcomp.com ) . in the 2-aminopyridine series , the presence of additional hydrogen bond donor functionality by virtue of the 2-amino substituent restricted our freedom to modulate physicochemical properties in the remainder of the molecule . thus , the choice of spirolactam isostere was diminished and we hypothesized that introduction of more lipophilic c5-pyridine substituents may be necessary to stay within our desired physicochemical property range and maximize the chances of low efflux ratios . thus , we introduced an isopropylpyrazole at c-5 in combination with the spirolactam at c-4 of the 2-aminopyridine scaffold . this tactic led to suboptimal human clint and an efflux ratio of 7 ( table 8 , entry 1 ) . we then combined the spirolactam and spirocarbamate with a methylindazole ( table 8 , entries 2 and 3 ) . the spirolactam derivative 109 ( measured log d = 2.5 ) was potent ( cdk8 ic50 = 4.9 0.6 nm , cdk19 ic50 = 2.6 0.4 nm with residence times of 53 and 86 min , respectively , in the reporter displacement assay ) , soluble , and stable with a low efflux ratio and acceptable in vivo mouse pharmacokinetics ( cl = 0.61 l / h / kg , f = 30% , table 9 ) ; however , the carbamate matched pair 110 was less potent and subject to increased efflux . to further improve the metabolic stability , we prepared compound 111 bearing an n - isopropylindazole ; however , this compound was less stable in all species . crystallization of 109 in cdk8/cyclin c ( figure 6 ) demonstrated a binding mode consistent with those previously observed , and pleasingly , a new hydrogen bond interaction between the exocyclic nitrogen of the 2-aminopyridine scaffold and the backbone carbonyl of asp98 was observed ( 3.0 ) . x - ray crystal structure of 109 in cdk8/cyclin c , pdb code 5hbj . compound 109 was further profiled in rat and dog pharmacokinetics ( table 9 ) . moderate clearance was observed in both species ; furthermore , the human pharmacokinetic prediction for 109 was significantly better than for compound 6 ( tables 9 and 1 , respectively , 31% of liver blood flow for 109 compared to 76% for 6 ) . pleasingly , compound 109 had acceptable aqueous solubility ( table 8 , entry 2 ) and demonstrated minimal activity when tested in a panel of 55 receptors , ion channels , and enzymes at 1 m ( tables s2 and s3 ) and in a panel of 279 kinases ( table s4 ) ; weak inhibition of cyps was observed ( table s5 ) . consistent with the profile of chemical probe 6 , compound 109 demonstrated potent inhibition of reporter - based readouts measuring basal wnt pathway activity in human cancer cell lines that have constitutively activated wnt pathway signaling : ls174 t ( -catenin mutant ) , sw480 and colo205 ( apc mutant ) or pa-1 human teratocarcinoma cells that are wnt ligand dependent ( table 10 ) compound 109 was then assessed in the in vivo apc - mutant sw620 human colorectal carcinoma xenograft model , treating established tumors in female ncr athymic mice . mice were treated orally ( 30 mg / kg q.d . ) for 15 days ; a 54.2% reduction in tumor weight was observed at day 15 ( figure 7 , panels a and b ) . we monitored inhibition of stat1 phosphorylation , which we have previously demonstrated to be an in vitro cell - based and in vivo pharmacodynamic biomarker of cdk8 inhibition . reduced stat1 phosphorylation was maintained for more than 6 h after the last dose ( figure s2 ) consistent with measured free plasma and tumor exposures that remained above cdk8 ic50 ( figure 7 , panel c , figure s3 , and table s6 ) . in light of its potent and selective profile coupled with good oral pharmacokinetics and duration of in vivo target engagement on oral dosing , compound 109 ( cct251921 ) has been selected for progression into further preclinical in vivo efficacy and safety studies . reduction of ( a ) tumor volume and ( b ) tumor weight versus vehicle - treated controls after chronic oral dosing ( 30 mg / kg q.d . ) of 109 to an apc - mutant sw620 human colorectal carcinoma xenograft animal model . ( c ) free plasma and free tumor exposure ( nm ) at 1 , 2 , 6 , and 24 h after the last dose in the same experiment . literature evidence and our own studies point to a role for the mediator complex - associated kinases cdk8 and cdk19 in human disease , particularly in colorectal cancer where cdk8 has been reported as a putative oncogene . cdk19 , a paralogue of cdk8 , is relatively unexplored , and our previous studies demonstrate that selectivity for cdk8 over cdk19 with a small molecule ligand is likely to be challenging . indeed , in our recently reported discovery of cct251545 ( 6 ) , a potent and selective chemical probe for the further exploration of cdk8 and cdk19 pharmacology , we show strong correlation of cdk8 and cdk19 binding affinity . here , we have further optimized the small molecule cdk8/19-selective chemical probe 6 to give compound 109 by improving oral pharmacokinetics and pharmaceutical properties in order to facilitate further in vivo evaluation of cdk8/19 pharmacology and progression into preclinical in vivo efficacy and safety studies . chemical probe 6 , although a high affinity ligand for cdk8 and cdk19 , displays moderate in vivo clearance in preclinical species resulting in a clearance prediction to man that may preclude consistent target engagement for extended periods of time ; in addition , aqueous solubility was suboptimal and we anticipated that this may limit the maximum absorbable dose . in attempting to reduce oxidative metabolism by reducing lipophilicity while maintaining key interactions within the cdk8 kinase domain , we benefited from detailed knowledge of the binding mode of 6 and analogs through protein we were able to improve in vitro metabolic stability by introducing a c-2 amino substituent to the pyridine scaffold which reinforced interactions with the kinase hinge region and reduced compound lipophilicity . however , we identified transporter - mediated hepatic uptake as a component of in vivo clearance . in particular , we noted that a significant number of compounds in both the pyridine and 2-aminopyridine series exhibited higher measured in vivo clearance than predicted by experimental in vitro clearance assessment . through careful correlation of in vitro caco-2 efflux ratios with physicochemical properties and subsequent medicinal chemistry design within desirable physicochemical property ranges , we were able to identify compounds that demonstrated reduced clearance without increased susceptibility to active hepatic uptake . compound 109 was identified as the best compromise of in vitro biochemical and pharmacokinetic properties that demonstrated acceptable in vivo pharmacokinetics suitable for progression to in vivo animal models of cancer . commercially available starting materials , reagents , and dry solvents were used as supplied . column chromatography was performed on a biotage sp1 purification system using thomson or biotage flash silica cartridges or on a companion purification system using interchim silica cartridges . ion exchange chromatography was performed using acidic isolute flash scx - ii columns or basic isolute flash nh2 columns . a , injections of the sample were made onto a sunfire c18 obd column ( 100 , 5 m , 30 mm 100 mm ) . chromatographic separation at room temperature was carried out using agilent tehnologies , 1260 infinity , acetonitrile / water gradient ( both modified with 0.1% formic acid ) at a flow rate of 50 ml / min . for method b , injections of the sample were made onto a phenomenex gemini column ( 10 m , 250 mm 21.2 mm , c18 , phenomenex , torrance , ca , usa ) . chromatographic separation at room temperature was carried out using gilson gx-281 liquid handler system combined with a gilson 322 hplc pump ( gilson , middleton , wi , usa ) over a 15 min gradient elution from 40:60 to 100:0 meoh / water ( both modified with 0.1% formic acid ) at a flow rate of 20 ml / min . h nmr spectra were recorded on a bruker avance 500 , bruker avance 400 , or avance ii 400 . samples were prepared as solutions in a deuterated solvent and referenced to the appropriate internal nondeuterated solvent peak . the following internal references were used : cdcl3 ( c 77.2 ) , cd3od ( c 49.0 ) , and dmso - d6 ( c 39.5 ) ; unobserved resonances for quaternary carbon atoms are denoted by lc / ms and hrms analyses were performed on an agilent 1200 series hplc and diode array detector coupled to a 6210 time - of - flight mass spectrometer with dual multimode apci / esi source . a , analytical separation was carried out on a chromolith speed rod column ( rp-18e , 50 mm 4.6 mm ) using a flow rate of 2.4 ml / min in a 3.9 min gradient elution with detection at 220 nm . the mobile phase was a mixture of water containing 0.05% formic acid ( solvent a ) and acetonitrile containing 0.04% formic acid ( solvent b ) . gradient elution was as follows : 95:5 ( a / b ) to 0:100 ( a / b ) over 2.8 min , 0:100 ( a / b ) for 0.5 min , and then reversion back to 95:5 ( a / b ) over 0.1 min , finally 95:5 ( a / b ) for 0.5 min . for method b , analytical separation was carried out on a chromolith performance column ( rp-18e , 100 mm 3 mm ) using a flow rate of 2.0 ml / min in a 4.8 min gradient elution with detection at 220 nm . the mobile phase was a mixture of water ( solvent a ) and acetonitrile ( solvent b ) both containing 0.1% tfa . gradient elution was as follows : 99:1 ( a / b ) over 0.2 min , then 99:1 to 0:100 ( a / b ) over 3.6 min , 0:100 ( a / b ) for 0.4 min , and then reversion back to 99:1 ( a / b ) over 0.1 min and finally 99:1 ( a / b ) for 0.5 min . for method c , analytical separation was carried out at 30 c on a merck purospher star column ( rp-18e , 30 mm 4 mm ) using a flow rate of 1.5 ml / min in a 4 min gradient elution with detection at 254 nm . the mobile phase was a mixture of meoh ( solvent a ) and water ( solvent b ) , both containing 0.1% formic acid . gradient elution was as follows : 1:9 ( a / b ) to 9:1 ( a / b ) over 2.5 min , 9:1 ( a / b ) for 1 min , and then reversion back to 1:9 ( a / b ) over 0.3 min , finally 1:9 ( a / b ) for 0.2 min . for method d , analytical separation was carried out at 30 c on a merck purospher star column ( rp-18e , 30 mm 4 mm ) using a flow rate of 1.5 ml / min in a 4 min gradient elution with detection at 220 nm . the mobile phase was a mixture of meoh ( solvent a ) and water ( solvent b ) , both containing 0.1% formic acid . gradient elution was as follows : 1:9 ( a / b ) to 9:1 ( a / b ) over 2.5 min , 9:1 ( a / b ) for 1 min , and then reversion back to 1:9 ( a / b ) over 0.3 min , finally 1:9 ( a / b ) for 0.2 min . for method e , analytical separation was carried out on xbridge c8 column ( 50 mm 4.6 mm , 3.5 m ) using a flow rate of 2.0 ml / min in a 10 min gradient elution with detection at 254 nm . the mobile phase was a mixture of acetonitrile ( solvent a ) and water ( solvent b ) , both containing 0.1% tfa . gradient elution was as follows : 5:95 ( a / b ) to 100:0 ( a / b ) over 8 min , 100:0 ( a / b ) for 0.1 min , and then reversion back to 5:95 ( a / b ) over 0.4 min , finally 5:95 ( a / b ) for 1.5 min . the following reference masses were used for hrms analysis : caffeine [ m + h ] 195.087 652 ; ( hexakis(1h,1h,3h - tetrafluoropentoxy)phosphazene [ m + h ] 922.009 798 ) and hexakis(2,2-difluoroethoxy)phosphazene [ m + h ] 622.028 96 or reserpine all compounds submitted for biological testing were determined to be > 95% pure by method a , b , c , d , or e unless stated otherwise . 2-chlorobenzylsulfonyl chloride ( 1.86 g , 8.26 mmol ) was dissolved in acetone ( 27 ml ) , and then ammonium hydroxide ( 18 ml ) was added . the reaction mixture was stirred for 2.5 h at rt , and the solvent was evaporated . the two layers were separated , and the aqueous layer was extracted with etoac . the crude product was purified by biotage column chromatography ( cyclohexane / acetone 90:10 to 60:40 ) to afford ( 2-chlorophenyl)methanesulfonamide as a white solid ( 1.6 g , 94% yield ) . h nmr ( 500 mhz , cdcl3 ) 7.567.53 ( m , 1h ) , 7.477.44 ( m , 1h ) , 7.367.30 ( m , 2h ) , 4.66 ( bs , 2h ) , 4.57 ( s , 2h ) ; lc ms ( method d , esi , m / z ) tr = 1.77 min , parent does not ionize . ( 2-chlorophenyl)methanesulfonamide ( 450 mg , 2.19 mmol ) , tris(dibenzylideneacetone)dipalladium ( 100 mg , 0.109 mmol ) , 2-di - tert - butylphosphino-2,4,6-triisopropylbiphenyl ( 186 mg , 0.438 mmol ) , and potassium carbonate ( 605 mg , 4.38 mmol ) were loaded in a microwave vial , and thf ( 8.8 ml ) was added . the reaction mixture was stirred at 80 c in an oil bath for 13 h before being quenched with a sat . the solvent was then evaporated and the residue was purified by biotage column chromatography ( cyclohexane / acetone 95:5 to 60:40 ) to afford 1,3-dihydrobenzo[c]isothiazole 2,2-dioxide as a white solid ( 296 mg , 80% yield ) . h nmr ( 500 mhz , cdcl3 ) 7.317.26 ( m , 1h ) , 7.267.23 ( m , 1h ) , 7.07 ( td , j = 7.6 , 0.9 hz , 1h ) , 6.90 ( d , j = 8.0 hz , 1h ) , 6.48 ( bs , 1h ) , 4.39 ( s , 2h ) ; lc ms ( method d , esi , m / z ) tr = 1.69 min , parent does not ionize . to a suspension of 1,3-dihydrobenzo[c]isothiazole 2,2-dioxide ( 280 mg , 1.66 mmol ) and potassium carbonate ( 229 mg , 1.66 mmol ) in dmf ( 5 ml ) was added iodomethane ( 414 l , 6.62 mmol ) . the reaction mixture was stirred for 6 h at rt and was then quenched with a sat . the reaction mixture was concentrated and purified by biotage column chromatography ( cyclohexane / acetone 90:10 to 70:30 ) to afford 1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide as a white solid ( 270 mg , 89% yield ) . h nmr ( 500 mhz , cdcl3 ) 7.377.32 ( m , 1h ) , 7.277.24 ( m , 1h ) , 7.02 ( td , j = 7.6 , 1.0 hz , 1h ) , 6.73 ( d , j = 8.0 hz , 1h ) , 4.34 ( s , 2h ) , 3.14 ( s , 3h ) ; lc ms ( method d , esi , m / z ) tr = 2.07 min , parent does not ionize . 1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide ( 272 mg , 1.49 mmol ) was dissolved in dmf ( 1.5 ml ) , and n - bromosuccinimide ( 264 mg , 1.49 mmol ) was added . the reaction mixture was stirred at rt for 4 h. after addition of water , the reaction mixture was concentrated . the residue was purified by biotage column chromatography ( cyclohexane / acetone 90:10 to 70:30 ) to afford 5-bromo-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide as a white solid ( 330 mg , 85% yield ) . h nmr ( 500 mhz , cdcl3 ) 7.457.41 ( m , 1h ) , 7.377.35 ( m , 1h ) , 6.59 ( d , j = 8.5 hz , 1h ) , 4.30 ( s , 2h ) , 3.09 ( s , 3h ) ; lc ms ( method c , esi , m / z ) tr = 2.46 min , parent does not ionize . 5-bromo-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide ( 267 mg , 1.02 mmol ) , bis(pinacolato)diboron ( 388 mg , 1.53 mmol ) , potassium acetate ( 300 mg , 3.06 mmol ) , and pd(dppf)cl2ch2cl2 ( 42 mg , 0.051 mmol ) were loaded in a microwave vial , and dme ( 7.4 ml ) was added . the reaction was concentrated and purified by biotage column chromatography ( cyclohexane / acetone 97:3 to 85:15 ) to afford 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 as a white solid ( 290 mg , 92% yield ) . h nmr ( 500 mhz , cdcl3 ) 7.807.77 ( m , 1h ) , 7.697.67 ( m , 1h ) , 6.71 ( d , j = 8.0 hz , 1h ) , 4.32 ( s , 2h ) , 3.15 ( s , 3h ) , 1.33 ( s , 12h ) ; lc ms ( method c , esi , m / z ) tr = 2.82 min , 309/310 ( m + h ) . 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 58 mg , 0.19 mmol ) , 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one8 ( 54 mg , 0.16 mmol ) , and pd(dppf)cl2ch2cl2 ( 6.4 mg , 7.9 mol ) were loaded in a microwave vial , and then 0.5 m sodium carbonate in water ( 440 l , 0.220 mmol ) and acetonitrile ( 2.8 ml ) were added . the solvent was evaporated and the crude material was purified by biotage column chromatography ( dcm / etoh 99:1 to 85:15 ) to give 8-(3-chloro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 23 as a white solid ( 57 mg , 81% yield ) . h nmr ( 500 mhz , cdcl3 ) 8.42 ( s , 1h ) , 8.17 ( s , 1h ) , 7.297.24 ( m , 2h ) , 6.81 ( d , j = 8.1 hz , 1h ) , 6.36 ( s , 1h ) , 4.48 ( s , 2h ) , 3.31 ( t , j = 6.8 hz , 2h ) , 3.18 ( s , 3h ) , 3.163.10 ( m , 2h ) , 2.81 ( t , j = 11.2 hz , 2h ) , 1.98 ( t , j = 6.8 hz , 2h ) , 1.911.80 ( m , 2h ) , 1.391.33 ( m , 2h ) ; c nmr ( 126 mhz , cdcl3 ) 181.6 , 153.0 , 150.4 , 149.9 , 141.5 , 133.2 , 131.0 , 130.3 , 128.3 , 126.6 , 118.0 , 109.1 , 50.8 , 47.7 , 41.6 , 38.8 , 32.4 , 32.2 , 26.6 ; lc ms ( method c , esi , m / z ) tr = 2.00 min , 447/449 ( m + h ) ; esi - hrms calcd for c21h24cln4o3s ( m + h ) 447.1252 , found 447.1247 . in a screw - capped vessel , 6-bromo-1h - indazol-3-amine ( 95% purity , 500 mg , 2.36 mmol ) and 4-(dimethylamino)pyridine ( 58 mg , 0.47 mmol ) were dissolved in thf ( 10 ml ) . di - tert - butyl dicarbonate ( 2.52 ml , 11.8 mmol ) and triethylamine ( 3.27 ml , 23.6 mmol ) were added , and the reaction solution was stirred for 3 days at rt . the reaction mixture was diluted with water ( 100 ml ) and the aqueous layer extracted with etoac . the organic layer was washed with water , dried , filtered , and evaporated to dryness to give tert - butyl 3-[bis(tert - butoxycarbonyl)amino]-6-bromoindazole-1-carboxylate ( 1.42 g , 73% pure , 86% corrected yield ) as a colorless oil , which was used without further purification . lc ms ( method b , esi , m / z ) tr = 3.91 min , 534/536 ( m + na ) . in a screw - capped vessel , tert - butyl 3-[bis(tert - butoxycarbonyl)amino]-6-bromoindazole-1-carboxylate ( 73% pure , 1.34 g , 1.91 mmol ) was dissolved in thf ( 16 ml ) . bis(pinacolato)diboron ( 486 mg , 1.91 mmol ) , potassium acetate ( 375 mg , 3.83 mmol ) , and pd(dppf)cl2ch2cl2 ( 78 mg , 0.096 mmol ) were added , and the red reaction mixture was stirred for 15 h at 70 c . further bis(pinacolato)diboron ( 486 mg , 1.91 mmol ) , potassium acetate ( 130 mg , 1.33 mmol ) , and pd(dppf)cl2ch2cl2 ( 78 mg , 0.096 mmol ) were added , and stirring was continued at 70 c for additional 4 h. the black reaction mixture was treated with etoac , filtered , and evaporated to dryness under reduced pressure . the dark brown residue was purified by flash chromatography ( heptane / dcm , gradient ) to give tert - butyl 3-[bis(tert - butoxycarbonyl)amino]-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole-1-carboxylate ( 1.00 g , 90% pure , 84% corrected yield ) as a yellow solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 3.94 min , 559/560 ( m + h ) . tert - butyl 3-[bis(tert - butoxycarbonyl)amino]-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole-1-carboxylate ( 90% pure , 1.00 g , 1.61 mmol ) was treated with hcl in dioxane ( 25 ml ) . the pale yellow solution was stirred at rt for 15 h. the solution was evaporated to dryness and the residue was treated with diethyl ether to obtain a beige solid . the mixture was filtered and the residue was washed with diethyl ether to afford ( 3-amino-1h - indazol-6-yl)boronic acid hydrochloride 16 ( 351 mg , 95% pure , 97% yield ) as a beige solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 1.34 min , 177/178 ( m + h ) . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one8 ( 92% pure , 180 mg , 0.480 mmol ) and ( 3-amino-1h - indazol-6-yl)boronic acid hydrochloride 16 ( 95% pure , 162 mg , 0.720 mmol ) were dissolved in acetonitrile ( 8 ml ) , and 0.5 m sodium carbonate in water ( 2.8 ml , 1.4 mmol ) and pd(dppf)cl2ch2cl2 ( 20 mg , 0.020 mmol ) were added . the reaction mixture was stirred for 1 h at 120 c under microwave irradiation , diluted with acetonitrile ( 10 ml ) , filtered and the filtrate evaporated to dryness . the residue was purified by preparative hplc ( method a , 20 min gradient elution from 2:98 to 20:80 acetonitrile / water ) . the pure fractions were combined and evaporated down to a volume of 20 ml . the solution was neutralized with solid nahco3 and extracted with etoac ( 3 30 ml ) . the residue was suspended in diethyl ether and filtered under vacuum to afford 8-(3-(3-amino-1h - indazol-6-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 25 ( 85 mg , 45% yield ) as a colorless solid . h nmr ( 500 mhz , cdcl3 ) 8.45 ( s , 1h ) , 8.25 ( s , 1h ) , 7.63 ( dd , j = 8.2 , 0.9 hz , 1h ) , 7.29 ( s , 1h ) , 6.97 ( dd , j = 8.2 , 1.4 hz , 1h ) , 5.66 ( s , 1h ) , 3.26 ( t , j = 6.8 hz , 2h ) , 3.213.13 ( m , 2h ) , 2.74 ( t , j = 11.7 hz , 2h ) , 1.971.87 ( m , 4h ) , 1.371.29 ( m , 2h ) ; c nmr ( 126 mhz , cdcl3 ) 181.4 , 153.2 , 150.9 , 149.9 , 148.9 , 142.5 , 137.1 , 134.0 , 128.2 , 121.6 , 119.6 , 114.2 , 110.3 , 47.7 , 41.5 , 38.7 , 32.5 , 32.2 ; lc ms ( method c , esi , m / z ) tr = 1.58 min , 397/399 ( m + h ) ; esi - hrms calcd for c20h22cln6o ( m + h ) 397.1538 , found 397.1522 . 4-aminomethyl-1-benzylpiperidin-4-ol ( 25.0 g , 113 mmol ) was suspended in dcm ( 400 ml ) , and a solution of triphosgene ( 32.8 g , 110 mmol ) dissolved in dcm ( 200 ml ) was added dropwise while maintaining the temperature between 30 and 35 c . to the reaction mixture 1 n naoh solution ( 200 ml ) was added , and the organic phase was separated , washed with water , dried over sodium sulfate , filtered , and evaporated to dryness . the residue was triturated with diethyl ether , the solid obtained was washed with diethyl ether and dried in vacuo to give 13.2 g ( 82% pure , 39% corrected yield ) of 8-benzyl-1-oxa-3,8-diazaspiro[4.5]decan-2-one as a yellow solid which was used in the next step without further purification . lc ms ( method b , esi , m / z ) tr = 1.49 min , 247 ( m + h ) . 8-benzyl-1-oxa-3,8-diazaspiro[4.5]decan-2-one ( 82% pure , 13.2 g , 43.9 mmol ) was dissolved in meoh ( 60 ml ) and thf ( 60 ml ) . pd / c 5% ( 54.1% h2o , 3.00 g ) was added , and the reaction mixture was stirred at rt under hydrogen for 18 h. since the reaction was incomplete , additional pd / c 5% ( 54.1% h2o , 6.00 g ) was added and the reaction mixture was stirred at rt for another 18 h. another portion of palladium was added and the reaction was stirred for 18 h. the catalyst was then filtered off and the filtrate was evaporated to dryness . the residue was triturated with diethyl ether , filtered , and dried in vacuum to yield in 5.39 g ( 78% yield ) of 1-oxa-3,8-diazaspiro[4.5]decan-2-one 38 as a brown solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 0.39 min , 157 ( m + h ) . in a microwave vial 3-bromo-4,5-dichloropyridine7 ( 2.00 g , 8.80 mmol ) was dissolved in nmp ( 15 ml ) . 1-oxa-3,8-diazaspiro[4.5]decan-2-one 38 ( 1.65 g , 10.6 mmol ) and triethylamine ( 2.44 ml , 17.6 mmol ) were added . the beige precipitate was filtered and washed with water and diethyl ether to give 8-(3-bromo-5-chloropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 27 ( 2.34 g , 77% yield ) as a beige solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 2.01 min , 346/348/350 ( m + h ) . 8-(3-bromo-5-chloropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 27 ( 2.54 g , 7.33 mmol ) was suspended in acetonitrile ( 60 ml ) . 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 2.72 g , 8.79 mmol ) , 0.5 m sodium carbonate in water ( 29.3 ml , 14.6 mmol ) , and ( pd(dppf)cl2ch2cl2 complex ( 299 mg , 0.366 mmol ) were added . the reaction mixture was stirred at 80 c for 3 h. after addition of etoac and water , the organic layer was separated and washed with water , dried over mgso4 , filtered , and evaporated to dryness . the crude material was purified by flash chromatography ( companion , dcm / meoh 100:0 to 90:10 ) to yield in 1.61 g ( 49% yield ) of 8-(3-chloro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 42 as a colorless solid . h nmr ( 500 mhz , dmso - d6 ) 8.46 ( s , 1h ) , 8.17 ( s , 1h ) , 7.50 ( s , 1h ) , 7.37 ( s , 1h ) , 7.32 ( dd , j = 8.2 , 1.9 hz , 1h ) , 7.08 ( d , j = 8.2 hz , 1h ) , 4.72 ( s , 2h ) , 3.20 ( s , 2h ) , 3.10 ( s , 3h ) , 2.962.82 ( m , 4h ) , 1.771.66 ( m , 4h ) ; c nmr ( 126 mhz , dmso - d6 ) 157.7 , 152.1 , 150.6 , 148.8 , 141.2 , 133.2 , 130.2 , 130.0 , 127.3 , 126.0 , 118.7 , 109.5 , 78.7 , 50.1 , 50.0 , 47.1 , 35.8 , 26.1 ; lc ms ( method c , esi , m / z ) tr = 1.87 min , 449/451 ( m + h ) ; esi - hrms calcd for c20h22cln4o4s ( m + h ) 449.1045 , found 449.1032 . to a suspension of 8-boc-1,8-diazaspiro[4.5]decane oxalate ( 202 mg , 0.839 mmol ) in acetonitrile ( 16.8 ml ) were added potassium carbonate ( 348 mg , 2.52 mmol ) , potassium iodide ( 139 mg , 0.839 mmol ) , and 2-bromoethyl methyl ether ( 95 l , 1.0 mmol ) . the filtrate was concentrated and the crude was purified by biotage column chromatography ( eluting with 18% meoh / aq nh3 ( 10:1 ) in dcm ) to give tert - butyl 1-(2-methoxyethyl)-1,8-diazaspiro[4.5]decane-8-carboxylate ( 160 mg , 64% yield ) as a colorless oil . h nmr ( 500 mhz , cd3od ) 4.11 ( br d , j = 14.2 hz , 2h ) , 3.53 ( t , j = 5.8 hz , 2h ) , 3.36 ( s , 3h ) , 3.052.67 ( m , 6h ) , 1.89 ( s , 4h ) , 1.671.57 ( m , 2h ) , 1.47 ( s , 9h ) , 1.441.37 ( m , 2h ) ; lc ms ( method c , esi , m / z ) tr = 1.75 min , 299 ( m + h ) . 3-bromo-4,5-dichloropyridine7 ( 70 mg , 0.31 mmol ) and tert - butyl 1-(2-methoxyethyl)-1,8-diazaspiro[4.5]decane-8-carboxylate ( 120 mg , 0.402 mmol ) were introduced in a microwave vial , and then 1-methoxy-2-propanol ( 773 l ) and triethylamine ( 130 l , 0.928 mmol ) were added . the reaction mixture was stirred for 2 h at 220 c under microwave irradiation . the solvent was evaporated and the crude material was purified by biotage column chromatography ( eluting with 25% meoh / aq nh3 ( 10:1 ) in dcm ) to give 8-(3-bromo-5-chloropyridin-4-yl)-1-(2-methoxyethyl)-1,8-diazaspiro[4.5]decane 36 ( 86 mg , 72% yield ) as a colorless oil . h nmr ( 500 mhz , cd3od ) 8.49 ( s , 1h ) , 8.36 ( s , 1h ) , 3.53 ( t , j = 6.0 hz , 2h ) , 3.45 ( td , j = 12.6 , 2.3 hz , 2h ) , 3.37 ( s , 3h ) , 3.343.28 ( m , 2h ) , 2.91 ( t , j = 6.6 hz , 2h ) , 2.76 ( t , j = 6.0 hz , 2h ) , 2.001.85 ( m , 6h ) , 1.461.40 ( m , 2h ) ; lc ms ( method c , esi , m / z ) tr = 1.72 min , 388/390/392 ( m + h ) . 8-(3-bromo-5-chloropyridin-4-yl)-1-(2-methoxyethyl)-1,8-diazaspiro[4.5]decane 36 ( 27 mg , 0.069 mmol ) , 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 21 mg , 0.069 mmol ) , and pd(dppf)cl2ch2cl2 ( 2.8 mg , 3.5 mol ) were loaded in a microwave vial , and then acetonitrile ( 1.2 ml ) and 0.5 m sodium carbonate in water ( 194 l , 0.0970 mmol ) were added . the solvents were evaporated and the crude material was purified by biotage column chromatography ( eluting with 15% meoh / aq nh3 ( 10:1 ) in dcm ) and further purified by scx-2 column chromatography ( loading with dcm : meoh , elution with 1 n nh3 in meoh ) to afford 5-(5-chloro-4-(1-(2-methoxyethyl)-1,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 54 ( 14 mg , 41% yield ) as a colorless oil . h nmr ( 500 mhz , acetone - d6 ) 8.38 ( s , 1h ) , 8.17 ( s , 1h ) , 7.45 ( s , 1h ) , 7.407.37 ( m , 1h ) , 7.05 ( d , j = 8.2 hz , 1h ) , 4.59 ( s , 2h ) , 3.37 ( t , j = 6.5 hz , 2h ) , 3.27 ( s , 3h ) , 3.16 ( s , 3h ) , 3.103.05 ( m , 2h ) , 2.882.73 ( m , 4h ) , 2.60 ( t , j = 6.5 hz , 2h ) , 1.731.59 ( m , 6h ) , 1.161.09 ( m , 2h ) ; c nmr ( 126 mhz , cd3od ) 153.8 , 149.6 , 148.6 , 141.8 , 134.2 , 130.6 , 130.3 , 128.0 , 126.2 , 118.5 , 109.0 , 71.5 , 57.6 , 50.8 , 49.1 , 47.2 , 32.3 , 31.0 , 25.1 , 20.0 ( cq and ch2 of the sultam not observed ) ; lc ms ( method c , esi , m / z ) tr = 1.65 min , 491/493 ( m + h ) ; esi - hrms calcd for c24h32cln4o3s ( m + h ) 491.1878 , found 491.1878 . 3-bromo-4-chloro-5-fluoropyridine 57 ( 300 mg , 1.43 mmol ) and tert - butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate ( 471 mg , 1.85 mmol ) were introduced in a microwave vial , and 1-methoxy-2-propanol ( 3.5 ml ) and triethylamine ( 601 l , 4.28 mmol ) were added . the reaction mixture was stirred for 1 h at 220 c under microwave irradiation . the solvent was evaporated and the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 92:8 ) to afford 8-(3-bromo-5-fluoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 58 ( 140 mg , 30% yield ) as a white solid . h nmr ( 500 mhz , cdcl3 ) 8.39 ( s , 1h ) , 8.22 ( d , j = 3.6 hz , 1h ) , 6.86 ( s , 1h ) , 3.513.43 ( m , 2h ) , 3.403.35 ( m , 2h ) , 3.243.16 ( m , 2h ) , 2.162.07 ( m , 4h ) , 1.571.50 ( m , 2h ) ; f nmr ( 500 mhz , cdcl3 ) 135 ; lc ms ( method c , esi , m / z ) tr = 2.46 min , 328/330 ( m + h ) . 8-(3-bromo-5-fluoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 58 ( 40 mg , 0.12 mmol ) , 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 49 mg , 0.16 mmol ) , and pd(dppf)cl2ch2cl2 ( 4.98 mg , 6.09 mol ) were loaded in a microwave vial , and acetonitrile ( 2.1 ml ) and 0.5 m sodium carbonate in water ( 341 l , 0.171 mmol ) were added . after evaporation of the solvents , the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 95:5 ) and by scx-2 column chromatography ( loading with dcm / meoh , elution with 1 n nh3 in meoh ) to give 8-(3-fluoro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 61 ( 33 mg , 63% yield ) as a cream solid . h nmr ( 500 mhz , cdcl3 ) 8.26 ( d , j = 4.2 hz , 1h ) , 8.09 ( s , 1h ) , 7.47 ( dd , j = 8.2 , 1.7 hz , 1h ) , 7.44 ( d , j = 1.7 hz , 1h ) , 6.83 ( d , j = 8.2 hz , 1h ) , 5.78 ( s , 1h ) , 4.46 ( s , 2h ) , 3.32 ( t , j = 6.9 hz , 2h ) , 3.263.19 ( m , 2h ) , 3.18 ( s , 3h ) , 3.032.94 ( m , 2h ) , 2.05 ( t , j = 6.8 hz , 2h ) , 1.81 ( ddd , j = 13.4 , 11.3 , 4.2 hz , 2h ) , 1.361.30 ( m , 2h ) ; f nmr ( 500 mhz , cdcl3 ) 139 ; c nmr ( 126 mhz , cdcl3 ) 181.2 , 154.6 ( d , j = 253.3 hz ) , 147.5 ( d , j = 3.9 hz ) , 143.8 , 141.6 , 138.1 ( d , j = 25.6 hz ) , 131.3 , 130.3 , 130.1 , 126.1 , 118.1 , 109.4 , 50.9 , 47.5 ( d , j = 5.0 hz ) , 41.5 , 38.7 , 32.3 , 31.8 , 26.6 ; lc ms ( method c , esi , m / z ) tr = 1.69 min , 431 ( m + h ) ; esi - hrms calcd for c21h24fn4o3s ( m + h ) 431.1548 , found 431.1590 . 8-benzyl-1-oxa-3,8-diaza - spiro[4.5]decan-2-one ( 7.40 g , 98% pure ) was dissolved in meoh ( 34 ml ) , thf ( 17 ml ) , and acetic acid ( 8 ml ) . pd / c 5% ( 54.1% h2o , 4.00 g ) was added , and the reaction mixture was stirred at rt under hydrogen for 16 h. the catalyst was then filtered off and the filtrate was evaporated to dryness . the residue was triturated with diethyl ether / diethyl ether , filtered and dried in vacuum to yield 6.16 g ( 97% yield ) of 1-oxa-3,8-diazaspiro[4.5]decan-2-one acetate 38 as a white solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 0.39 min , 157 ( m + h ) . 3-bromo-4-chloro-5-fluoropyridine 57 ( 656 mg , 3.12 mmol ) and 1-oxa-3,8-diazaspiro[4.5]decan-2-one acetate 38 ( 1.34 g , 6.23 mmol ) were introduced in a microwave vial , and 1-methoxy-2-propanol ( 7.8 ml ) and triethylamine ( 1.34 ml , 9.35 mmol ) were added , and the reaction mixture was stirred at 220 c for 1 h under microwave irradiation . the solvent was evaporated and the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 95:5 ) to afford 8-(3-bromo-5-fluoropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 59 ( 380 mg , 37% yield ) as a cream solid . h nmr ( 500 mhz , cdcl3 ) 8.42 ( s , 1h ) , 8.25 ( d , j = 3.4 hz , 1h ) , 6.20 ( s , 1h ) , 3.583.50 ( m , 2h ) , 3.42 ( s , 2h ) , 3.313.24 ( m , 2h ) , 2.122.06 ( m , 2h ) , 1.991.92 ( m , 2h ) ; f nmr ( 500 mhz , cdcl3 ) 135 ; lc ms ( method c , esi , m / z ) tr = 2.32 min , 330/332 ( m + h ) . 8-(3-bromo-5-fluoropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 59 ( 50 mg , 0.15 mmol ) , 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 52 mg , 0.17 mmol ) , and pd(dppf)cl2ch2cl2 ( 6.2 mg , 7.6 mol ) were loaded in a microwave vial , and acetonitrile ( 2.6 ml ) and 0.5 m sodium carbonate in water ( 424 l , 0.212 mmol ) were added . after evaporation of the solvent , the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 95:5 ) and by scx-2 column chromatography ( loading with dcm / meoh , elution with 1 n nh3 in meoh ) to afford 8-(3-fluoro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 63 ( 44 mg , 67% yield ) as white solid . h nmr ( 500 mhz , cdcl3 ) 8.26 ( d , j = 4.1 hz , 1h ) , 8.08 ( s , 1h ) , 7.39 ( dd , j = 8.2 , 1.8 hz , 1h ) , 7.30 ( s , 1h ) , 6.81 ( d , j = 8.2 hz , 1h ) , 5.78 ( s , 1h ) , 4.40 ( s , 2h ) , 3.32 ( s , 2h ) , 3.283.16 ( m , 5h ) , 3.063.03 ( m , 2h ) , 1.921.81 ( m , 2h ) , 1.721.64 ( m , 2h ) ; f nmr ( 500 mhz , cdcl3 ) 139 ; c nmr ( 126 mhz , cdcl3 ) 158.5 , 154.4 ( d , j = 253.0 hz ) , 147.9 ( d , j = 4.0 hz ) , 143.1 ( d , j = 7.4 hz ) , 141.5 , 138.0 ( d , j = 25.5 hz ) , 131.0 , 130.3 , 130.2 , 125.6 , 117.9 , 109.3 , 79.9 , 51.3 , 50.7 , 47.0 ( d , j = 4.6 hz ) , 36.3 , 26.5 ; lc ms ( method c , esi , m / z ) tr = 1.70 min , 433 ( m + h ) ; esi - hrms calcd for c20h22fn4o4s ( m + h ) 431.1340 , found 433.1339 . in a 3 l three - necked round - bottom flask , 1,4-dihydropyridin-4-one ( 95% pure , 50 g , 0.50 mol ) and n - iodosuccinimide ( 97% pure , 232 g , 1.00 mmol ) were suspended in acetonitrile ( 1 l ) . the reaction mixture was heated under reflux for 3 h and then cooled with an ice bath , filtered and the filtrate washed with acetonitrile ( 150 ml ) . the light yellow solid was dried at 60 c under reduced pressure for 15 h to obtain 3,5-diiodopyridin-4-ol ( 165 g , 95% yield ) as a light yellow solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 1.34 min , 347 ( m + h ) . in a 3 l three - necked round - bottom flask , 3,5-diiodopyridin-4-ol ( 150 g , 432 mmol ) was suspended in dmf ( 1 l ) . this mixture was warmed to 70 c , and phosphoryl chloride ( 39.7 ml , 432 mmol ) was slowly added in a dropwise manner ( caution : slightly exothermic reaction ) . after completion of the addition of phosphoryl chloride , the reaction mixture was then heated to 95 c for 30 min . the dark brown mixture was cooled to rt and slowly poured into 6 l of ice water . a beige precipitate formed . the residue was washed with acetonitrile ( 100 ml ) and dried at 60 c under reduced pressure for 15 h to give 4-chloro-3,5-diiodopyridine64 as a yellow solid , which was used without further purification ( 142 g , 95% pure , 85% corrected yield ) . lc ms ( method b , esi , m / z ) tr = 3.06 min , 366/368 ( m + h ) . in a 50 ml round - bottom flask , 4-chloro-3,5-diiodopyridine 64 ( 500 mg , 1.37 mmol ) and 1-oxa-3,8-diazaspiro[4.5]decan-2-one acetate 38 ( 448 mg , 2.05 mmol ) were suspended in nmp ( 10 ml ) and triethylamine ( 570 l , 4.11 mmol ) . the reaction mixture was stirred at 240 c for 3 h and then cooled and poured into water ( 150 ml ) . the solid was dissolved in dcm ( 150 ml ) , the solution was dried over na2so4 , filtered , and concentrated to dryness . the crude residue was purified by flash column chromatography ( companion , dcm / meoh 100:0 to 90:10 ) to give 8-(3,5-diiodopyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one ( 196 mg , 30% yield ) 66 as a light brown solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 2.26 min , 486 ( m + h ) . into a 10 ml schlenck , silver(i ) fluoride ( 57 mg , 0.45 mmol ) was loaded and dmf ( 3 ml ) and ( trifluoromethyl)trimethylsilane ( 81 l , 0.54 mmol ) were added at rt , and the resulting brown suspension was stirred for 15 min at rt . then finely powdered copper ( 43 mg , 0.67 mmol ) was added and the resulting dark red suspension was stirred for 1.5h at rt . the reaction mixture turned green , and a silver precipitate formed on the vessel wall . 8-(3,5-diiodopyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 66 ( 196 mg , 0.403 mmol ) was added , and the suspension was stirred at 90 c for 3 h. the green suspension was diluted with dmf ( 10 ml ) and filtered over celite . the crude residue was dissolved in dcm ( 10 ml ) and treated with diethyl ether ( 35 ml ) . the resulting light brown precipitate was filtered and washed with diethyl ether ( 10 ml ) , the filtrate was evaporated to dryness to yield in 8-(3-iodo-5-trifluoromethylpyridin-4-yl)-1-oxa-3,8-diaza - spiro[4.5]decan-2-one 68 as a yellow oil ( 78 mg , 71% pure , 32% corrected yield ) which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 2.45 min , 428 ( m + h ) . to 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1h - pyrazole37 ( 126 mg , 0.390 mmol ) and pd(dppf)cl2ch2cl2 ( 5 mg , 0.01 mmol ) was added a solution of 8-(3-iodo-5-(trifluoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 68 ( 71% pure , 78 mg , 0.13 mmol ) in acetonitrile ( 4 ml ) and 0.5 m sodium carbonate solution ( 780 l , 0.390 mmol ) . the reaction mixture was stirred at 120 c for 1 h under microwave irradiation and diluted with acetonitrile ( 5 ml ) , filtered and the filtrate concentrated . the crude product was purified by preparative hplc ( method a , 20 min gradient elution from 2:98 to 25:75 acetonitrile / water to give 8-(3-(4-(1-methyl-1h - pyrazol-4-yl)phenyl)-5-(trifluoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 2,2,2-trifluoroacetate 72 ( 18 mg , 24% yield ) as a colorless solid . h nmr ( 500 mhz , dmso - d6 ) 8.82 ( s , 1h ) , 8.53 ( s , 1h ) , 8.24 ( d , j = 0.8 hz , 1h ) , 7.96 ( d , j = 0.8 hz , 1h ) , 7.71 ( d , j = 8.2 hz , 2h ) , 7.45 ( s , 1h ) , 7.40 ( d , j = 8.2 hz , 2h ) , 3.88 ( s , 3h ) , 3.13 ( s , 2h ) , 2.962.84 ( m , 4h ) , 1.691.56 ( m , 4h ) ; f nmr ( 500 mhz , dmso - d6 , tfa salt ) 74.5 ; c nmr ( 126 mhz , dmso - d6 , free base ) 157.7 , 155.9 , 155.3 , 147.7 ( d , j = 7.0 hz ) , 136.5 , 136.2 , 134.1 , 132.5 , 130.0 , 128.2 , 124.9 , 124.0 ( q , j = 273 hz ) , 121.3 , 78.5 , 50.0 , 48.2 , 38.8 , 35.5 ; lc ms ( method c , esi , m / z ) tr = 2.64 min , 458 ( m + h ) ; esi - hrms calcd for c23h23f3n5o2 ( m + h ) 458.1798 , found 458.1785 . n - bromosuccinimide ( 10.9 g , 61.3 mmol ) was added to a solution of 4-chloro-2-aminopyridine ( 7.50 g , 58.3 mmol ) in dry acetonitrile ( 130 ml ) at rt under a nitrogen atmosphere . the yellow solution was stirred for 3 h before the mixture was concentrated in vacuo . the resulting crude product was purified by biotage column chromatography ( cyclohexane / etoac 5:1 to 1:1 ) to give 5-bromo-4-chloropyridin-2-amine as a yellow solid ( 10.0 g , 83% yield ) . h nmr ( 500 mhz , cdcl3 ) 8.16 ( s , 1h ) , 6.62 ( s , 1h ) , 4.57 ( s , 2h ) ; lc ms ( method c , esi , m / z ) tr = 2.04 min , 207/209/211 ( m + h ) . n - chlorosuccinimide ( 6.11 g , 45.8 mmol ) was added portionwise to a solution of 5-bromo-4-chloropyridin-2-amine ( 10.0 g , 48.2 mmol ) in dry acetonitrile ( 180 ml ) at rt under a nitrogen atmosphere . the reaction mixture was heated to 95 c for 3 h and then cooled to rt . the resultant solid was filtered off and washed with acetonitrile to give the desired compound as a light brown solid . the filtrate was concentrated to 50 ml , and the resulting precipitate was filtered and the resultant solid collected . the two batches of solid material were combined and recrystallized from hot acetonitrile to give two batches of 5-bromo-3,4-dichloropyridin-2-amine 73 ( p1 , 6.8 g ; p2 , 2.0 g ; 75% yield ) which were used in the next step without further purification . h nmr ( 500 mhz , cdcl3 ) 8.10 ( s , 1h ) , 5.06 ( s , 2h ) ; lc ms ( method c , esi , m / z ) tr = 2.93 min , 241/243/245 ( m + h ) . a solution of 5-bromo-3,4-dichloropyridin-2-amine 73 ( 2.37 g , 4.91 mmol ) in dry dmf ( 50 ml ) at 0 c was treated portionwise with sodium hydride ( 60% in mineral oil ) ( 0.775 g , 18.6 mmol ) and the mixture stirred at 0 c for 15 min before a solution of p - methoxybenzyl chloride ( 2.10 ml , 15.5 mmol ) in dry dmf ( 1 ml ) was added . the combined organic layers were dried over mgso4 , filtered , and concentrated in vacuo . the resulting brown residue was purified by biotage column chromatography ( cyclohexane / dcm 100:0 to 75:25 ) to give 5-bromo-3,4-dichloro - n , n - bis(4-methoxybenzyl)pyridin-2-amine 81 ( 2.4 g , 80% yield ) as a colorless oil . h nmr ( 500 mhz , cdcl3 ) 8.26 ( s , 1h ) , 7.20 ( d , j = 8.6 hz , 4h ) , 6.85 ( d , j = 8.6 hz , 4h ) , 4.42 ( s , 4h ) , 3.81 ( s , 6h ) ; lc ms ( method c , esi , m / z ) tr = 3.51 min , 361/363/365 ( m + h - pmb ) . 5-bromo-3,4-dichloro - n , n - bis(4-methoxybenzyl)pyridin-2-amine 81 ( 5.00 g , 10.4 mmol ) , 8-boc-2,8-diaza - spiro-[4.5]decan-1-one ( 2.64 g , 10.4 mmol ) , and potassium fluoride ( 1.21 g , 20.7 mmol ) were loaded in a microwave vial . triethylamine ( 4.00 ml , 31.1 mmol ) and nmp ( 25 ml ) were added , and the light brown solution was degassed with argon . the reaction mixture was stirred at 220 c for 1 h under microwave irradiation . after the addition of water , the aqueous layer was extracted twice with etoac and the organic layer was washed with water , dried over mgso4 , and concentrated under vacuum . the resulting brown oil was purified by biotage column chromatography ( dcm / etoh , 97:3 to 90:10 ) to give 8-(2-(bis(4-methoxybenzyl)amino)-5-bromo-3-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 82 ( 3.0 g , 54% yield ) as a light brown solid . h nmr ( 500 mhz , cdcl3 ) 8.15 ( br s , 1h ) , 7.19 ( d , j = 8.7 hz , 4h ) , 6.84 ( d , j = 8.7 hz , 4h ) , 5.62 ( s , 1h ) , 4.38 ( s , 4h ) , 3.80 ( s , 6h ) , 3.423.32 ( m , 6h ) , 2.252.15 ( m , 4h ) , 1.681.46 ( m , 2h ) ; lc ms ( method c , esi , m / z ) tr = 3.68 min , 599/601 ( m + h ) . 8-(2-(bis(4-methoxybenzyl)amino)-5-bromo-3-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 82 ( 203 mg , 0.339 mmol ) and 2-methyl-1-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1h - pyrazol-1-yl)propan-2-ol 9 ( 73% pure , 190 mg , 0.407 mmol ) were dissolved in acetonitrile ( 5 ml ) . 0.5 m aqueous sodium carbonate ( 1.40 ml , 0.678 mmol ) and pd(dppf)cl2ch2cl2 ( 25 mg , 0.034 mmol ) were added . the reaction mixture was stirred at 120 c for 1 h under microwave irradiation , and the solvent was evaporated . the crude residue was purified by flash chromatography ( n - heptane / etoac , gradient and then with dcm / meoh , gradient ) to give 8-(2-(bis(4-methoxybenzyl)amino)-3-chloro-5-(4-(1-(2-hydroxy-2-methylpropyl)-1h - pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 81 mg , 60% pure , 19% corrected yield ) as a light - brown solid which was used directly in the next step . lc ms ( method a , esi , m / z ) tr = 2.59 min , 735/737 ( m + h ) . 8-(2-(bis(4-methoxybenzyl)amino)-3-chloro-5-(4-(1-(2-hydroxy-2-methylpropyl)-1h - pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 60% pure , 81 mg , 0.066 mmol ) was dissolved in tfa ( 1 ml ) and stirred at rt overnight . the crude mixture was concentrated , and the residue was treated with 1 n naoh . the mixture was extracted with etoac , and the organic layer was dried over na2so4 , filtered , and concentrated . the crude product was dissolved in acetonitrile , and the resultant solid precipitate was filtered and dried in vacuo to afford 8-(2-amino-3-chloro-5-(4-(1-(2-hydroxy-2-methylpropyl)-1h - pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 86 ( 31 mg , 94% yield ) as a beige solid . h nmr ( 500 mhz , dmso - d6 ) 8.12 ( s , 1h ) , 7.91 ( s , 1h ) , 7.64 ( s , 1h ) , 7.62 ( d , j = 8.2 hz , 2h ) , 7.51 ( s , 1h ) , 7.23 ( d , j = 8.2 hz , 2h ) , 6.13 ( s , 2h ) , 4.75 ( s , 1h ) , 4.04 ( s , 2h ) , 3.10 ( t , j = 6.8 hz , 2h ) , 3.012.93 ( m , 2h ) , 2.742.61 ( m , 2h ) , 1.82 ( t , j = 6.8 hz , 2h ) , 1.761.65 ( m , 2h ) , 1.251.18 ( m , 2h ) , 1.10 ( s , 6h ) ; c nmr ( 126 mhz , dmso - d6 ) 180.0 , 156.3 , 153.3 , 147.7 , 135.8 , 135.7 , 131.1 , 129.6 , 128.1 , 124.7 , 123.6 , 121.1 , 108.6 , 69.3 , 62.2 , 47.4 , 41.4 , 37.8 , 31.8 , 30.4 , 27.3 ; lc ms ( method c , esi , m / z ) tr = 1.99 min , 495/497 ( m + h ) ; esi - hrms calcd for c26h32cln6o2 ( m + h ) 495.2270 , found 495.2256 . to a solution of lda ( 2 m , 7.97 ml , 15.9 mmol ) in thf ( 31 ml ) at 78 c was added 5-chloro-3-fluoropyridin-2-amine ( 934 mg , 6.37 mmol ) in solution in thf ( 9 ml ) . after 50 min at 78 c , hexachloroethane ( 1.44 ml , 12.8 mmol ) in thf ( 9 ml ) was added . the reaction mixture was stirred for 40 min and then quenched with nh4cl and extracted with dcm . the crude material was purified by biotage column chromatography ( dcm 100% ) to afford 4,5-dichloro-3-fluoropyridin-2-amine 74 ( 950 mg , 82% yield ) as a cream solid . h nmr ( 500 mhz , cdcl3 ) 7.92 ( d , j = 1.0 hz , 1h ) , 4.74 ( s , 2h ) ; f nmr ( 500 mhz , cdcl3 ) 137 ; lc ms ( method c , esi , m / z ) tr = 2.66 min , 181/183/185 ( m + h ) . 4,5-dichloro-3-fluoropyridin-2-amine 74 ( 100 mg , 0.553 mmol ) and tert - butyl 1-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate ( 211 mg , 0.829 mmol ) were introduced in a microwave vial , and nmp ( 1.4 ml ) and triethylamine ( 233 l , 1.66 mmol ) were added . the reaction mixture was stirred for 2 1 h at 220 c under microwave irradiation . the reaction mixture was concentrated and the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 94:6 ) to afford 8-(2-amino-5-chloro-3-fluoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 77 ( 105 mg , 64% yield ) as a cream solid . h nmr ( 500 mhz , dmso - d6 ) 7.67 ( s , 1h ) , 7.59 ( s , 1h ) , 6.17 ( s , 2h ) , 3.353.27 ( m , 2h ) , 3.19 ( t , j = 6.8 hz , 2h ) , 3.123.05 ( m , 2h ) , 2.01 ( t , j = 6.8 hz , 2h ) , 1.79 ( td , j = 12.4 , 4.2 hz , 2h ) , 1.441.38 ( m , 2h ) ; f nmr ( 500 mhz , dmso - d6 ) 149 ; lc ms ( method c , esi , m / z ) tr = 1.73 min , 299/301 ( m + h ) . 8-(2-amino-5-chloro-3-fluoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 7 ( 20 mg , 0.067 mmol ) , 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 27 mg , 0.087 mmol ) , and pdcl2(pcy3)2 ( 2.5 mg , 3.4 mol ) were loaded in a microwave vial . acetonitrile ( 1.1 ml ) and 0.5 m sodium carbonate in water ( 187 l , 0.0940 mmol ) were added , and the reaction mixture was stirred at 150 c for 30 min under microwave irradiation . after evaporation of the solvent , the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 90:10 ) to afford 8-(2-amino-3-fluoro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 92 ( 17 mg , 57% yield ) as a cream solid . h nmr ( 500 mhz , dmso - d6 ) 7.54 ( s , 1h ) , 7.48 ( s , 1h ) , 7.45 ( d , j = 1.8 hz , 1h ) , 7.41 ( dd , j = 8.3 , 1.8 hz , 1h ) , 6.97 ( d , j = 8.3 hz , 1h ) , 6.02 ( s , 2h ) , 4.67 ( s , 2h ) , 3.12 ( t , j = 6.8 hz , 2h ) , 3.083.01 ( m , 2h ) , 3.06 ( s , 3h ) , 2.89 ( t , j = 12.2 hz , 2h ) , 1.91 ( t , j = 6.8 hz , 2h ) , 1.611.52 ( m , 2h ) , 1.211.15 ( m , 2h ) ; f nmr ( 500 mhz , dmso - d6 ) 153 ; c nmr ( 126 mhz , dmso - d6 ) 180.5 , 150.4 ( d , j = 12.8 hz ) , 143.9 ( d , j = 5.4 hz ) , 142.7 ( d , j = 4.3 hz ) , 141.1 ( d , j = 247 hz ) , 140.6 , 131.3 , 129.5 , 126.4 , 121.8 , 118.7 , 109.8 , 50.6 , 47.5 ( d , j = 4.6 hz ) , 41.6 , 38.3 , 32.1 , 31.1 , 26.6 ; lc ms ( method c , esi , m / z ) tr = 1.73 min , 446 ( m + h ) ; esi - hrms calcd for c21h25fn5o3s ( m + h ) 446.1657 , found 446.1656 . 3-(trifluoromethyl)pyridin-2-amine ( 35.0 g , 0.210 mol ) in dmf ( 350 ml ) was cooled to 05 c under a nitrogen atmosphere . n - bromosuccinimide ( 38.8 g , 0.210 mol ) was added portionwise , and the mixture was stirred at rt for 1 h. the reaction mixture was concentrated , and the light brown viscous liquid residue was triturated with ice - cold water ( 200 ml ) . the precipitate was filtered and dried to afford 5-bromo-3-(trifluoromethyl)pyridin-2-amine ( 45.0 g , 86% yield ) as an off - white solid that was used directly in the next step . to a stirred solution of 5-bromo-3-(trifluoromethyl)pyridin-2-amine ( 25.0 g , 0.100 mol ) in thf ( 250 ml ) was added sodium hydride ( 60% in mineral oil , 7.35 g , 0.180 mol ) portionwise at 05 c under a nitrogen atmosphere ; the reaction mixture was stirred for 1 h at 05 c . a solution of di - tert - butyl dicarbonate ( 25.8 ml , 0.110 mol ) in thf ( 50 ml ) was added dropwise at 05 c , and stirring was continued for 3 h. since the reaction was incomplete , sodium hydride ( 60% in mineral oil , 4.08 g , 0.100 mol ) was added portionwise at 05 c and stirring continued for 1 h. a solution of di - tert - butyl dicarbonate ( 12 ml , 0.050 mol ) in thf ( 20 ml ) was added dropwise , and the reaction mixture was stirred for an additional 2 h at 05 c . the mixture was poured onto ice cold water ( 250 ml ) and extracted with ethyl acetate ( 3 150 ml ) . the combined organic layers were washed with brine ( 100 ml ) , dried over na2so4 , and concentrated under reduced pressure to afford a brown viscous solid . the residue was purified by column chromatography ( petroleum ether / etoac 100:0 to 94:6 ) to give tert - butyl ( 5-bromo-3-(trifluoromethyl)pyridin-2-yl)carbamate ( 30 g , 74% yield ) as an off - white solid that was taken to the next step without further purification . to a stirred solution of diisoproylamine ( 13 ml , 0.090 mol ) in thf ( 220 ml ) cooled to 70 c under nitrogen atmosphere was added n - butyllithium ( 1.6 m solution in hexane , 54 ml , 0.090 mol ) dropwise over a period 0.5 h , and the mixture was stirred for additional 30 min at the same temperature . after cooling to 70 c a solution of ( tert - butyl ( 5-bromo-3-(trifluoromethyl)pyridin-2-yl)carbamate ( 22 g , 0.040 mol ) in thf ( 100 ml ) was added dropwise over a period 20 min at 10 c . the mixture was stirred for 30 min at 70 c , then a solution of 1,1,1,2,2,2-hexachloroethane ( 21 g , 0.090 mol ) in thf ( 100 ml ) was added dropwise over a period of 30 min and the reaction mixture stirred for a 1 h at the same temperature . the mixture was slowly warmed to rt and stirred for additional 20 min , poured onto ice - cold water ( 200 ml ) , and extracted with etoac ( 3 200 ml ) . the combined organic layers were washed with brine ( 100 ml ) , dried over na2so4 , and concentrated to give the crude product as a brown viscous solid . the crude product was purified by column chromatography ( petroleum ether / etoac 95:5 to 90:10 ) to afford tert - butyl ( 5-bromo-4-chloro-3-(trifluoromethyl)pyridin-2-yl)carbamate ( 9.5 g , 62% yield ) as an off - white solid which was used directly in the next step . tert - butyl ( 5-bromo-4-chloro-3-(trifluoromethyl)pyridin-2-yl)carbamate ( 9.5 g , 0.020 mol ) in 1,4-dioxane ( 48 ml ) was cooled to 05 c . the reaction mixture was stirred at rt for 4 h and then evaporated to give an off - white solid . the crude product was purified by column chromatography ( petroleum ether / etoac 100:0 to 90:10 ) to afford 5-bromo-4-chloro-3-(trifluoromethyl)pyridin-2-amine 75 ( 4.85 g , 70% yield ) as an off - white solid . h nmr ( 400 mhz , dmso - d6 ) 8.41 ( s , 1h ) , 6.95 ( s , 2h ) ; lc ms ( method e , esi , m / z ) tr = 4.38 min , 275/277/279 ( m + h ) . 5-bromo-4-chloro-3-(trifluoromethyl)pyridin-2-amine 75 ( 300 mg , 1.09 mmol ) and 1-oxa-3,8-diazaspiro[4.5]decan-2-one 38 ( 340 mg , 2.18 mmol ) were dissolved in nmp ( 3 ml ) , and triethylamine ( 0.453 ml , 3.27 mmol ) was added . the reaction mixture was stirred for 2 h at 220 c under microwave irradiation . the brown precipitate was filtered , washed with water and diethyl ether , and then dried to give 8-(2-amino-5-bromo-3-(trifluoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 80 ( 280 mg , 89% pure , 58% corrected yield ) as a pale brown solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 1.83 min , 395/397 ( m + h ) . 8-(2-amino-5-bromo-3-(trifluoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 80 ( 89% pure , 140 mg , 0.315 mmol ) was suspended in acetonitrile ( 10 ml ) . 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1h - pyrazole 37 ( 81% pure , 166 mg , 0.473 mmol ) , 0.5 m aqueous sodium carbonate ( 1.26 ml , 0.631 mmol ) , and pd(dppf)cl2ch2cl2 ( 13 mg , 0.016 mmol ) were added . the reaction mixture was stirred at 120 c for 1 h under microwave irradiation . after addition of etoac , the mixture was filtered and the filtrate was concentrated . the brown oily residue was purified by preparative hplc ( method a , gradient elution from 2:98 to 30:70 acetonitrile / water in 20 min ) to afford 8-(2-amino-5-(4-(1-methyl-1h - pyrazol-4-yl)phenyl)-3-(trifluoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 2,2,2-trifluoroacetate 95 ( 46 mg , 25% yield ) as a white fluffy solid . h nmr ( 500 mhz , dmso - d6 ) 8.20 ( d , j = 0.8 hz , 1h ) , 7.92 ( d , j = 0.8 hz , 1h ) , 7.76 ( s , 1h ) , 7.66 ( d , j = 8.2 hz , 2h ) , 7.48 ( s , 1h ) , 7.37 ( d , j = 8.2 hz , 2h ) , 3.87 ( s , 3h ) , 3.13 ( s , 2h ) , 3.123.01 ( m , 4h ) , 1.611.45 ( m , 4h ) ; c nmr ( 126 mhz , dmso - d6 ) 160.8 , 157.6 , 154.2 , 144.0 , 136.2 , 133.1 , 132.3 , 130.1 , 128.1 , 124.9 , 124.0 , 121.3 , 78.1 , 49.8 , 48.1 , 38.7 , 34.5 ( 1 cq not observed ) ; lc ms ( method c , esi , m / z ) tr = 2.05 min , 473 ( m + h ) ; esi - hrms calcd for c23h24f3n6o2 ( m + h ) 473.1907 , found 473.1893 . 6-bromo-1h - indazol-3-amine ( 95% pure , 1.00 g , 4.48 mmol ) was dissolved in pyridine ( 20 ml ) and the mixture cooled to 0 c ; phenyl chloroformate ( 0.620 ml , 4.93 mmol ) was added dropwise . and the reaction mixture was stirred at 0 c for 4 h. after addition of dcm ( 50 ml ) and water ( 50 ml ) , the layers were separated and the organic layer was washed with brine , dried over na2so4 , and evaporated to dryness . the residue was purified by flash chromatography ( n - heptane / etoac , gradient ) and by preparative hplc ( method a , gradient elution from 0:100 to 30:70 over 3 min and to 60:40 over 17 min acetonitrile / water ) to give first the desired phenyl ( 6-bromo-1h - indazol-3-yl)carbamate ( 54 mg , 3% yield ) as a white solid , which was used directly in the next step , and second undesired phenyl 3-amino-6-bromo-1h - indazole-1-carboxylate ( 346 mg ) as a white solid . lc ms ( method b , esi , m / z ) tr = 2.62 min , 332/334 ( m + h ) . phenyl ( 6-bromo-1h - indazol-3-yl)carbamate ( 54 mg , 0.16 mmol ) was dissolved in thf ( 3 ml ) , and lithium aluminum hydride solution 1.0 m in thf ( 325 l , 0.330 mmol ) was added dropwise . the mixture was stirred at rt for 2 h and then diluted with water ( 2 ml ) and thf ( 8 ml ) . the suspension was filtered over celite and the mother liquor was evaporated to dryness to give 6-bromo - n - methyl-1h - indazol-3-amine ( 41 mg , 89% pure , 99% corrected yield ) as a pale brown solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 1.95 min , 226/228 ( m + h ) . 6-bromo - n - methyl-1h - indazol-3-amine ( 89% pure , 41 mg , 0.16 mmol ) , bis(pinacolato)diboron ( 82 mg , 0.32 mmol ) , potassium acetate ( 48 mg , 0.48 mmol ) , and pd(dppf)cl2ch2cl2 ( 13 mg , 0.020 mmol ) were loaded in a microwave vial , and acetonitrile was added ( 4 ml ) . the reaction mixture was stirred for 1 h at 120 c under microwave irradiation and then diluted with acetonitrile ( 10 ml ) , filtered and the filtrate evaporated to dryness . the crude residue 97 ( 230 mg , 13% pure , 100% corrected yield ) was used in the next step without purification . lc ms ( method b , esi , m / z ) tr = 1.79 min , 191/192 ( m + h ) . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one8 ( 70.3 mg , 0.20 mmol ) , ( 3-(methylamino)-1h - indazol-6-yl)boronic acid 97 ( 13% pure , 230 mg , 0.160 mmol ) were loaded in a microwave vial , and acetonitrile ( 4 ml ) , 0.5 m aqueous sodium carbonate ( 1.23 ml , 0.610 mmol ) and pd(dppf)cl2ch2cl2 ( 8 mg , 0.01 mmol ) were added . the reaction mixture was stirred for 1 h at 120 c under microwave irradiation . after dilution with acetonitrile ( 5 ml ) , the mixture was filtered and the filtrate was concentrated . the crude product was purified by preparative hplc ( method a , 20 min gradient elution from 2:98 to 20:80 acetonitrile / water ) to give 8-(3-chloro-5-(3-(methylamino)-1h - indazol-6-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 100 ( 13 mg , 12% yield ) as a white solid . h nmr ( 500 mhz , cd3od ) 8.56 ( s , 1h ) , 8.28 ( s , 1h ) , 7.89 ( dd , j = 8.4 , 0.8 hz , 1h ) , 7.417.37 ( m , 1h ) , 7.07 ( dd , j = 8.4 , 1.4 hz , 1h ) , 3.413.35 ( m , 2h ) , 3.27 ( t , j = 6.9 hz , 2h ) , 3.06 ( s , 3h ) , 2.972.89 ( m , 2h ) , 2.00 ( t , j = 6.9 hz , 2h ) , 1.921.84 ( m , 2h ) , 1.41 ( d , j = 13.3 hz , 2h ) ; lc ms ( method c , esi , m / z ) tr = 1.74 min , 411/413 ( m + h ) ; esi - hrms calcd for c21h24cln6o ( m + h ) 411.1695 , found 411.1680 . 6-bromo-1-methyl-1h - indazol-3-amine ( 500 mg , 2.21 mmol ) and n , n - dimethylpyridin-4-amine ( 54 mg , 0.44 mmol ) were dissolved in dcm ( 15 ml ) . triethylamine ( 1.74 ml , 13.3 mmol ) and di - tert - butyl dicarbonate ( 1.42 ml , 6.64 mmol ) were were added , and the reaction solution was stirred overnight at rt . after addition of water and dcm , the layers were separated and the aqueous layer was extracted with dcm . the organic layer was washed with water , dried , filtered , and evaporated to dryness . the brown solid was purified by flash - chromatography ( companion , n - heptane / etoac gradient ) to give tert - butyl n-(6-bromo-1-methyl - indazol-3-yl)-n - tert - butoxycarbonylcarbamate ( 150 mg , 16% yield ) as a colorless solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 3.29 min , 448/450 ( m + na ) . tert - butyl n-(6-bromo-1-methylindazol-3-yl)-n - tert - butoxycarbonylcarbamate ( 150 mg , 0.352 mmol ) was dissolved in thf ( 5 ml ) . bis(pinacolato)diboron ( 179 mg , 0.704 mmol ) , potassium acetate ( 104 mg , 1.06 mmol ) , and pd(dppf)cl2ch2cl2 ( 29 mg , 0.035 mmol ) were added , and the red reaction mixture was stirred overnight at 70 c . the resultant black reaction mixture was treated with etoac , filtered and the filtrate evaporated under vacuum . the dark brown residue was purified by flash chromatography ( companion , heptane / dcm gradient ) to give a mixture of [ 3-[bis(tert - butoxycarbonyl)amino]-1-methyl - indazol-6-yl]boronic acid and tert - butyl n - tert - butoxycarbonyl - n-[1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazol-3-yl]carbamate ( 2:3 ratio , 182 mg , quant ) as a pale yellow solid 98 which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 2.59 min , 413/414 ( m + na ) and tr = 3.49 min , 495/496 ( m + na ) . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one8 ( 84.4 mg , 0.25 mmol ) was suspended in acetonitrile ( 2 ml ) . a mixture of tert - butyl n - tert - butoxycarbonyl - n-[1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazol-3-yl]carbamate and [ 3-[bis(tert - butoxycarbonyl)amino]-1-methyl - indazol-6-yl]boronic acid 98 ( 3:2 ratio , 174 mg , 0.368 mmol ) , 0.5 m aqueous sodium carbonate ( 982 ul , 0.491 mmol ) , and pd(dppf)cl2ch2cl2 ( 18 mg , 0.025 mmol ) were added . after addition of etoac , the mixture was filtered and the filtrate was concentrated . the brown residue was purified by flash chromatography ( companion , dcm / meoh gradient ) to give tert - butyl ( 6-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-1h - indazol-3-yl)carbamate ( 47 mg , 71% pure , 27% corrected yield ) as a colorless solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 2.19 min , 511/513 ( m + h ) . tert - butyl ( 6-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-1h - indazol-3-yl)carbamate ( 71% pure , 47 mg , 0.066 mmol ) was dissolved in 5 n hydrogen chloride in dioxane ( 5 ml ) and meoh ( 1 ml ) . the pale brown solution was stirred at rt for 4 h , and the solvents were evaporated . the residue was purified by preparative hplc ( method a , 20 min gradient elution from 2:98 to 25:75 acetonitrile / water ) to give 8-(3-(3-amino-1-methyl-1h - indazol-6-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 2,2,2-trifluoroacetate 101 ( 15 mg , 43% yield ) as a yellow solid . h nmr ( 500 mhz , cd3od ) 8.66 ( s , 1h ) , 8.35 ( s , 1h ) , 7.88 ( d , j = 8.4 hz , 1h ) , 7.47 ( s , 1h ) , 7.04 ( d , j = 8.4 hz , 1h ) , 3.89 ( s , 3h ) , 3.553.47 ( m , 2h ) , 3.27 ( t , j = 6.9 hz , 2h ) , 3.022.95 ( m , 2h ) , 2.01 ( t , j = 6.9 hz , 2h ) , 1.951.87 ( m , 2h ) , 1.501.42 ( m , 2h ) ; c nmr ( 126 mhz , cd3od ) 183.1 , 159.7 , 148.3 , 144.4 , 143.3 , 143.1 , 136.6 , 134.7 , 127.8 , 122.5 , 121.2 , 115.5 , 110.6 , 49.8 , 43.1 , 39.7 , 35.1 , 33.4 , 32.6 ; lc ms ( method c , esi , m / z ) tr = 1.86 min , 411/413 ( m + h ) ; esi - hrms calcd for c21h24cln6o ( m + h ) 411.1695 , found 411.1684 . 4-bromo-2-fluorobenzonitrile ( 1.0 g , 5.0 mmol ) was dissolved in ethanol ( 11 ml ) , and methylhydrazine ( 0.756 ml , 14.4 mmol ) was added . the reaction mixture was stirred under microwave irradiation at 120 c for 50 min . the reaction was quenched with water , and the product was extracted with etoac three times . the combined organic layers were dried over mgso4 and concentrated to give 6-bromo-1-methyl-1h - indazol-3-amine ( 940 mg , 83% yield ) as a brown solid . h nmr ( 500 mhz , cdcl3 ) 7.47 ( dd , j = 8.7 , 0.7 hz , 1h ) , 7.38 ( dd , j = 1.5 , 0.7 hz , 1h ) , 7.13 ( dd , j = 8.7 , 1.5 hz , 1h ) , 3.80 ( s , 3h ) ; lc ms ( method c , esi , m / z ) tr = 2.50 min , 226/228 ( m + h ) . to 6-bromo-1-methyl-1h - indazol-3-amine ( 736 mg , 3.26 mmol ) in formic acid ( 3.7 ml , 98 mmol ) was added formaldehyde 37% in water ( 897 l , 32.6 mmol ) . the reaction mixture was heated at 100 c for 2 h and concentrated . the crude material was purified by biotage column chromatography ( cyclohexane / etoac 95:5 to 82:18 ) to afford 6-bromo - n , n,1-trimethyl-1h - indazol-3-amine ( 85 mg , 10% yield ) as a brown solid . h nmr ( 500 mhz , cdcl3 ) 7.60 ( dd , j = 8.7 , 0.6 hz , 1h ) , 7.37 ( dd , j = 1.5 , 0.6 hz , 1h ) , 7.06 ( dd , j = 8.7 , 1.5 hz , 1h ) , 3.82 ( s , 3h ) , 3.07 ( s , 6h ) ; lc ms ( method c , esi , m / z ) tr = 3.11 min , 254/256 ( m + h ) . 6-bromo - n , n,1-trimethyl-1h - indazol-3-amine ( 100 mg , 0.394 mmol ) , bis(pinacolato)diboron ( 150 mg , 0.590 mmol ) , potassium acetate ( 116 mg , 1.18 mmol ) , and pd(dppf)cl2ch2cl2 ( 16 mg , 0.020 mmol ) were loaded in a flask , and dme ( 2.8 ml ) was added . the crude material was purified by biotage column chromatography ( cylohexane / etoac 95:5 to 70:30 ) to give n , n,1-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h - indazol-3-amine 99 ( 100 mg , 84% yield ) . h nmr ( 500 mhz , cdcl3 ) 7.75 ( dd , j = 8.1 , 0.9 hz , 1h ) , 7.72 ( s , 1h ) , 7.39 ( d , j = 8.1 hz , 1h ) , 3.91 ( s , 3h ) , 3.09 ( s , 6h ) , 1.38 ( s , 12h ) ; lc ms ( method c , esi , m / z ) tr = 3.19 min , 301/302 ( m + h ) . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one8 ( 13 mg , 0.036 mmol ) , n , n,1-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h - indazol-3-amine 99 ( 12 mg , 0.040 mmol ) , and pd(dppf)cl2ch2cl2 ( 1.5 mg , 1.8 mol ) were loaded in a microwave vial , and acetonitrile ( 624 l ) and 0.5 m sodium carbonate in water ( 101 l , 0.0510 mmol ) were added . the solvent was evaporated , and the crude material was purified by biotage column chromatography ( dcm / etoh 98:2 to 91:9 ) and then by preparative hplc ( method b ) . fractions were concentrated under vacuum and the product was solubilized in dcm and filtered on a basic isolute flash nh2 column to afford 8-(3-chloro-5-(3-(dimethylamino)-1-methyl-1h - indazol-6-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 102 ( 8 mg , 50% yield ) . h nmr ( 500 mhz , cdcl3 ) 8.46 ( s , 1h ) , 8.27 ( s , 1h ) , 7.82 ( dd , j = 8.4 , 0.8 hz , 1h ) , 7.13 ( dd , j = 1.4 , 0.8 hz , 1h ) , 6.88 ( dd , j = 8.4 , 1.4 hz , 1h ) , 5.68 ( s , 1h ) , 3.92 ( s , 3h ) , 3.283.23 ( m , 2h ) , 3.213.15 ( m , 2h ) , 3.13 ( s , 6h ) , 2.71 ( t , j = 11.8 hz , 2h ) , 2.001.90 ( m , 4h ) , 1.361.29 ( m , 2h ) ; lc ms ( method c , esi , m / z ) tr = 2.40 min , 439/441 ( m + h ) ; esi - hrms calcd for c23h28cln6o ( m + h ) 439.2008 , found 439.2004 . into a 500 ml screw cap jar , 8-(2-(bis(4-methoxybenzyl)amino)-5-bromo-3-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 82 ( 82% pure , 13.4 g , 18.4 mmol ) , 1-methylindazole-5-boronic acid 39 ( 97% pure , 4.99 g , 27.5 mmol ) , and pd(dppf)cl2ch2cl2 ( 749 mg , 0.920 mmol ) were weighed in and suspended in acetonitrile ( 300 ml ) and sodium carbonate solution ( 0.5 m , 73.4 ml , 36.7 mmol ) . the mixture was diluted with acetonitrile ( 200 ml ) , filtered , and evaporated to dryness . the crude residue was purified by flash chromatography ( dcm / meoh , gradient ) . the isolated product was suspended in acetonitrile ( 100 ml ) and treated with diethyl ether ( 200 ml ) . the resultant beige precipitate was filtered , washed with diethyl ether ( 100 ml ) , and dried under vacuum . the mother liquor was evaporated to 50 ml volume and treated with diethyl ether ( 100 ml ) . the resulting precipitate was filtered , washed with diethyl ether ( 40 ml ) , and dried under vacuum . the two batches of compound were combined to yield in 8-(2-(bis(4-methoxybenzyl)amino)-3-chloro-5-(1-methyl-1h - indazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 7.05 g , 59% yield ) as a colorless solid which was used directly in the next step . lc ms ( method b , esi , m / z ) tr = 2.78 min , 651/653 ( m + h ) . 8-(2-(bis(4-methoxybenzyl)amino)-3-chloro-5-(1-methyl-1h - indazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 7.05 g , 10.8 mmol ) was dissolved in trifluoroacetic acid ( 70 ml ) and the reaction mixture stirred at rt for 4 h. the solution was evaporated to dryness . the crude residue was suspended in dcm and treated with diethyl ether ( 350 ml ) . the white precipitate was filtered and washed with diethyl ether ( 100 ml ) and dried under vacuum overnight . the residue was dissolved in dmso ( 50 ml ) and poured into sodium carbonate solution ( 0.25 m , 500 ml ) . the resulting light yellow precipitate was filtered , washed with water ( 150 ml ) , and dried at 70 c under vacuum overnight to give 8-(2-amino-3-chloro-5-(1-methyl-1h - indazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 109 ( 3.34 g , 75% yield ) as a light yellow solid . h nmr ( 500 mhz , dmso - d6 ) 8.05 ( d , j = 1.0 hz , 1h ) , 7.697.64 ( m , 2h ) , 7.637.60 ( m , 1h ) , 7.49 ( s , 1h ) , 7.27 ( dd , j = 8.6 , 1.6 hz , 1h ) , 6.10 ( s , 2h ) , 4.07 ( s , 3h ) , 3.07 ( t , j = 6.7 hz , 2h ) , 2.982.92 ( m , 2h ) , 2.672.55 ( m , 2h ) , 1.74 ( t , j = 6.7 hz , 2h ) , 1.711.61 ( m , 2h ) , 1.16 ( d , j = 12.6 hz , 2h ) ; c nmr ( 126 mhz , dmso - d6 ) 180.0 , 156.2 , 153.5 , 148.2 , 138.7 , 132.4 , 130.6 , 128.4 , 124.2 , 123.7 , 120.3 , 109.2 , 108.8 , 47.3 , 41.4 , 37.8 , 35.5 , 31.8 , 30.3 ; lc ms ( method c , esi , m / z ) tr = 1.79 min , 411/413 ( m + h ) . esi - hrms calcd for c21h24cln6o ( m + h ) 411.1695 , found 411.1681 . microsomes ( final concentration 0.5 mg / ml ) , 50 mm phosphate buffer ph 7.4 , and compound ( final concentration 1 m ) were added to the assay plate and allowed to preincubate for 5 min at 37 c . the reaction was initiated by the addition of nadph ( final concentration 1.5 mm ) , and the plate was shaken at 800 rpm at 37 c . after 0 , 5 , 10 , 20 , and 30 min aliquots were taken and the reaction was stopped using cold acetonitrile . the samples were centrifuged at 4000 rpm for 30 min at 4 c and analyzed by lc caco-2 cells were maintained in dmem in an atmosphere of 8.5% co2 . for transport experiments 0.125 10 cells / well of were seeded on polycarbonate filter inserts and allowed to grow and differentiate for 14 1 days before the cell monolayers were used for experiments . drug transport experiments were carried out using a cocktail approach in a four - dimensional setting . apparent permeability coefficients were determined for a b and b a directions with and without the presence of cyclosporine a as a transporter inhibitor . up to five test items and reference compounds were dissolved in hank s balanced salt solution at ph 7.4 to yield a final concentration of 1 m . the assays were performed in hbss containing 25 mm hepes ( ph 7.4 ) in an atmosphere of 5% co2 at 37 c . prior to the study , the monolayers were washed in prewarmed hbss . at the start of the experiments prewarmed hbss containing the test items was added to the donor side of the monolayer and hbss without test items was added to the receiver side . after 2 h the transwell insert containing the monolayer was carefully removed and placed in a new plate and aliquots of both the receiver and donor sides were taken and diluted with an equal volume of acetonitrile containing the internal standard . the apparent permeability coefficients ( papp ) were calculated using the formula : papp = [ vrec/(a c0,donor)](dcrec / dt ) 10 with dcrev / dt being the change in concentration in the receiver compartment with time , vrec the volume of the sample in the receiver compartment , c0,donor the concentration in the donor compartment at time 0 , and a the area of the compartment with the cells . female nmri mice ( n = 5 ) received either a single intravenous ( bolus ) injection or single oral administration ( by gavage ) of the compound in a cocktail preparation . doses of 0.2 mg / kg ( per compound , intravenous administration ) and 0.5 mg / kg ( per compound , perorally ) were given as solutions in dmso / peg 200/water . consecutive blood samples were taken retroorbitally under isofluorane inhalation from n = 3 animals per route of administration after 0.1 ( iv only ) , 0.25 ( po only ) , 0.5 , 1 , 2 , 4 , and 6 h and were further processed to obtain plasma . in addition , feces of 2 mice per route of administration were collected over the time interval from 0 to 24 h ( pooled for analysis , no blood sampling ) . male wistar rats ( n = 6 ) received either a single intravenous ( bolus ) injection or a single oral administration ( by gavage ) of the compound in a cocktail preparation . doses of 0.2 mg / kg ( pre compound , intravenous administration ) and 0.5 mg / kg ( per compound , perorally ) were given as solutions in dmso / peg 200/water . consecutive blood samples were taken sublingually under isofluorane inhalation from n = 3 animals per route of administration after 0.1 ( iv only ) , 0.25 ( po only ) , 0.5 , 1 , 2 , 4 , and 6 h and were further processed to obtain plasma . in addition , feces and urine of 3 rats per route of administration were collected over the time interval from 0 to 24 h ( pooled for analysis , no blood sampling ) . female beagle dogs received either a single intravenous ( bolus ) injection or an oral administration ( by gavage ) of the compound in a cocktail preparation . doses of 0.2 and 0.5 mg / kg were given intravenously and per orally as a solution in dmso / peg 200/water , respectively . consecutive blood samples were taken by puncture of the vena cephalica from n = 2 animals per route of administration ( crossover design ) predose and after 0.1 ( only ( iv ) , 0.25 ( only po ) , 0.5 , 1 , 2 , 4 , 6 , 24 , and 48 h and were further processed to obtain plasma . the institute of cancer research does not use non - rodent species in research and , where this is deemed essential , require ethical approval for use by organizations with whom we collaborate . pharmacokinetic analysis of compounds 6 and 109 in dogs , necessary for prediction of human pharmacokinetics , was approved by the icr ethics committee . studies were sponsored and conducted in full compliance with national regulations at an association for assessment and accreditation of laboratory animal care ( aaalac ) accredited site of merck biopharma . the concentrations of compound in plasma and feces were quantified using an uplc method with tandem mass spectrometric detection ( lc ms / ms ) . ms system consisted of a waters acquity uplc coupled to an ab sciex mass spectrometer api 5500 q - trap . the uplc separation was carried out on a reversed phase column ( acquity uplc bec c8 , 1.7 m , 2.1 mm 50 mm ) using a mobile phase gradient with 0.1% formic acid and acetonitrile as eluents . the detection of the drug was performed using multiple reaction monitoring in the positive ionization mode . plasma samples were spiked with internal standard , and the analyte was extracted from the matrix using tert - butyl methyl ether ( tbme ) . feces samples were homogenized with 4 times their volume ethanol / water ( 4:1 ) . aliquots of the aqueous ethanolic extracts were further diluted with acetonitrile/0.1% formic acid , spiked with internal standard , and directly injected into the system . allometric methods used for clearance prediction to human were simple allometry ( 3-species ) , allometry with ( a ) correction by fu , ( b ) with correction by fu and mlp ( maximum life span ) , ( c ) with correction by fu and brw ( brain weight ) ; in addition , in vitro to in vivo ( iviv ) scaling from liver microsomes and hepatocytes was used for clearance prediction ( for compounds 6 and 109 , iviv from hepatocytes of 0.53 and 0.215 l / h / kg may be included in the mean calculation ) . for compound 6 , the clearance prediction ranged from 1.45 l / h / kg ( simple allometry ) to 0.485 l / h / kg ( brw ) . allometry with correction for fu and mlp was chosen because of the highest r value ( 0.9992 ) in the linear regression plot ; the mean of all methods gives a clearance prediction of 0.88 l / h / kg . for volume allometry , values ranged between 0.7 and 1.0 l / kg ; the selected method of choice was allometry with correction by fu ( vss = 0.85 l / kg ) , which represents roughly the mean of the values . for compound 109 the corresponding values were as follows : clearance prediction ; mlp = 0.511 l / h / kg , brw = 0.343 l / h / kg . allometry with correction for fu and scaling from liver microsomes and hepatocytes was included ; the mean of all methods gave a clearance prediction of 0.36 l / h / kg . for volume allometry , methods included 3 species allometry with correction for and human dog proportionality . values ranged between 1.3 and 1.5 l / kg ; the mean of all methods gave a volume prediction of 1.4 l / kg . 7df3 cells were seeded at 5000 per well in 50 l of dulbecco s modified eagle medium ( dmem ) with 4 mm supplemental l - glutamine in 384-well white tissue culture plates ( corning 3570 , usa ) . after incubating overnight at 37 c/5% co2 , compounds ranging in final concentration from 90 m to 0.3 nm were added in duplicate to the wells . after 2 h of further incubation as above the cells were incubated for 24 h at 37 c/5% co2 , and then 25 l of luciferase reagent ( steadyglo , promega , usa ) was added and mixed . after leaving the plate for 60 min at room temperature , luminescence was read on a plate luminescence reader ( topcount , perkinelmer , usa ) . percentage inhibition for each compound concentration was calculated using total counts ( no compound ) and low counts ( no -oestradiol ) as highs and lows , respectively . ic50 was subsequently determined using a curve - fitting software package in excel ( excelfit , idbs , u.k . ) . data are reported as average values of at least two determinations with standard deviations unless otherwise noted . a dye - labeled atp competitive tracer served as a fret acceptor upon binding to cdk8 . the fret donor was a strepavidin - eu - chelate bound to a biotinylated anti - his antibody . when the tracer is competed by an inhibitor , the respective fret signal is affected . the cdk8 used for this assay was a protein coexpressed with cycc . the assay procedure for assays in the 384-well plate format ( greiner ps , 784075 ) was the following : 2 l of cdk8/biotin - anti - his ab / sa - eu mix in assay buffer was pipetted into the wells of the microplates . then 1 l compounds in 20 mm hepes buffer/5% dmso was added . the plates were shaken for 30 s and incubated for 20 min at rt . then 2 l of alexa647 tracer in assay buffer was added . the plates were shaken for 30 s and incubated for 60 min at rt in the dark . finally the plates were read out on a perkinelmer envision 2104 ( mode lance / trf , excitation 340 nm , emissions at 615 and 615 nm ) . for evaluation the emission ratios ( 615 nm/655 nm ) were calculated . these were normalized to data from control wells containing vehicle only or 6 at 30 um . the final concentrations of the reaction components in 5 l total assay volume were 1% dmso ( merck no . pv4402 ) , 2 nm biotin - a - his ab ( invitrogen no . data are reported as average values of at least two determinations with standard deviations unless otherwise noted . interaction of compounds with human cdk8(aa1 - 464)/cyclin c(aa1 - 283 ) and human cdk19(aa1 - 502)/cyclin c(aa1 - 283 ) was analyzed using the reporter displacement assay provided by proteros biostructures gmbh . in brief , the reporter displacement assay is based on reporter probes that are designed to bind to the site of interest of the target protein . the proximity between reporter and protein results in the emission of an optical signal . compounds that bind to the same site as the reporter probe displace the probe , causing signal diminution . reporter displacement is measured over time after addition of compounds at various concentrations . for ic50 determination , percent probe displacement values are calculated for the last time point at which the system has reached equilibrium . for each compound concentration , percent probe displacement values are calculated and plotted against the compound concentration . ic50-like values ( corresponding to 50% probe displacement ) are calculated using standard fitting algorithms . data are reported as average values of at least two determinations with standard deviations unless otherwise noted . female athymic nude mice crl : nu(ncr)-foxn1 supplied by charles river laboratories were acclimatized for 1 week prior to use . the human colorectal adenocarcinoma cell line , sw620 ( atcc , no . ccl-227 ) , was expanded in culture , and 5 million cells were injected sc into the right flanks of animals ( n = 12 per group ) under isofluorothane anesthesia . tumors were established for 7 days when dosing commenced ( day 0 ) and continued for 16 days . the compound was weighed into a glass 30 ml universal tube , and methocel solution ( 0.5% methocel , 0.25% tween 20 in sterile phosphate buffered saline ) was added to provide a final concentration of 3 mg / ml 109 . the preparation was agitated at room temperature by magnetic stirrer to bring it into a fine suspension before use . animals were dosed orally by gavage every 24 h at 0.1 ml per 10 g body weight . tumors were measured three times weekly by vernier calipers and body weights recorded . at the end of the study , animals were culled at intervals : 3 control and 3 treated at 1 , 2 , 6 , and 24 h after the final dose . heparinized blood was collected by cardiac puncture , spun , and plasma snap frozen for analysis of compound exposure . tumors were excised , weighed and samples snap frozen for compound quantification and pd analyses . tumor growth was significantly inhibited ( t / c = 45.8 ; p = 0.0241 final tumor weights ) . ms / ms on a water tqs following a separation on a waters acquity beh c18 column ( 50 mm 2.1 mm ; 1.7 m ) with conditions of 0.1% formic acid ( mobile phase a ) and methanol ( mobile phase b ) . the column was equilibrated at initial condition of 95% a and 5% b , linear gradient over 3 min to 100% b , held over 1 min , followed by linear gradient back to 5% b over 0.1 min , at 0.6 ml / min flow rate . detection was achieved in positive electrospray ionization mode by multiple reaction monitoring , 412.17 > 376.40 at 33 ev for compound 109 , and 299.19 > 91.24 at 33 ev , 299.19 > 177.29 at 26 ev for the internal standard olomoucine . samples were quantified by external standard calibration ( 8-point calibration ranging from 2 nm to 50 000 nm ) . quality controls were included at 25 , 250 , 2500 , and 7500 nm in duplicate at the beginning and the end of the analytical run . tumors were excised , immediately snap - frozen in liquid nitrogen , and stored at 80 c until use . samples were transferred to mk28 reinforced homogenizing tubes with metal beads ( stretton scientific ) , and cdk lysis buffer ( 50 mm tris - hcl , ph7.4 , 1 mm edta , 1% triton x-100 ( v / v ) , 150 mm nacl , 1 mm activated sodium orthovanandate , 1 mm pmsf , protease cocktail ( sigma p8340 1:100 ) , and phosphatase inhibitors ( sigma p5726 and p0044 1:50 dilution ) were added immediately . the samples were ground using a precellys 24 at 6000 rpm , 2 20 s ( stretton scientific ) and lysates frozen at 80 c until use . for analysis , samples were debanked and sonicated for 3 min , on ice for 10 min . tubes were spun at 14 000 rpm at 4 c for 10 min , and supernatants were collected , aliquoted , and frozen at 80 c until use . tumor lysates were diluted 1:5 in lysis buffer and the concentrations determined using a direct detect spectrometer ( merck millipore ) . samples were aliquoted , then boiled in electrophoresis sample buffer and loaded on sds-4 - 12% page gels . after transfer to pvdf membranes , blots were blocked at room temperature for 1 h in blocking buffer ( 5% dry milk in tnt : 1 m tris - hcl , ph 8 , 5 m nacl , 0.1% tween 20 ) and then incubated at 4 c overnight with the appropriate antibody ( table 11 ) . after washing with tnt , blots were incubated with hrp - conjugated secondary antibodies in blocking buffer at room temperature for 1 h , and bands were visualized using the enhanced chemoluminescence ( ecl prime ) method . membranes were cut and incubated at the same time for phospho - stat ( 84 , 91 kda ) , total stat1 ( 84 , 91 kda ) , and gapdh ( 37 kda ) .

the mediator complex - associated cyclin - dependent kinase cdk8 has been implicated in human disease , particularly in colorectal cancer where it has been reported as a putative oncogene . here we report the discovery of 109 ( cct251921 ) , a potent , selective , and orally bioavailable inhibitor of cdk8 with equipotent affinity for cdk19 . we describe a structure - based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance , minimize transporter - mediated biliary elimination while maintaining acceptable aqueous solubility . compound 109 affords the optimal compromise of in vitro biochemical , pharmacokinetic , and physicochemical properties and is suitable for progression to animal models of cancer .

Introduction Chemistry Results and Discussion Conclusions Experimental Section

the mediator complex is a multiprotein assembly comprising at least 30 subunits that functions as a regulator of gene transcription in multiple contexts including stem cell function , the immune response , inflammation , cell adhesion , the epithelial to mesenchymal transition and development . the mediator complex - associated kinase cdk8 and its paralog cdk19 are cyclin c - dependent enzymes that , along with med12 and med13 , form the kinase module of the mediator complex . cdk8 has been reported to regulate basal transcription by phosphorylation of rna polymerase ii and to phosphorylate e2f1 , thereby activating wnt signaling . consistent with these reports , cdk8 is located in a region of chromosome 13 known to undergo copy number gain in 60% of colorectal cancers and inducible shrna - mediated knockdown of cdk8 protein reduces the growth of ht29 and colo205 colorectal cancer human tumor xenograft animal models harboring cdk8 gene amplification . cdk19 has been reported to form mediator complexes independent of cdk8 ; however their context - dependent roles are the subject of ongoing study . the marketed kinase inhibitor sorafenib ( 2 ) has been cocrystallized with cdk8/cyclin c , and subsequently the same group reported a fragment - based approach to cdk8 ligands building from the urea moiety associated with the type ii binding mode of sorafenib . recently , a cell - based hts campaign seeking inhibitors of p21-activated transcription was reported ; this effort led to the discovery of aminoquinazoline - based cdk8/19 ligands , exemplified by senexin b ( 5 ) ( chart 1 ) . we have previously reported the discovery of 6 ( cct251545 ) , a potent , orally bioavailable small molecule inhibitor of wnt signaling from a cell - based pathway screen ( chart 1 ) . here we describe the medicinal chemistry optimization of 6 to compound 109 , a potent , selective , and orally bioavailable inhibitor of cdk8 with equipotent affinity for cdk19 that demonstrates potent cell - based activity together with improved pharmacokinetic and pharmaceutical properties . compound 6 is a high affinity ligand for cdk8 and cdk19 ( ic50 of 7.2 1.4 and 6.0 1.0 nm , respectively ) and demonstrates potent inhibition of wnt - dependent signaling using our previously described inducible luciferase reporter assay in human embryonic kidney cells ( hek293 ) that contains both estrogen receptor - dishevelled ( dvl2 ) and tcf - luciferase - ires - gfp constructs ( 7df3 ic50 = 5.0 2.0 nm , table 2 ) ; it also demonstrates potent activity in ls174 t human colorectal carcinoma cells that harbor a reporter - based readout measuring constitutive -catenin mutation - driven wnt pathway activity ( ic50 = 23 11 nm ) . in the course of improving physicochemical , pharmaceutical , and pharmacokinetic properties , we monitored in vitro biochemical affinity for cdk8 and cdk19 which we found to consistently predict for cell - based activity in both the reporter - based 7df3 and ls174 t assays ; we also observed consistent sar in these two reporter - based cellular assays for compounds described in this manuscript ( figure s1 in supporting information ) . introduction of a basic center resulted in a 4-fold drop in potency in both biochemical and cell - based assays , suggesting that permeability is not responsible for the observed decrease ( table 2 , entries 46 ) . furthermore , the thermodynamic aqueous solubility of 22 was improved by 120-fold ; however a 23-fold drop in cell - based potency was also observed . the aminoindazole 25 gave an acceptable in vitro clearance profile across all species and high aqueous solubility ( table 2 , entry 10 ) ; however , as for most of the derivatives in table 2 , we observed low oral bioavailability and no improvement of the in vivo mouse clearance compared to 6 . notably , binding to the hinge via the pyridine is conserved despite the introduction of a potential alternative 3-aminoindazole hinge - binding motif ; pleasingly , the only difference we observe between the binding modes of compounds 6 and 25 is the presence of a dmso molecule in a small pocket adjacent to 25 ( see figure 1 , panel a ) . compound 42 ( table 3 , entry 5 ) , which combines the optimal carbamate isomer and an n - methyl sultam , gave very potent cdk8/19 affinity with improved metabolic stability compared to 6 . pleasingly , all derivatives were significantly more stable in microsomal clearance assays ; however , translation to cell - based potency was eroded for the imidazolidine-2,4-dione , 2-methylimidazole-5-one , and piperazine-2,5-dione derivatives ( table 3 , entries 811 ) and no examples demonstrated significant improvement in in vivo mouse clearance ( table 3 , entries 711 ) . given that the lower dynamic range of our in vitro biochemical assays for cdk8 and cdk19 is approximately 3 nm due to the concentration of enzyme ( 5 and 6.7 nm , respectively ) , it is possible that the exquisite cell - based potency for compound 49 is on - target . as noted above , a dmso - filled pocket was observed in the x - ray crystal structure of 25 ( figure 1 , panel a ) , and we postulated that potency could be regained by targeting this region . despite its high in vivo clearance , the spirocarbamate 42 ( table 3 , entry 5 ) was attractive from its in vitro profile and we considered spirocarbamate as a potential replacement for the spirolactam in our further optimization . the protein ligand crystal structure of 25 in cdk8/cyclin c demonstrates that the c2-h of the pyridine is proximal to the backbone carbonyl of asp98 ( distance of 2.3 , figure 1 ) , an observation consistent with our previously reported crystal structure of 6 in cdk8/cyclin c. we hypothesized that introduction of a hydrogen bond donor at the 2 position of the pyridine could form a favorable interaction with the backbone carbonyl of asp98 . thus , in vitro clint did not appear to be a sufficient predictor of in vivo clearance for this series . taken together , these data are consistent with the notion that transporter - mediated hepatic uptake and elimination contribute to the overall clearance of some compounds in this series . analysis of physicochemical properties versus efflux ratio in a madin darby canine kidney epithelial cell line ( mdck ) , transfected to express human p - gp ( mdr - mdck ) , led to broadly similar trends . analysis of caco-2 data in the 2-aminopyridine series indicated that physicochemical properties in the range hbd 3 and tpsa < 100 or hbd 3 and log p > 3 are necessary for low efflux in this chemical series , and a combination of hbd 3 , tpsa < 100 , and log p > 3 is more likely to lead to a low efflux ratio . these observations are consistent with previously published studies demonstrating that a fine balance of physicochemical properties is often required to achieve potency , metabolic stability , and low efflux in a single molecule . the spirolactam derivative 109 ( measured log d = 2.5 ) was potent ( cdk8 ic50 = 4.9 0.6 nm , cdk19 ic50 = 2.6 0.4 nm with residence times of 53 and 86 min , respectively , in the reporter displacement assay ) , soluble , and stable with a low efflux ratio and acceptable in vivo mouse pharmacokinetics ( cl = 0.61 l / h / kg , f = 30% , table 9 ) ; however , the carbamate matched pair 110 was less potent and subject to increased efflux . crystallization of 109 in cdk8/cyclin c ( figure 6 ) demonstrated a binding mode consistent with those previously observed , and pleasingly , a new hydrogen bond interaction between the exocyclic nitrogen of the 2-aminopyridine scaffold and the backbone carbonyl of asp98 was observed ( 3.0 ) . pleasingly , compound 109 had acceptable aqueous solubility ( table 8 , entry 2 ) and demonstrated minimal activity when tested in a panel of 55 receptors , ion channels , and enzymes at 1 m ( tables s2 and s3 ) and in a panel of 279 kinases ( table s4 ) ; weak inhibition of cyps was observed ( table s5 ) . consistent with the profile of chemical probe 6 , compound 109 demonstrated potent inhibition of reporter - based readouts measuring basal wnt pathway activity in human cancer cell lines that have constitutively activated wnt pathway signaling : ls174 t ( -catenin mutant ) , sw480 and colo205 ( apc mutant ) or pa-1 human teratocarcinoma cells that are wnt ligand dependent ( table 10 ) compound 109 was then assessed in the in vivo apc - mutant sw620 human colorectal carcinoma xenograft model , treating established tumors in female ncr athymic mice . we monitored inhibition of stat1 phosphorylation , which we have previously demonstrated to be an in vitro cell - based and in vivo pharmacodynamic biomarker of cdk8 inhibition . in light of its potent and selective profile coupled with good oral pharmacokinetics and duration of in vivo target engagement on oral dosing , compound 109 ( cct251921 ) has been selected for progression into further preclinical in vivo efficacy and safety studies . literature evidence and our own studies point to a role for the mediator complex - associated kinases cdk8 and cdk19 in human disease , particularly in colorectal cancer where cdk8 has been reported as a putative oncogene . cdk19 , a paralogue of cdk8 , is relatively unexplored , and our previous studies demonstrate that selectivity for cdk8 over cdk19 with a small molecule ligand is likely to be challenging . indeed , in our recently reported discovery of cct251545 ( 6 ) , a potent and selective chemical probe for the further exploration of cdk8 and cdk19 pharmacology , we show strong correlation of cdk8 and cdk19 binding affinity . here , we have further optimized the small molecule cdk8/19-selective chemical probe 6 to give compound 109 by improving oral pharmacokinetics and pharmaceutical properties in order to facilitate further in vivo evaluation of cdk8/19 pharmacology and progression into preclinical in vivo efficacy and safety studies . in attempting to reduce oxidative metabolism by reducing lipophilicity while maintaining key interactions within the cdk8 kinase domain , we benefited from detailed knowledge of the binding mode of 6 and analogs through protein we were able to improve in vitro metabolic stability by introducing a c-2 amino substituent to the pyridine scaffold which reinforced interactions with the kinase hinge region and reduced compound lipophilicity . however , we identified transporter - mediated hepatic uptake as a component of in vivo clearance . through careful correlation of in vitro caco-2 efflux ratios with physicochemical properties and subsequent medicinal chemistry design within desirable physicochemical property ranges , we were able to identify compounds that demonstrated reduced clearance without increased susceptibility to active hepatic uptake . compound 109 was identified as the best compromise of in vitro biochemical and pharmacokinetic properties that demonstrated acceptable in vivo pharmacokinetics suitable for progression to in vivo animal models of cancer . 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 ( 58 mg , 0.19 mmol ) , 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one8 ( 54 mg , 0.16 mmol ) , and pd(dppf)cl2ch2cl2 ( 6.4 mg , 7.9 mol ) were loaded in a microwave vial , and then 0.5 m sodium carbonate in water ( 440 l , 0.220 mmol ) and acetonitrile ( 2.8 ml ) were added . the organic layer was washed with water , dried , filtered , and evaporated to dryness to give tert - butyl 3-[bis(tert - butoxycarbonyl)amino]-6-bromoindazole-1-carboxylate ( 1.42 g , 73% pure , 86% corrected yield ) as a colorless oil , which was used without further purification . bis(pinacolato)diboron ( 486 mg , 1.91 mmol ) , potassium acetate ( 375 mg , 3.83 mmol ) , and pd(dppf)cl2ch2cl2 ( 78 mg , 0.096 mmol ) were added , and the red reaction mixture was stirred for 15 h at 70 c . 4-aminomethyl-1-benzylpiperidin-4-ol ( 25.0 g , 113 mmol ) was suspended in dcm ( 400 ml ) , and a solution of triphosgene ( 32.8 g , 110 mmol ) dissolved in dcm ( 200 ml ) was added dropwise while maintaining the temperature between 30 and 35 c . to the reaction mixture 1 n naoh solution ( 200 ml ) was added , and the organic phase was separated , washed with water , dried over sodium sulfate , filtered , and evaporated to dryness . the combined organic layers were washed with brine ( 100 ml ) , dried over na2so4 , and concentrated to give the crude product as a brown viscous solid . 5-bromo-4-chloro-3-(trifluoromethyl)pyridin-2-amine 75 ( 300 mg , 1.09 mmol ) and 1-oxa-3,8-diazaspiro[4.5]decan-2-one 38 ( 340 mg , 2.18 mmol ) were dissolved in nmp ( 3 ml ) , and triethylamine ( 0.453 ml , 3.27 mmol ) was added . the residue was purified by flash chromatography ( n - heptane / etoac , gradient ) and by preparative hplc ( method a , gradient elution from 0:100 to 30:70 over 3 min and to 60:40 over 17 min acetonitrile / water ) to give first the desired phenyl ( 6-bromo-1h - indazol-3-yl)carbamate ( 54 mg , 3% yield ) as a white solid , which was used directly in the next step , and second undesired phenyl 3-amino-6-bromo-1h - indazole-1-carboxylate ( 346 mg ) as a white solid . bis(pinacolato)diboron ( 179 mg , 0.704 mmol ) , potassium acetate ( 104 mg , 1.06 mmol ) , and pd(dppf)cl2ch2cl2 ( 29 mg , 0.035 mmol ) were added , and the red reaction mixture was stirred overnight at 70 c . 6-bromo - n , n,1-trimethyl-1h - indazol-3-amine ( 100 mg , 0.394 mmol ) , bis(pinacolato)diboron ( 150 mg , 0.590 mmol ) , potassium acetate ( 116 mg , 1.18 mmol ) , and pd(dppf)cl2ch2cl2 ( 16 mg , 0.020 mmol ) were loaded in a flask , and dme ( 2.8 ml ) was added . the resulting light yellow precipitate was filtered , washed with water ( 150 ml ) , and dried at 70 c under vacuum overnight to give 8-(2-amino-3-chloro-5-(1-methyl-1h - indazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 109 ( 3.34 g , 75% yield ) as a light yellow solid . microsomes ( final concentration 0.5 mg / ml ) , 50 mm phosphate buffer ph 7.4 , and compound ( final concentration 1 m ) were added to the assay plate and allowed to preincubate for 5 min at 37 c . allometric methods used for clearance prediction to human were simple allometry ( 3-species ) , allometry with ( a ) correction by fu , ( b ) with correction by fu and mlp ( maximum life span ) , ( c ) with correction by fu and brw ( brain weight ) ; in addition , in vitro to in vivo ( iviv ) scaling from liver microsomes and hepatocytes was used for clearance prediction ( for compounds 6 and 109 , iviv from hepatocytes of 0.53 and 0.215 l / h / kg may be included in the mean calculation ) . ccl-227 ) , was expanded in culture , and 5 million cells were injected sc into the right flanks of animals ( n = 12 per group ) under isofluorothane anesthesia . membranes were cut and incubated at the same time for phospho - stat ( 84 , 91 kda ) , total stat1 ( 84 , 91 kda ) , and gapdh ( 37 kda ) .

more than 85 to 90% of all women report premenstrual syndrome ( pms ) , which includes psychosomatic changes , including irritation , depression , food cravings , and breast pain before and during menstruation ( 1 , 2 ) . approximately 24 - 40% of reproductive - aged women experience pms ( 3 , 4 ) . this syndrome has been reported to account for 3 - 16% of the economic loss from workforce degradation ( 5 ) . thus , pms is not only a personal issue , but also a social concern . multiple physiological and psychosocial factors such as ovarian hormones , abnormalities in neurotransmitter physiology , lifestyle habits and stress are related to pms ( 6 , 7 ) . relieving the syndrome through self - care is currently one of the therapeutic approaches available for pms because its exact pathology is unknown , and no effective therapy has been discovered . some studies have shown that stress is associated with pms ( 3 , 4 , 8) . most measurements of the stress response in women with pms have used perceived stress , and no studies quantifying stress based on biochemical parameters have been conducted . measures of stress response include : 1 ) perceived stress , using tools such as questionnaires ; 2 ) physiological parameters , including blood pressure and heart rate variability ; and 3 ) biochemical parameters such as blood cortisol levels ( 9 ) . the influence of the stress due to pain caused by blood sampling on biochemical parameters has long been regarded as a problem in medicine . it is an important biological defense hormone , along with catecholamines , and it has been measured as a classic stress parameter due to its response to acute psychological / physical stress ( 10 ) . in the recent years , the evaluation of stress using easy , noninvasive measurements of salivary substances such as secretory immunoglobulin a ( s - iga ) and cortisol levels has become available ( 11 , 12 ) . the aim of this study was to analyze the association between pms and stress across two menstrual cycles using biochemical parameters and questionnaire - based reports . the sample size was calculated based on power analysis by g*power ( 13 , 14 ) , with power of 0.80 and an effect size of 0.40 at p < 0.05 with analysis of variance with repeated measures . a sample size of 12 was estimated for each group . the subjects were recruited using the following process : the subjects were required to be residents of the kansai region in japan , so that the researchers could meet with them to explain the research methods . the general method of recruitment was through the investigators personal networks , including social groups , a junior high school parent - teacher association , and a university nursing school . subjects were included only if they were not pregnant or breast - feeding , had no chronic health conditions or psychiatric conditions , and were not currently using oral contraceptives . , the following subjects withdrew from participation during the study : two subjects who were unable to have their stress levels evaluated on saliva testing due to irregular menstrual cycles ; one subject who discovered she was pregnant ; and four subjects who were unable to continue taking basal body temperature measurements . the final study set comprised 24 subjects ( age range of 21 to 38 years ) . these subjects had regular self - reported menstrual cycle lengths of 25 - 38 days . among the subjects , 13 reported having premenstrual syndrome ( pms group ) , and 11 reported not having premenstrual syndrome ( n - pms group ) . based on published diagnostic criteria for pms ( 1 ) , the subjects were classified in the two mentioned groups based on both their basal body temperature during three to five menstrual cycles and subjective records kept in a menstrual symptom journal , the pms memory diary ( 15 ) . the pms memory diary recorded 17 listed symptoms and the respondents perceived symptoms at four different levels ( blank or 0 , no symptoms ; 1 ) moderate symptoms that did not affect everyday activities ; 2 ) symptoms that affected everyday activities ; and 3 ) severe symptoms ) . cumulative scores were calculated based on the severity and frequency of symptoms in each phase . day 1 was defined as the day of the onset of menses , which was recorded as the start of the menstrual cycle . cycle phases were defined as follows : premenstrual phase ( one to seven days before menstruation ) ; menstrual phase ( days one to seven after the start of menstruation ) ; and postmenstrual phase ( days eight to fourteen after the start of menstruation ) . perceived stress and biochemical parameters were measured twice over two complete menstrual cycles , with two measurements for each item , to ensure reproducibility . before stress measurements were taken , premenstrual , menstrual and postmenstrual phases salivary s - iga concentrations ( g / ml ) and secretion rates ( g / min ) , as well as cortisol concentrations ( g / dl ) , were measured as biochemical parameters . cortisol concentrations are high in the morning and decrease in the evening , indicating circadian variations typical of humans ( 16 ) . given this circadian pattern , collection early in the morning was avoided , and the effects of subjects everyday activities were taken into account . saliva samples were collected using a salimetrics oral swab ( salimetrics llc , state college , pa ) by a medical technologist . to standardize collection conditions , samples were collected between 5:00 p.m. and 8:00 p.m. in addition , the difference in collection time between menstrual phases was strictly kept to less than 30 minutes . the oral cavity of each subject was rinsed with water 10 minutes before sample collection . the oral swab was placed under the tongue for two minutes and then transferred to a swab storage tube ( salimetrics llc ) . the volume of saliva collected through the oral swab was determined by weighing the device along with the storage tube before and after collection . collected samples were frozen immediately at -20c and then thawed completely , vortexed , and centrifuged at 1500 g for 15 minutes . the supernatants were stored at 80c until analysis ( within six months ) . the cortisol levels and both the s - iga concentrations and secretion rates sampling methods and laboratory procedures were followed in accordance with previous studies , and it is believed that the reliability of test values was confirmed , since reference values were adopted from previous studies ( 17 , 18 ) . the levels of perceived stress were assessed using the japanese version of the stress checklist km ( scl - km ) ( 19 ) . the scl - km uses a binary - choice format for 30 items of psychosomatic complaints that can develop to a stress response . each item is a two - point response scale ( 0=not at all or 1=yes ) . one point is given to any item reflecting high evaluation of stress ; thus , the possible point range is from 0 to 30 . abbreviations : bbt , basal body temperature ; scl - km , stress checklist km ; s - iga , secretory immunoglobulin a. day 1 , onset of menses and start of the menstrual cycle . the spss software ( version 20.0 , ibm , tokyo , japan ) was used for data analysis . the mann - whitney nonparametric u test was used to examine any differences in the present age and age at menarche between the pms and n - pms groups . chi - square tests were used to examine differences in marital status , parity , smoking status , and alcohol use between the groups . outcome data were not normally distributed and were transformed to log 10 values prior to statistical analyses . a two - way repeated measures analysis of variance was used for the groups ( pms and n - pms ) and the menstrual cycle phases ( premenstrual , menstrual and postmenstrual ) . after confirming that there was no significant interaction between group and menstrual cycle phase , analysis of variance with repeated measures after confirmation of the reasonably normal distribution of the two - paired samples , student s t - test was used to evaluate differences between groups . correlation testing between perceived stress and biochemical parameters was conducted using pearson s correlation coefficient for both groups combined . the objectives of the study were explained , and informed consent was obtained from each subject included in the study . the kobe university graduate school of medicine , medical ethics committee approved the study ( no . the sample size was calculated based on power analysis by g*power ( 13 , 14 ) , with power of 0.80 and an effect size of 0.40 at p < 0.05 with analysis of variance with repeated measures . a sample size of 12 was estimated for each group . the subjects were recruited using the following process : the subjects were required to be residents of the kansai region in japan , so that the researchers could meet with them to explain the research methods . the general method of recruitment was through the investigators personal networks , including social groups , a junior high school parent - teacher association , and a university nursing school . subjects were included only if they were not pregnant or breast - feeding , had no chronic health conditions or psychiatric conditions , and were not currently using oral contraceptives . , the following subjects withdrew from participation during the study : two subjects who were unable to have their stress levels evaluated on saliva testing due to irregular menstrual cycles ; one subject who discovered she was pregnant ; and four subjects who were unable to continue taking basal body temperature measurements . the final study set comprised 24 subjects ( age range of 21 to 38 years ) . these subjects had regular self - reported menstrual cycle lengths of 25 - 38 days . among the subjects , 13 reported having premenstrual syndrome ( pms group ) , and 11 reported not having premenstrual syndrome ( n - pms group ) . based on published diagnostic criteria for pms ( 1 ) , the subjects were classified in the two mentioned groups based on both their basal body temperature during three to five menstrual cycles and subjective records kept in a menstrual symptom journal , the pms memory diary ( 15 ) . the pms memory diary recorded 17 listed symptoms and the respondents perceived symptoms at four different levels ( blank or 0 , no symptoms ; 1 ) moderate symptoms that did not affect everyday activities ; 2 ) symptoms that affected everyday activities ; and 3 ) severe symptoms ) . cumulative scores were calculated based on the severity and frequency of symptoms in each phase . day 1 was defined as the day of the onset of menses , which was recorded as the start of the menstrual cycle . cycle phases were defined as follows : premenstrual phase ( one to seven days before menstruation ) ; menstrual phase ( days one to seven after the start of menstruation ) ; and postmenstrual phase ( days eight to fourteen after the start of menstruation ) . perceived stress and biochemical parameters were measured twice over two complete menstrual cycles , with two measurements for each item , to ensure reproducibility . before stress measurements were taken , premenstrual , menstrual and postmenstrual phases were confirmed based on records of basal body temperature taken across two menstrual cycles . salivary s - iga concentrations ( g / ml ) and secretion rates ( g / min ) , as well as cortisol concentrations ( g / dl ) , were measured as biochemical parameters . cortisol concentrations are high in the morning and decrease in the evening , indicating circadian variations typical of humans ( 16 ) . given this circadian pattern , collection early in the morning was avoided , and the effects of subjects everyday activities were taken into account . saliva samples were collected using a salimetrics oral swab ( salimetrics llc , state college , pa ) by a medical technologist . to standardize collection conditions , samples were collected between 5:00 p.m. and 8:00 p.m. in addition , the difference in collection time between menstrual phases was strictly kept to less than 30 minutes . the oral cavity of each subject was rinsed with water 10 minutes before sample collection . the oral swab was placed under the tongue for two minutes and then transferred to a swab storage tube ( salimetrics llc ) . the volume of saliva collected through the oral swab was determined by weighing the device along with the storage tube before and after collection . collected samples were frozen immediately at -20c and then thawed completely , vortexed , and centrifuged at 1500 g for 15 minutes . the cortisol levels and both the s - iga concentrations and secretion rates were determined using enzyme - linked immunoassay kits ( salimetrics llc ) . sampling methods and laboratory procedures were followed in accordance with previous studies , and it is believed that the reliability of test values was confirmed , since reference values were adopted from previous studies ( 17 , 18 ) . the levels of perceived stress were assessed using the japanese version of the stress checklist km ( scl - km ) ( 19 ) . the scl - km uses a binary - choice format for 30 items of psychosomatic complaints that can develop to a stress response . each item is a two - point response scale ( 0=not at all or 1=yes ) . one point is given to any item reflecting high evaluation of stress ; thus , the possible point range is from 0 to 30 . abbreviations : bbt , basal body temperature ; scl - km , stress checklist km ; s - iga , secretory immunoglobulin a. day 1 , onset of menses and start of the menstrual cycle . the spss software ( version 20.0 , ibm , tokyo , japan ) was used for data analysis . the mann - whitney nonparametric u test was used to examine any differences in the present age and age at menarche between the pms and n - pms groups . chi - square tests were used to examine differences in marital status , parity , smoking status , and alcohol use between the groups . outcome data were not normally distributed and were transformed to log 10 values prior to statistical analyses . a two - way repeated measures analysis of variance was used for the groups ( pms and n - pms ) and the menstrual cycle phases ( premenstrual , menstrual and postmenstrual ) . after confirming that there was no significant interaction between group and menstrual cycle phase , analysis of variance with repeated measures after confirmation of the reasonably normal distribution of the two - paired samples , student s t - test was used to evaluate differences between groups . correlation testing between perceived stress and biochemical parameters was conducted using pearson s correlation coefficient for both groups combined . the objectives of the study were explained , and informed consent was obtained from each subject included in the study . the kobe university graduate school of medicine , medical ethics committee approved the study ( no . the pms and n - pms groups did not differ significantly regarding age , marital status or parity . all subjects were nonsmokers , and their alcohol drinking frequency was less than one drink a week for most subjects ( table 1 ) . abbreviations : pms , having premenstrual syndrome ; n - pms , not having premenstrual syndrome . two - way analysis of variance showed no interaction between groups and menstrual cycle phases . no significant differences were found in the s - iga concentration and secretion rate and the cortisol level between the groups ( figures 2 and 3 ) . no significant differences were observed in the s - iga concentration and secretion rate among menstrual cycle phases in the n - pms group . the s - iga concentrations in the first [ f ( 2 , 36 ) = 9.330 , p < 0.001 ] and second [ f ( 2 , 36 ) = 6.009 , p < 0.01 ] measurements were significantly higher in the premenstrual phase than in the postmenstrual phase in the pms group . moreover , the s - iga concentration in the first measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group . the s - iga secretion rate in the second measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group [ f ( 2 , 36 ) = 5.178 , p < 0.05 ] . postmenstrual s - iga concentration ( t ( 22 ) = 2.090 , p < 0.05 , 95% confidence interval , ci , [ 0.012 0.485 ] ; figure 2 ) and secretion rate ( t ( 22 ) = 2.129 , p < 0.05 , 95% confidence interval , ci , [ 0.009 0.651 ] ; figure 2 ) were significantly lower at the second measurement in the pms group when compared to the n - pms group . no significant differences in the cortisol level were seen in either group at any phase ( figure 3 ) . n - pms-1 , n - pms-2 : the first and second measurements of the n - pms group . no significant changes in the scl - km score were observed among menstrual cycle phases in either group . premenstrual ( t ( 22)=3.812 , p < 0.001 , 95% ci [ 2.5 , 8.4 ] ; t ( 22)=2.733 , p < 0.05 , 95% ci [ 5.9 , 19.7 ] ) and postmenstrual phase ( t ( 22)=2.347 , p < 0.05 , 95% ci [ 1.8 , 7.9 ] ; t ( 22)=2.098 , p < 0.05 , 95% ci [ 1.7 , 7.1 ] ) scl - km scores were significantly higher in the pms group than in the n - pms group ( for both measurements ) . menstrual phase scl - km scores in the second measurement were significantly higher in the pms group than in the n - pms group ( t ( 22)=3.034 , p < 0.01 , 95% ci [ 1.3 , 6.9 ] ; figure 4 ) . a significant negative correlation was observed between s - iga [ concentration ( r = 0.491 , p < 0.05 ) ; secretion rate ( r = 0.543 , p < 0.01 ) ] and scl - km scores at the second measurement in the postmenstrual phase . furthermore , no significant correlations were found between the cortisol level and scl - km scores . the pms and n - pms groups did not differ significantly regarding age , marital status or parity . all subjects were nonsmokers , and their alcohol drinking frequency was less than one drink a week for most subjects ( table 1 ) . abbreviations : pms , having premenstrual syndrome ; n - pms , not having premenstrual syndrome . two - way analysis of variance showed no interaction between groups and menstrual cycle phases . no significant differences were found in the s - iga concentration and secretion rate and the cortisol level between the groups ( figures 2 and 3 ) . no significant differences were observed in the s - iga concentration and secretion rate among menstrual cycle phases in the n - pms group . the s - iga concentrations in the first [ f ( 2 , 36 ) = 9.330 , p < 0.001 ] and second [ f ( 2 , 36 ) = 6.009 , p < 0.01 ] measurements were significantly higher in the premenstrual phase than in the postmenstrual phase in the pms group . moreover , the s - iga concentration in the first measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group . the s - iga secretion rate in the second measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group [ f ( 2 , 36 ) = 5.178 , p < 0.05 ] . postmenstrual s - iga concentration ( t ( 22 ) = 2.090 , p < 0.05 , 95% confidence interval , ci , [ 0.012 0.485 ] ; figure 2 ) and secretion rate ( t ( 22 ) = 2.129 , p < 0.05 , 95% confidence interval , ci , [ 0.009 0.651 ] ; figure 2 ) were significantly lower at the second measurement in the pms group when compared to the n - pms group . no significant differences in the cortisol level were seen in either group at any phase ( figure 3 ) . n - pms-1 , n - pms-2 : the first and second measurements of the n - pms group . no significant changes in the scl - km score were observed among menstrual cycle phases in either group . premenstrual ( t ( 22)=3.812 , p < 0.001 , 95% ci [ 2.5 , 8.4 ] ; t ( 22)=2.733 , p < 0.05 , 95% ci [ 5.9 , 19.7 ] ) and postmenstrual phase ( t ( 22)=2.347 , p < 0.05 , 95% ci [ 1.8 , 7.9 ] ; t ( 22)=2.098 , p < 0.05 , 95% ci [ 1.7 , 7.1 ] ) scl - km scores were significantly higher in the pms group than in the n - pms group ( for both measurements ) . menstrual phase scl - km scores in the second measurement were significantly higher in the pms group than in the n - pms group ( t ( 22)=3.034 , p < 0.01 , 95% ci [ 1.3 , 6.9 ] ; figure 4 ) . a significant negative correlation was observed between s - iga [ concentration ( r = 0.491 , p < 0.05 ) ; secretion rate ( r = 0.543 , p < 0.01 ) ] and scl - km scores at the second measurement in the postmenstrual phase . furthermore , no significant correlations were found between the cortisol level and scl - km scores . this study examined changes in biochemical parameters of stress , and measured perceived stress during the menstrual cycle in women with pms . the findings of this study indicated that the postmenstrual s - iga concentration and secretion rate were significantly lower at the second measurement in the pms group than in the n - pms group . the s - iga is a protein that plays a major role in the local immune system of the mucosal epithelium . evidence indicates that its secretion sensitively reflects changes in the immune system ( 10 ) . secretory iga has been reported to decrease in response to chronic stress and general life events , and it has been suggested to be a chronic stress marker ( 20 , 21 ) . the present study results suggest that lower s - iga levels indicate chronic stress in the postmenstrual phase , even without the psychosomatic burden caused by pms . conversely , the findings of this study indicated that s - iga concentration was significantly higher during the premenstrual or menstrual phase than the postmenstrual phase in the pms group . significantly higher levels of s - iga were reported during the menstrual phase in women with dysmenorrhea compared with women without dysmenorrhea ; this was likely to be a result of pain ( 17 ) . in addition , elevated s - iga levels have been shown to indicate a transient acute stress response ( 22 , 23 ) . this suggests that , accompanied by chronic stress conditions , psychosomatic changes associated with pms increase stress responses in women with premenstrual syndrome . cortisol has been reported to be secreted in response to stronger stress loads than s - iga ( 24 , 25 ) . stresses from everyday activities have a greater effect on pms than unusual severe stress ( 26 ) . in the present study , changes in cortisol levels were not observed , presumably because the subjects in this study did not have a severe stress load . the scl - km score was significantly higher in the pms group than in the n - pms group in both the premenstrual and menstrual phases . in addition , perceived stress scores were similar among menstrual cycle phases in both groups , with or without pms . this suggests a high perceived stress condition in women with pms , supporting the results of previous studies ( 27 ) . higher levels of nervous tension , anxiety , depression , and anger have been reported to be caused by either pms ( 28 ) or perceived stress ( 29 ) , primarily in the premenstrual phase . however , other studies reported no differences in stress responses among menstrual cycle phases ( 30 ) . since these studies used different scales and found no differences among menstrual phases in small studies with 21 - 25 subjects , further studies are necessary to verify the methods of measuring perceived stress . furthermore , it has been reported that 27% of the perceived stress response varies depending on the subject s status at the time of measurement ( 27 ) ; changing stress levels across the two menstrual cycles were reflected in salivary biochemical parameters and perceived stress scores . these might have affected the agreement between the results of the first and second measurements . there were negative correlations in the postmenstrual phase between the s - iga concentration and secretion rate and the scl - km score . these results indicate that s - iga can be used for the quantitative evaluation of chronic stress in women with pms . conversely , no significant correlations were observed between cortisol levels and the scl - km scores . this result is compatible with a previous study that used daily urine samples , in which no correlation was observed between cortisol and perceived stress ( 31 ) . in conclusion , whether cortisol would respond to the perceived stress analyzed in the present study remains to be clarified . the absence of significant correlations between cortisol levels and perceived stress scores might imply a fundamental difference between these two types of stress . first , the subjects were from a limited area in japan and were recruited through the researchers personal networks . therefore , the study was not randomized , which limits the generalizability of the results . second , the number of subjects was small , which limits the power of the analysis of the results of s - iga concentration and secretion rate from different menstrual cycle phases . third , the difference in sample size between the two groups increased type 1 error , which could potentially account for differences in marital status and parity between the groups , as well as for cortisol and perceived stress not being detected as statistically significant . for future research , further analyses of the associations of salivary s - iga and cortisol with pmsare needed with more subjects and experimental stress loads to test the usefulness of these biochemical parameters for quantitative stress measurements . the results of the present study suggest that measurements of s - iga , rather than cortisol or subjective responses to stress , may be most closely associated with pms . salivary s - iga can be used as a highly sensitive method for stress evaluation in women with pms in the general population . in addition , it can be a very helpful tool for evaluating the progress and success of stress management and reduction of pms symptoms .

background : several studies have demonstrated the associations between premenstrual syndrome and perceived stress , and no studies quantifying stress based on biochemical parameters have been conducted.objectives:the objective of this study was to examine the changes in biochemical parameters of stress and measured perceived stress during the menstrual cycle of women with premenstrual syndrome.patients and methods : a longitudinal observational study was conducted in 2010 in the kansai region of japan . thirteen women with premenstrual syndrome and 11 controls , all with regular menstrual cycles , participated in this study . salivary secretory immunoglobulin a ( s - iga ) and cortisol levels were measured as biochemical parameters , and scores on the stress check list km ( scl - km ) ( cronbach s in this study ranged from 0.76 to 0.84 ) were used to indicate perceived stress through two complete menstrual cycles . before stress measurements were taken , premenstrual , menstrual and postmenstrual phases were confirmed based on records of basal body temperature across two menstrual cycles . data analysis was performed using the student s t - test , analysis of variance with repeated measures , and pearson s correlation coefficient , as appropriate.results:both the postmenstrual s - iga concentration and secretion rate were significantly lower in the group with premenstrual syndrome than in controls ( p < 0.05 ) . premenstrual s - iga concentrations were significantly higher than postmenstrual levels in the group with premenstrual syndrome ( p < 0.05 ) . no significant differences in cortisol levels were seen in either group during any phase . premenstrual and postmenstrual phase scl - km scores were significantly higher in the group with premenstrual syndrome than in controls ( p < 0.05 ) . no significant changes in the scl - km scores were observed among menstrual cycle phases in either group . postmenstrual s - iga levels were negatively correlated with the scl - km score ( p < 0.05).conclusions : the stress due to psychosomatic changes in the menstrual cycle is associated with premenstrual syndrome . measures of s - iga , rather than cortisol or subjective responses to stress , may be most closely associated with pms .

1. Background 2. Objectives 3. Patients and Methods 3.1. Subjects 3.2. Measures 3.3. Statistical Analysis 3.4. Ethical Considerations 4. Results 4.1. Subjects Characteristics 4.2. Premenstrual Syndrome and Biochemical Parameters:Salivary Secretory IgA (Concentration and Secretion Rate) and Cortisol 4.3. Premenstrual Syndrome and Perceived Stress 4.4. Correlations Between Salivary Biochemical Parameters and Perceived Stress Scores 5. Discussion

more than 85 to 90% of all women report premenstrual syndrome ( pms ) , which includes psychosomatic changes , including irritation , depression , food cravings , and breast pain before and during menstruation ( 1 , 2 ) . relieving the syndrome through self - care is currently one of the therapeutic approaches available for pms because its exact pathology is unknown , and no effective therapy has been discovered . some studies have shown that stress is associated with pms ( 3 , 4 , 8) . most measurements of the stress response in women with pms have used perceived stress , and no studies quantifying stress based on biochemical parameters have been conducted . measures of stress response include : 1 ) perceived stress , using tools such as questionnaires ; 2 ) physiological parameters , including blood pressure and heart rate variability ; and 3 ) biochemical parameters such as blood cortisol levels ( 9 ) . the influence of the stress due to pain caused by blood sampling on biochemical parameters has long been regarded as a problem in medicine . it is an important biological defense hormone , along with catecholamines , and it has been measured as a classic stress parameter due to its response to acute psychological / physical stress ( 10 ) . in the recent years , the evaluation of stress using easy , noninvasive measurements of salivary substances such as secretory immunoglobulin a ( s - iga ) and cortisol levels has become available ( 11 , 12 ) . the aim of this study was to analyze the association between pms and stress across two menstrual cycles using biochemical parameters and questionnaire - based reports . the sample size was calculated based on power analysis by g*power ( 13 , 14 ) , with power of 0.80 and an effect size of 0.40 at p < 0.05 with analysis of variance with repeated measures . the subjects were recruited using the following process : the subjects were required to be residents of the kansai region in japan , so that the researchers could meet with them to explain the research methods . , the following subjects withdrew from participation during the study : two subjects who were unable to have their stress levels evaluated on saliva testing due to irregular menstrual cycles ; one subject who discovered she was pregnant ; and four subjects who were unable to continue taking basal body temperature measurements . among the subjects , 13 reported having premenstrual syndrome ( pms group ) , and 11 reported not having premenstrual syndrome ( n - pms group ) . based on published diagnostic criteria for pms ( 1 ) , the subjects were classified in the two mentioned groups based on both their basal body temperature during three to five menstrual cycles and subjective records kept in a menstrual symptom journal , the pms memory diary ( 15 ) . cumulative scores were calculated based on the severity and frequency of symptoms in each phase . cycle phases were defined as follows : premenstrual phase ( one to seven days before menstruation ) ; menstrual phase ( days one to seven after the start of menstruation ) ; and postmenstrual phase ( days eight to fourteen after the start of menstruation ) . perceived stress and biochemical parameters were measured twice over two complete menstrual cycles , with two measurements for each item , to ensure reproducibility . before stress measurements were taken , premenstrual , menstrual and postmenstrual phases salivary s - iga concentrations ( g / ml ) and secretion rates ( g / min ) , as well as cortisol concentrations ( g / dl ) , were measured as biochemical parameters . given this circadian pattern , collection early in the morning was avoided , and the effects of subjects everyday activities were taken into account . the cortisol levels and both the s - iga concentrations and secretion rates sampling methods and laboratory procedures were followed in accordance with previous studies , and it is believed that the reliability of test values was confirmed , since reference values were adopted from previous studies ( 17 , 18 ) . the levels of perceived stress were assessed using the japanese version of the stress checklist km ( scl - km ) ( 19 ) . the scl - km uses a binary - choice format for 30 items of psychosomatic complaints that can develop to a stress response . abbreviations : bbt , basal body temperature ; scl - km , stress checklist km ; s - iga , secretory immunoglobulin a. day 1 , onset of menses and start of the menstrual cycle . the mann - whitney nonparametric u test was used to examine any differences in the present age and age at menarche between the pms and n - pms groups . chi - square tests were used to examine differences in marital status , parity , smoking status , and alcohol use between the groups . a two - way repeated measures analysis of variance was used for the groups ( pms and n - pms ) and the menstrual cycle phases ( premenstrual , menstrual and postmenstrual ) . after confirming that there was no significant interaction between group and menstrual cycle phase , analysis of variance with repeated measures after confirmation of the reasonably normal distribution of the two - paired samples , student s t - test was used to evaluate differences between groups . correlation testing between perceived stress and biochemical parameters was conducted using pearson s correlation coefficient for both groups combined . the sample size was calculated based on power analysis by g*power ( 13 , 14 ) , with power of 0.80 and an effect size of 0.40 at p < 0.05 with analysis of variance with repeated measures . the subjects were recruited using the following process : the subjects were required to be residents of the kansai region in japan , so that the researchers could meet with them to explain the research methods . , the following subjects withdrew from participation during the study : two subjects who were unable to have their stress levels evaluated on saliva testing due to irregular menstrual cycles ; one subject who discovered she was pregnant ; and four subjects who were unable to continue taking basal body temperature measurements . among the subjects , 13 reported having premenstrual syndrome ( pms group ) , and 11 reported not having premenstrual syndrome ( n - pms group ) . based on published diagnostic criteria for pms ( 1 ) , the subjects were classified in the two mentioned groups based on both their basal body temperature during three to five menstrual cycles and subjective records kept in a menstrual symptom journal , the pms memory diary ( 15 ) . cumulative scores were calculated based on the severity and frequency of symptoms in each phase . cycle phases were defined as follows : premenstrual phase ( one to seven days before menstruation ) ; menstrual phase ( days one to seven after the start of menstruation ) ; and postmenstrual phase ( days eight to fourteen after the start of menstruation ) . perceived stress and biochemical parameters were measured twice over two complete menstrual cycles , with two measurements for each item , to ensure reproducibility . before stress measurements were taken , premenstrual , menstrual and postmenstrual phases were confirmed based on records of basal body temperature taken across two menstrual cycles . salivary s - iga concentrations ( g / ml ) and secretion rates ( g / min ) , as well as cortisol concentrations ( g / dl ) , were measured as biochemical parameters . given this circadian pattern , collection early in the morning was avoided , and the effects of subjects everyday activities were taken into account . the cortisol levels and both the s - iga concentrations and secretion rates were determined using enzyme - linked immunoassay kits ( salimetrics llc ) . the levels of perceived stress were assessed using the japanese version of the stress checklist km ( scl - km ) ( 19 ) . the scl - km uses a binary - choice format for 30 items of psychosomatic complaints that can develop to a stress response . one point is given to any item reflecting high evaluation of stress ; thus , the possible point range is from 0 to 30 . abbreviations : bbt , basal body temperature ; scl - km , stress checklist km ; s - iga , secretory immunoglobulin a. day 1 , onset of menses and start of the menstrual cycle . the mann - whitney nonparametric u test was used to examine any differences in the present age and age at menarche between the pms and n - pms groups . chi - square tests were used to examine differences in marital status , parity , smoking status , and alcohol use between the groups . a two - way repeated measures analysis of variance was used for the groups ( pms and n - pms ) and the menstrual cycle phases ( premenstrual , menstrual and postmenstrual ) . after confirming that there was no significant interaction between group and menstrual cycle phase , analysis of variance with repeated measures after confirmation of the reasonably normal distribution of the two - paired samples , student s t - test was used to evaluate differences between groups . correlation testing between perceived stress and biochemical parameters was conducted using pearson s correlation coefficient for both groups combined . the objectives of the study were explained , and informed consent was obtained from each subject included in the study . two - way analysis of variance showed no interaction between groups and menstrual cycle phases . no significant differences were found in the s - iga concentration and secretion rate and the cortisol level between the groups ( figures 2 and 3 ) . no significant differences were observed in the s - iga concentration and secretion rate among menstrual cycle phases in the n - pms group . the s - iga concentrations in the first [ f ( 2 , 36 ) = 9.330 , p < 0.001 ] and second [ f ( 2 , 36 ) = 6.009 , p < 0.01 ] measurements were significantly higher in the premenstrual phase than in the postmenstrual phase in the pms group . moreover , the s - iga concentration in the first measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group . the s - iga secretion rate in the second measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group [ f ( 2 , 36 ) = 5.178 , p < 0.05 ] . postmenstrual s - iga concentration ( t ( 22 ) = 2.090 , p < 0.05 , 95% confidence interval , ci , [ 0.012 0.485 ] ; figure 2 ) and secretion rate ( t ( 22 ) = 2.129 , p < 0.05 , 95% confidence interval , ci , [ 0.009 0.651 ] ; figure 2 ) were significantly lower at the second measurement in the pms group when compared to the n - pms group . no significant differences in the cortisol level were seen in either group at any phase ( figure 3 ) . no significant changes in the scl - km score were observed among menstrual cycle phases in either group . premenstrual ( t ( 22)=3.812 , p < 0.001 , 95% ci [ 2.5 , 8.4 ] ; t ( 22)=2.733 , p < 0.05 , 95% ci [ 5.9 , 19.7 ] ) and postmenstrual phase ( t ( 22)=2.347 , p < 0.05 , 95% ci [ 1.8 , 7.9 ] ; t ( 22)=2.098 , p < 0.05 , 95% ci [ 1.7 , 7.1 ] ) scl - km scores were significantly higher in the pms group than in the n - pms group ( for both measurements ) . menstrual phase scl - km scores in the second measurement were significantly higher in the pms group than in the n - pms group ( t ( 22)=3.034 , p < 0.01 , 95% ci [ 1.3 , 6.9 ] ; figure 4 ) . a significant negative correlation was observed between s - iga [ concentration ( r = 0.491 , p < 0.05 ) ; secretion rate ( r = 0.543 , p < 0.01 ) ] and scl - km scores at the second measurement in the postmenstrual phase . furthermore , no significant correlations were found between the cortisol level and scl - km scores . two - way analysis of variance showed no interaction between groups and menstrual cycle phases . no significant differences were found in the s - iga concentration and secretion rate and the cortisol level between the groups ( figures 2 and 3 ) . no significant differences were observed in the s - iga concentration and secretion rate among menstrual cycle phases in the n - pms group . the s - iga concentrations in the first [ f ( 2 , 36 ) = 9.330 , p < 0.001 ] and second [ f ( 2 , 36 ) = 6.009 , p < 0.01 ] measurements were significantly higher in the premenstrual phase than in the postmenstrual phase in the pms group . moreover , the s - iga concentration in the first measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group . the s - iga secretion rate in the second measurement was significantly higher in the menstrual phase than in the postmenstrual phase in the pms group [ f ( 2 , 36 ) = 5.178 , p < 0.05 ] . postmenstrual s - iga concentration ( t ( 22 ) = 2.090 , p < 0.05 , 95% confidence interval , ci , [ 0.012 0.485 ] ; figure 2 ) and secretion rate ( t ( 22 ) = 2.129 , p < 0.05 , 95% confidence interval , ci , [ 0.009 0.651 ] ; figure 2 ) were significantly lower at the second measurement in the pms group when compared to the n - pms group . no significant differences in the cortisol level were seen in either group at any phase ( figure 3 ) . no significant changes in the scl - km score were observed among menstrual cycle phases in either group . premenstrual ( t ( 22)=3.812 , p < 0.001 , 95% ci [ 2.5 , 8.4 ] ; t ( 22)=2.733 , p < 0.05 , 95% ci [ 5.9 , 19.7 ] ) and postmenstrual phase ( t ( 22)=2.347 , p < 0.05 , 95% ci [ 1.8 , 7.9 ] ; t ( 22)=2.098 , p < 0.05 , 95% ci [ 1.7 , 7.1 ] ) scl - km scores were significantly higher in the pms group than in the n - pms group ( for both measurements ) . menstrual phase scl - km scores in the second measurement were significantly higher in the pms group than in the n - pms group ( t ( 22)=3.034 , p < 0.01 , 95% ci [ 1.3 , 6.9 ] ; figure 4 ) . a significant negative correlation was observed between s - iga [ concentration ( r = 0.491 , p < 0.05 ) ; secretion rate ( r = 0.543 , p < 0.01 ) ] and scl - km scores at the second measurement in the postmenstrual phase . furthermore , no significant correlations were found between the cortisol level and scl - km scores . this study examined changes in biochemical parameters of stress , and measured perceived stress during the menstrual cycle in women with pms . the findings of this study indicated that the postmenstrual s - iga concentration and secretion rate were significantly lower at the second measurement in the pms group than in the n - pms group . the s - iga is a protein that plays a major role in the local immune system of the mucosal epithelium . evidence indicates that its secretion sensitively reflects changes in the immune system ( 10 ) . secretory iga has been reported to decrease in response to chronic stress and general life events , and it has been suggested to be a chronic stress marker ( 20 , 21 ) . the present study results suggest that lower s - iga levels indicate chronic stress in the postmenstrual phase , even without the psychosomatic burden caused by pms . conversely , the findings of this study indicated that s - iga concentration was significantly higher during the premenstrual or menstrual phase than the postmenstrual phase in the pms group . significantly higher levels of s - iga were reported during the menstrual phase in women with dysmenorrhea compared with women without dysmenorrhea ; this was likely to be a result of pain ( 17 ) . in addition , elevated s - iga levels have been shown to indicate a transient acute stress response ( 22 , 23 ) . this suggests that , accompanied by chronic stress conditions , psychosomatic changes associated with pms increase stress responses in women with premenstrual syndrome . in the present study , changes in cortisol levels were not observed , presumably because the subjects in this study did not have a severe stress load . the scl - km score was significantly higher in the pms group than in the n - pms group in both the premenstrual and menstrual phases . in addition , perceived stress scores were similar among menstrual cycle phases in both groups , with or without pms . this suggests a high perceived stress condition in women with pms , supporting the results of previous studies ( 27 ) . higher levels of nervous tension , anxiety , depression , and anger have been reported to be caused by either pms ( 28 ) or perceived stress ( 29 ) , primarily in the premenstrual phase . however , other studies reported no differences in stress responses among menstrual cycle phases ( 30 ) . since these studies used different scales and found no differences among menstrual phases in small studies with 21 - 25 subjects , further studies are necessary to verify the methods of measuring perceived stress . furthermore , it has been reported that 27% of the perceived stress response varies depending on the subject s status at the time of measurement ( 27 ) ; changing stress levels across the two menstrual cycles were reflected in salivary biochemical parameters and perceived stress scores . there were negative correlations in the postmenstrual phase between the s - iga concentration and secretion rate and the scl - km score . these results indicate that s - iga can be used for the quantitative evaluation of chronic stress in women with pms . conversely , no significant correlations were observed between cortisol levels and the scl - km scores . this result is compatible with a previous study that used daily urine samples , in which no correlation was observed between cortisol and perceived stress ( 31 ) . in conclusion , whether cortisol would respond to the perceived stress analyzed in the present study remains to be clarified . the absence of significant correlations between cortisol levels and perceived stress scores might imply a fundamental difference between these two types of stress . second , the number of subjects was small , which limits the power of the analysis of the results of s - iga concentration and secretion rate from different menstrual cycle phases . third , the difference in sample size between the two groups increased type 1 error , which could potentially account for differences in marital status and parity between the groups , as well as for cortisol and perceived stress not being detected as statistically significant . for future research , further analyses of the associations of salivary s - iga and cortisol with pmsare needed with more subjects and experimental stress loads to test the usefulness of these biochemical parameters for quantitative stress measurements . the results of the present study suggest that measurements of s - iga , rather than cortisol or subjective responses to stress , may be most closely associated with pms . salivary s - iga can be used as a highly sensitive method for stress evaluation in women with pms in the general population .

mammalian cells have evolved a variety of mechanisms to remove defective and genomically unstable cells . although it does not constitute a bona fide cell death mechanism in itself , mitotic catastrophe precedes and uses antiproliferative measures including apoptosis , necrosis , and senescence to prevent the proliferation of defective mitotic cells [ 2 , 3 ] . mitotic catastrophe is characterised by unique nuclear alterations that lead to multinucleation and/or micronucleation and are used as morphological markers for detection . giant multinucleated cells arise from clusters of missegregated uncondensed chromosomes , whereas micronucleated cells arise from lagging chromosomes or chromosome fragments during anaphase that are left outside the daughter nuclei formed during telophase , thereby giving rise to a micronucleus in addition to the main nucleus . figure 1 illustrates the morphological features following normal cell division ( a ) and a multinucleated cell formed during mitotic catastrophe ( b ) . failure of the mitotic catastrophe antiproliferative process leads to persistent genome instability and aneuploidy ( c f ) . furthermore , as a result of the various antiproliferative pathways adopted by mitotic catastrophe it is often accompanied by morphological and biochemical features of apoptosis and necrosis [ 2 , 3 ] . the detection and removal of mitotically defective cells are important steps in the prevention of genome instability . defective or failed mitosis leads to the generation of aneuploid or tetraploid cells , which are a common feature of tumour cells [ 5 , 6 ] . it was postulated by theodor boveri more than 100 year ago that abnormality in chromosome segregation during mitosis could promote tumour formation . it is now known that aneuploidy is present in approximately 90% of solid human tumours and > 50% of haematopoietic cancer . some aneuploid tumours have a minor imbalance in chromosome number whereas others are characterised by a large amount of aneuploidy and contain a near tetraploid chromosome number . during mitosis , the loss or gain of chromosomes can occur through a variety of mechanisms including mitotic checkpoints defects , chromosome cohesion defects that lead to sister chromatid missegregation , and centrosome amplification that promotes multipolar mitosis . the hyperstabilisation of kinetochore - microtubule attachments can also prevent the correction of previous attachment defects . on the other hand , tetraploid cells have twice the normal diploid chromosome content , which can arise due to mitotic slippage , cytokinesis failure , cell fusion , and endoreplication . tetraploid cells also contain twice the normal centrosome content , which promotes multipolar mitosis and whole chromosome missegregation , and provides a mechanism for the transition of cells from a tetraploid state to an aneuploid state . multipolar mitotic divisions generally lead to catastrophic chromosome missegregation events that are incompatible with survival ; however , cancer cells can avoid such catastrophic events and suppress multipolar mitosis by clustering centrosomes into two groups thereby allowing division to occur in a bipolar fashion . while there has been much debate over the role of aneuploidy and tetraploidy in tumour onset , mounting evidence suggests that tetraploid cells can trigger cellular transformation and tumour formation [ 5 , 6 ] . for example , p53 tetraploid mouse cells formed tumours when transplanted into immunocompromised mice , which was not detected with the isogenic diploid cells . tetraploid cells generated by virus induced cell - cell fusion can proliferate and induce transformation [ 11 , 12 ] . mutation of adenomatous polyposis coli ( apc ) in colorectal cancer resulted in tetraploid genomes in vivo due to cytokinesis failure . furthermore , tetraploidy was identified as an early event during cervical carcinoma , and tetraploid cells formed following cytokinesis failure induced transformation in vivo [ 15 , 16 ] . in these cases transformation was coupled with extensive genome instability with abnormalities in the number and structure of chromosomes , providing evidence that tetraploidy represents an intermediate stage to promote aneuploidy and genome instability . moreover , the loss of two tumour suppressor genes breast cancer susceptibility gene 2 ( brca2 ) or the lats1 tumour suppressor is accompanied by cytokinesis defects , suggesting a role for these tumour suppressors during cytokinesis [ 17 , 18 ] . aneuploidy increases the rate of both spontaneous and carcinogen - induced tumour formation ; however , paradoxically , cases where aneuploidy does not promote tumourigenesis or where it suppresses tumourigenesis have also been reported . it is clear that aneuploidy alters the path of tumour development , and a variety of factors influence the final outcome including the combination of chromosomes involved , cell type , genetic context , for example , the presence of additional cooperating mutations in key regulatory genes , as well as the microenvironment within different tissue . this context driven outcome is illustrated in patients with down syndrome who carry an extra copy of chromosome 21 and have increased incidence of haematological malignancies but reduced incidence of solid tumours [ 20 , 21 ] . more recently it was suggested that the rate of chromosome missegregation will determine whether aneuploidy will promote or suppress tumour growth , where low rates of chromosome missegregation can promote tumourigenesis , and high rates lead to cell death and thereby prevent tumour growth . in each scenario , the final outcome will be influenced by the functional status of damage sensing mitotic catastrophe signals as well as the cell survival and death machinery . thus , mitotic catastrophe represents an important part of our genome maintenance machinery and abrogated or compromised signals will contribute to tumour onset . understanding the molecular mechanism that dictates mitotic catastrophe here we provide an update on current knowledge about the mechanism of mitotic catastrophe induction and signalling and highlight approaches to target and exploit the process in cancer treatment . the cell cycle represents a highly coordinated process whereby a cell is divided into two genetically identical daughter cells . pioneering work over the past four decades has revealed the molecular components that control the cell cycle in eukaryotes . the mammalian cell cycle can be divided into distinct phases , dna replication ( synthesis ( s ) phase ) and division ( mitosis ( m ) phase ) , which are separated by gap phases ( g1 and g2 ) . mitosis is subdivided into prophase , prometaphase , metaphase , anaphase , telophase and cytokinesis , which together regulate nuclear envelope breakdown , chromosome attachment to spindle microtubules , alignment along the metaphase plate , sister chromatid separation , and finally , the coordinated plasma membrane remodelling and cytoplasmic division to produce two daughter cells . transition through the cell cycle is controlled by the interplay between cyclin - dependent kinases ( cdks ) and their respective cyclin binding partners [ 23 , 24 ] . activation of cdk1 , which occurs upon formation of a cdk1/cyclin b complex , regulates entry and progression through mitosis . active cdk1/cyclin b phosphorylates substrates involved in nuclear envelope breakdown , assembly of the mitotic spindle , chromosome condensation , and activation of the spindle assembly checkpoint [ 23 , 24 ] . during metaphase the mitotic chromosomes , which are composed of sister chromatids held together by cohesion , are aligned on the mitotic spindle by stable microtubule attachment through their kinetochores . properly aligned chromosomes are separated during anaphase and move towards opposite ends of the spindle . a narrow region of overlapping nonkinetochore microtubules forms the central spindle at the midzone between separating chromosomes . this is followed by formation of centralspindlin comprised of mklp1 and cyk4 and containing a gtpase - activating protein ( gap ) domain , and the chromosomal passenger complex ( cpc ) composed of aurora b and three additional proteins , incenp , survivn , and borealin that are required for aurora b regulation [ 2629 ] . central spindle recruits ect2 , a rhogtpase leading to rhoa activation and assembly of an actinomyosin contractile ring around the central core of the cell . the contractile ring constricts to form a cleavage furrow that ingresses and packs the midzone microtubules to form the dense region termed the midbody at the centre of a long intercellular bridge holding daughter cells together . during cytokinesis , the midbody acts as a platform for components required during abscission of the plasma membrane and eventual daughter cell separation [ 2629 ] . as well as the coordinated activation and inactivation of cdk1 that controls mitotic progression , the fidelity of the process is maintained by an independent and evolutionary conserved checkpoint known as the spindle assembly checkpoint ( sac ) . the sac is a surveillance process at the transition from metaphase to anaphase that monitors the attachment of chromosomes to the kinetochore spindles and halts progression of anaphase until all chromosomes are correctly attached to the bipolar spindle . upon proper attachment , the sac is switched off and cdc20 activates the e3 ubiquitin ligase , anaphase promoting complex ( apc ) , leading to ubiquitination and proteolytic degradation of two substrates , cyclin b , which maintains cdk1 in an active form , and securin , which inhibits separase . following degradation of securin , the liberated separase targets cohesion causing sister chromatid separation , and , anaphase onset . furthermore , apc - mediated degradation of cyclin b leads to inactivation of cdk1 and signals mitotic exit . a single unattached or incorrectly attached chromosome is sufficient to block progression to anaphase by inhibition of apc activity , thereby leading to mitotic arrest . mitotic catastrophe senses mitotic damage and directs the defective cell to one of three possible antiproliferative fates ( figure 2 ) . defective mitotic cells can engage the cell death machinery and undergo death in mitosis , when cyclin b levels remain high . alternatively , defective cells can exit mitosis , known as slippage , and undergo cell death execution during g1 in the subsequent cell cycle . finally , defective cells can exit mitosis and undergo senescence [ 2 , 3 ] . it is clear that mitotic catastrophe is always accompanied by mitotic arrest ; however , the mechanisms that dictate cell fate following mitotic catastrophe remain unclear [ 2 , 3 ] . it was originally proposed that death signals gradually accumulate during mitotic arrest , and therefore the length of mitotic arrest determines cell fate . since then a model has been proposed whereby cell fate is dictated by two independent , yet competing networks ; one involves activation of prodeath signals and the other protects against cyclin b degradation . both pathways work in opposite directions during prolonged mitosis ; that is , cell death signals accumulate and cyclin b levels decline . both pathways have a threshold and the fate of the cell is determined by which threshold is breached first . it is known that cyclin b levels slowly decline during prolonged mitotic arrest even in presence of an active sac ; thus , if levels fall below the threshold that dictates mitotic exit , slippage occurs , whereas if the death threshold is met first the cell will undergo death in mitosis . additional work has focused on determining the molecular events that govern each network and its threshold in order to understand how cells respond to mitotic stress . the activity of the bcl-2 protein family is a key determinant of fate following mitotic catastrophe [ 6771 ] and phosphorylation mediated by cdk1 is an important signal that controls bcl-2 family activity [ 67 , 68 , 72 , 73 ] . the family is comprised of multidomain prosurvival proteins ( bcl-2 , bcl - xl , bcl - w , mcl1 , a1 , and bcl - b ) and multidomain proapoptotic effector proteins ( bax , bak , and bok ) as well as bh3-only proteins ( bim , puma , bad , noxa , bik , hrk , bmf , and tbid ) . the multidomain members of the family ( the prosurvival proteins and the effectors bak , bax , and bok ) contain four bcl-2 homology regions ( bh1bh4 ) , whereas the bh3-only proteins contain only a bh3 domain , which is important in mediating their interaction with the multidomain members . various models are proposed to describe how prosurvival and proapoptotic bcl-2 proteins interact together to control apoptosis . active cyclin b / cdk1 directly phosphorylates bcl-2 , bcl - xl , and mcl-1 during mitosis and negatively regulates their activity [ 67 , 68 ] . cdk1 phosphorylation of bcl-2 and bcl - xl blocks heterodimer formation with proapoptotic members , bax and bak , promoting their oligomerization at the outer mitochondrial membrane , release of cytochrome c , and thereby apoptosis [ 75 , 76 ] . in contrast , cdk1-mediated phosphorylation of mcl-1 during mitosis controls protein stability by ubiquitination and degradation via the proteasome . harley et al . demonstrated that phosphorylation of mcl-1 by cdk1/cyclin b initiates its degradation during mitotic arrest in a cdc20/apc-3 dependent manner . like bcl-2 and bcl - xl , loss of antiapoptotic mcl-1 promotes the oligomerisation of bax and bak and thus death during prolonged mitotic arrest . thus , it is proposed that during a typical mitosis the transient phosphorylation of mcl-1 by cdk1/cyclin b is not sufficient to drive cell death before cyclin b levels drop sufficiently to inactive cdk1 . this transient effect ensures that normal cells are not subject to a fate of apoptosis during normal mitosis . in contrast , however , the sustained cdk1 activity that occurs during mitotic arrest leads to a significant drop in mcl-1 levels thereby suppressing its antiapoptotic effect and triggering cell death before mitotic exit . phosphorylation of mcl-1 also controls interaction with the fbw7 tumour - suppressor and subsequent degradation by the skp - cullin - f - box ( scf ) complex during mitotic arrest . collectively , these reports highlight a role for posttranslational phosphorylation and ubiquitination in the crosstalk between the mitotic and apoptotic machinery to control cell fate during mitotic arrest . mac fhearraigh and mc gee demonstrated that two bim isoforms , bimel and biml , undergo transient phosphorylation during normal mitosis ; however , hyperphosphorylation was evident during sustained mitotic arrest . furthermore , bim directly binds cyclin b , which acts as a molecular bridge for cdk1 phosphorylation , and serine 44 within biml was identified as a novel cdk-1 phosphorylation site . it is suggested that cdk-1-mediated phosphorylation of bim alters its heterodimer formation with bcl-2 , leading to enhanced activation of bak and mitochondrial cell death , consistent with the view that mitochondrial proapoptotic signalling entails the interplay between pro- and antiapoptotic bcl-2 proteins . in contrast , cdk1-dependent phosphorylation of bimel promotes its polyubiquitylation and degradation via the proteasome during mitotic arrest . thus , based on the competing model that was proposed by gascoigne and taylor , the gradual loss of cyclin b during prolonged mitotic arrest will lead to the loss of cdk-1-mediated bim phosphorylation , which will alter its cell death activity during prolonged mitosis and following slippage , either through stability or heterodimer formation . this may partially explain the contradictory reports that mitotic catastrophe - induced death occurs via bim - dependent and bim - independent mechanisms [ 7982 ] . cdk1 also phosphorylates members of the proteolytic caspase family , specifically caspase-2 , caspase-8 , and caspase-9 , leading to inhibition of their apoptotic activity , which is believed to be a cytoprotective measure during normal mitosis [ 8385 ] . furthermore , caspase activity is not required for spindle assembly checkpoint function or mitotic slippage following mitotic catastrophe ; however , the downstream cell death can manifest in a caspase - dependent or caspase - independent manner . cell death can involve mitochondrial perturbations including mitochondrial outer membrane permeabilisation ( momp ) and cytochrome c release induced following bax / bak oligomerisation and pore formation on the outer mitochondrial membrane , leading to caspase - dependent apoptosis . alternatively cell death can occur through the permeability transition pore complex ( ptpc ) , a large complex that bridges the junction between the inner and outer mitochondrial membranes . a sudden increase in permeability of the inner mitochondrial membrane to small solutes leads to ca overload , oxidative stress , and mitochondrial permeability transition- ( mpt- ) induced death that is independent of caspase activity . although details of the mitotic and cell death processes are well characterised , the molecular signals that link these events during mitotic catastrophe remain poorly understood and are the focus of intense investigation . two interesting candidates are mad2 and survivin that are reported to have dual functions in regulating spindle checkpoint and cell death [ 58 , 89 ] . furthermore , it was recently shown that mitochondrial protein tyrosine phosphatase 1b ( ptp1b ) undergoes coordinate phosphorylation by cdk1 and plk1 during mitotic arrest . phosphorylation of mitochondrial ptp1b increases its phosphatase activity and sensitises cells to antimitotic agents representing a new molecular link between the mitotic machinery and the mitochondrion during mitotic catastrophe . the identification of ptp1b substrates at the mitochondria will provide better insight into its precise function and help delineate the downstream execution events . it was also recently demonstrated that cdk1 can directly phosphorylate and regulate numerous mitochondrial proteins , including subunits of the respiratory chain to regulate respiration in a cell cycle specific manner [ 91 , 92 ] , providing a further link between cdk1 activity and mitochondrial function . faithful mitotic progression requires the proper function of various cell components including microtubules , mitotic enzymes , motor proteins , and protein complexes [ 34 , 93 ] . microtubules are essential cytoskeletal components composed of subunits of -tubulin and -tubulin that dimerise to form linear protofilaments , which together form microtubules . the dynamic nature of microtubule plus ends , which undergo continuous polymerisation and depolymerisation , allow them to form cell structure and enable motility and intracellular transport . during mitosis microtubules form a bipolar spindle array that emanates from the centrosomes located at opposite sides of the cell . unattached or incorrectly attached kinetochores , such as merotelic or syntelic attachments , initiate a network of signals to recruit mitotic checkpoint components including mad1 , mad2 , bub1 , bub3 , bubr1 , cenp - e , and mps1 to kinetochores [ 25 , 9496 ] . the formation of an inhibitory complex termed the mitotic checkpoint complex ( mcc ) , consisting of three sac components , mad2 , bubr1 , and bub3 as well as cdc20 , acts as the sac effector that is enriched at unattached kinetochores . mcc binds to and potently inhibits apc by sequestering cdc20 , thereby preventing mitotic exit [ 25 , 9496 ] ( figure 2 ) . biorientated attachment of all sister chromatid pairs to their kinetochore microtubules promotes displacement of the sac proteins , allowing release of cdc20 from the mcc . improper kinetochore - microtubule attachment also causes reduced tension across the spindle apparatus which inhibits the apc through a mechanism involving aurora b kinase [ 97 , 98 ] . most cancer cells display a certain level of aneuploidy [ 5 , 6 , 19 ] , and it was proposed that mechanisms that induce additional instability constitute a therapeutic strategy . consistent with that , cancer cells are more susceptible to cell death following mitotic damage in comparison to nontransformed cells , and a number of mitotic targets have been identified . these include mitotic kinases such as aurora kinases , monopolar spindle 1 ( mps1 ) , and polo - like kinases ( plks ) that play key roles during faithful chromosome segregation . the aurora kinase family of serine / threonine protein kinases includes aurora a , aurora b and aurora c , each with a distinct expression pattern , subcellular localisation pattern , and function [ 101 , 102 ] . aurora a localises to centrosomes during interphase and to spindles poles and spindle microtubules during mitosis , where it regulates mitotic entry , centrosome maturation , and spindle formation . aurora b localises to kinetochores and forms part of the chromosome passenger complex that plays critical roles during chromosome condensation , biorientation , and cytokinesis . aurora c is mainly expressed in testes and required for spermatogenesis and mouse embryogenesis . dysregulated aurora kinase activity generates mitotic abnormalities and cytokinesis failure [ 103 , 104 ] . thus , the critical role of aurora kinases during mitosis makes them indispensible with faithful mitosis . mps1 forms a core component of the spindle assembly checkpoint ( sac ) and functions in the alignment and orientation of chromosomes during metaphase . polo - like kinases ( plks ) also play critical roles during mitotic progression [ 102 , 106 ] . the most widely studied is plk1 , which is involved in assembly of the mitotic spindle , maturation of centrosomes , activation of the sac , chromosome segregation , and cytokinesis [ 102 , 106 ] . deregulation of the centrosome cycle leading to supernumerary centrosomes generates multipolar mitosis that promotes genome instability , sac activation , and mitotic catastrophe [ 107 , 108 ] . faithful mitosis is also dependent on microtubule motor proteins such as eg5 , a plus - end directed motor from the kinesin superfamily that is responsible for mitotic spindle formation and function . disruption of eg5 function during mitosis leads to monopolar spindles and activation of the sac . furthermore , the centromere - associated motor protein ( cenp - e ) is a component of the kinetochore corona fibres of mammalian centromeres and is required for chromosome biorientated attachment and proper mitotic checkpoint signalling [ 109111 ] . in addition to disrupting chromosome segregation , inhibition of cytoplasmic division following anaphase onset will generate genome instability and stimulate mitotic catastrophe in the next cell cycle [ 109 , 112 ] ( figure 2 ) . the development of pharmacological agents that induce mitotic catastrophe via disruption of bipolar spindle function or faithful chromosome segregation is discussed in more details later . failure to repair the dna damage leads to mutations and genome instability that ultimately contributes to diseases including cancer [ 113 , 114 ] . the dna damage response ( ddr ) is a complex mechanism to sense various types of dna damage and respond appropriately to maintain genomic integrity . following dna damage the phosphatidylinositol 3-kinase - related kinases atm ( ataxia - telangiectasia mutated ) and atr ( atm and rad-3 related ) are activated and coordinate activation of the dna damage checkpoints through the phosphorylation of numerous downstream substrates . checkpoint kinase-1 ( chk1 ) and checkpoint kinase-2 ( chk2 ) are serine threonine kinases that transduce the dna damage signal downstream . chk2 , which is expressed throughout the cell cycle , undergoes phosphorylation and activation by atm , whereas chk1 is preferentially expressed in s and g2 and is phosphorylated by atr . in addition to chk1 and chk2 , mapk - activated protein kinase-2 ( mk2 ) regulates cell cycle checkpoint activation [ 113 , 114 ] . genomic stress activates the g1 checkpoint , which prevents s phase entry by inhibition of dna replication . at this point , chk2 , which is activated by atm , phosphorylates and suppresses the phosphatase cdc25-a , thereby preventing activation of cyclin e / cdk2 and thus halting the cell cycle . the s phase checkpoint is activated in response to replication errors and dna damage that occurs during s phase , whereas the g2 checkpoint deals with cells that have either undergone dna damage in g2 , or they have escaped the g1 and s phase checkpoints . cdk1 activity and mitotic entry are tightly regulated and balanced by inactivating phosphorylation by the protein kinases wee1 and myelin transcription factor 1 ( myt1 ) , together with the activating cdc25 phosphatase . thus , wee1 and cdc25 act as a central switch for mitosis and are regulated by posttranslational alterations thereby enabling rapid switching . at g2 , chk1 , which is activated by atr , phosphorylates and suppresses cdc25-a , -b , and -c thereby preventing cyclin b / cdk1 activation and causing g2 arrest [ 113115 ] . thus , the g2 checkpoint is the last opportunity to halt the cycle and repair dna damage in cells that have escaped the g1 and s phase checkpoints . abrogated or compromised g2 checkpoint will allow premature mitotic entry of defective cells that fail to undergo proper chromosome segregation thereby leading to mitotic catastrophe ( figure 2 ) . in support of this , the fusion of interphase and mitotic cells led to mitotic catastrophe which was due to the cyclin b / cdk1 driven - premature entry of cells into mitosis before they had completed s or g2 . furthermore , knockout of the cytoplasmic binding protein 14 - 3 - 3 in colorectal cancer cells resulted in failure to sequester cyclin b1 and prevented g2 arrest following dna damage , culminating in mitotic catastrophe . inhibition of chk2 also abrogates the g2 checkpoint leading to mitotic catastrophe following dna damage . . in contrast , cells that harbour dna damage and undergo death in interphase do not constitute an example of mitotic catastrophe [ 2 , 3 , 113 ] . furthermore , while an abrogated or defective g2 checkpoint is essential for dna damage - induced mitotic catastrophe , the eventual mode of cell death induced is determined by whether p53 is present or absent . for example , dna damage induces two distinct forms of cell death in ovarian carcinoma . functional p53 triggered apoptosis in ovarian carcinoma cells following mitotic catastrophe whereas loss of p53 in these cells triggered necrosis . the exact mechanism of p53 activation during or after mitotic catastrophe remains to be elucidated ; however , it was shown that phosphorylated h2ax - atm - p53 pathway dictates an apoptotic outcome following mitotic catastrophe . loss of p53 or depletion of atm protected against apoptosis and instead led to necrosis . thus it is proposed that the initiation of mitotic catastrophe occurs independently of p53 status and caspase activity ; however , the presence of functional p53 is required for a caspase - mediated apoptotic response . mitotic catastrophe is induced by a variety of agents classified as those that disrupt mitotic progression or directly damage dna ( figure 2 and table 1 ) . the best known antimitotic agents are the microtubule targeting agents ( mtas ) , also known as spindle poisons . mtas are grouped into two families : the microtubule polymerisers which include the taxanes ( paclitaxel and docetaxel ) and the microtubule depolymerisers which include the vinca alkaloids ( vinblastine and vincristine ) . the suppression of microtubule dynamics by both groups precludes normal bipolar spindle formation and prevents chromosome biorientation , leading to mitotic arrest and cell death . taxol ( paclitaxel ) originally isolated in 1967 from a yew tree ( taxus brevifolia ) , was approved for clinical use in 1995 and is widely used across a range of malignancies . for example , taxanes have been used in the treatment of kaposi 's sarcoma , non - small - cell lung cancer , breast cancer , ovarian cancer , and prostate cancer , whereas vinca alkaloids are used to treat haematological malignancies [ 34 , 121 ] . although they have been used clinically for decades , microtubule targeting agents lack specificity towards cancer cells and disrupt other important microtubule - dependent functions leading to severe side effects including neuropathy . furthermore , the development of drug resistance limits their use , which can be ascribed to drug efflux pumps , overexpression of prosurvival bcl-2 proteins , and mutations in tubulin that abrogates drug binding [ 122 , 123 ] . resistance may also occur as a result of mitotic slippage . thus , research efforts have focused on the development of non - microtubule antimitotic therapeutics , such as those targeted at mitotic kinases and spindle motor proteins , with the hope that that they would overcome some of the drawbacks associated with microtubule targeting agents . moreover , their depletion or inhibition impairs the proliferation of cancer cells , thus , making them an attractive target for cancer treatment [ 35 , 36 ] . a number of aurora kinase inhibitors have been developed that target the enzymes atp binding domain . early inhibitors did not display specificity towards a family member ; however , in recent years work has focused on development of selective inhibitors and a number are in various stages of clinical evaluation including alisertib ( mln8237 ) that has displayed promising antitumour properties and is currently in phases i and ii trials . mps1 kinase inhibitors have been developed which induce mitotic defects and death in cancer cells , across a variety of preclinical models , either alone or in combination with microtubule inhibitors [ 4951 ] . plk1 is upregulated in a range of human tumours ; thus targeting plk1 is an attractive therapeutic strategy and a number of inhibitors are under clinical evaluation including bi2536 [ 46 , 47 ] . furthermore , agents that disrupt the centrosome cycle in tumour cells , through centrosome amplification and centrosome declustering promote multipolar mitosis , genome instability , and mitotic catastrophe [ 5557 ] . a number of kinesin motor protein inhibitors have been developed [ 37 , 124 ] . monastrol is a selective inhibitor of the kinesin-5 motor protein ( ksp , also termed eg5 ) . eg5 inhibition leads to mitotic arrest and death in tumour cells in culture and in xenograft models . furthermore , they were found to be free from severe cytotoxic effects and are generally well tolerated . eg5 inhibitors under clinical development include azd4877 , ispinesib [ 39 , 40 ] , and arry-520 . cenp - e ( centrosome - associated protein - e ) is a microtubule motor that plays a role in mitosis . the small molecule cenp - e inhibitor , gsk923295 , induces defective mitosis and displays antiproliferative effects in vivo and has recently entered clinical trial . cancer cells often harbour a deficient or defective g1 checkpoint due to aberrant p53 signalling , which ultimately leads to increased dna damage at the g2 checkpoint compared to normal cells . based on this , abrogation of the g2 checkpoint allows cells with unrepaired dna damage to proceed into a premature m phase . thus , cancer cells that are defective in g1 and g2 checkpoints will undergo mitotic catastrophe following dna damage induced by radiation , alkylating agents , and doxorubicin . the chk1 kinase inhibitors , ucn-01 and azd7762 , abrogate the g2 checkpoint and potentiate death in p53-deficient tumours [ 58 , 60 , 61 ] and are currently in clinical development [ 62 , 63 ] . evaluation of a panel of therapeutic agents in combination with chk1 inhibition highlights that the precise drug combinations are important and influence the outcome in a particular genetic background and when treating a certain tumour type . in addition to chk1 , targeting wee1 kinase activity together with dna damage can effectively induce mitotic catastrophe . furthermore , histone deacetylase ( hdac ) inhibitors promote mitotic catastrophe and cell death and have shown promise in multiple myeloma and glioma treatments [ 6466 ] . despite the promising preclinical data displayed by new generation antimitotic agents , their clinical efficacy has been disappointing in comparison to microtubule targeting agents [ 37 , 128 ] . this may be explained by the shorter doubling time of cells in culture compared to patients and by differences in drug retention times . it has also been suggested that the success of microtubule targeting agents may be due to nonmitotic function of microtubules . furthermore , the development of resistance to antimitotic agents represents a major challenge that occurs following mitotic slippage when defective cells adapt and survive , although in some cases mitotic slippage is required for cell death . based on this observation , recent research has focused on strategies to block slippage and mitotic exit in order to maximise mitotic arrest - induced death . such approaches include targeting apc - cdc20 to prevent cyclin b degradation and mitotic exit that have shown a very promising response [ 5254 ] . an alternative approach is the inhibition of cytokinesis which blocks mitotic exit in postanaphase cells and may be an attractive strategy to overcome resistance in slippage prone cells . small molecule dynamin gtpase inhibitors have shown antiproliferative effects and induce cytokinesis failure and cell death in cancer cells . it is clear that mitotic catastrophe is an important anticancer strategy that is achieved by a variety of mechanisms that target the cell cycle . although these approaches target proteins that are upregulated in cancer cells , thereby providing a therapeutic window to preferentially kill the cancer cells , they are not specific to cancer cells and are likely to be accompanied by some side effects . recent reports highlight that the myc oncogene regulates mitotic events to support its oncogenic program [ 134 , 135 ] and one way that this may occur is through transcriptional regulation of aurora kinase expression [ 136 , 137 ] . moreover , loss of myc activity due to inhibition of sumoylation or transcriptional inactivation by omomyc led to mitotic catastrophe and cell death in in vivo models of breast cancer and glioma , respectively . thus , targeting myc activity , using approaches that inhibit sumoylation and/or mimic omomyc action , represents new approaches to selectively induce mitotic catastrophe in cancer cells . furthermore , a better understanding of the postmitotic signals that connect to the cell death and senescence pathways will reveal new approaches to push cells down a defined antiproliferative route and is likely to synergise with current antimitotic drugs to kill cancer cells before adaption and the development of drug resistance . these new approaches may provide more effective strategies to exploit mitotic catastrophe in cancer prevention and treatment .

mitotic catastrophe , as defined in 2012 by the international nomenclature committee on cell death , is a bona fide intrinsic oncosuppressive mechanism that senses mitotic failure and responds by driving a cell to an irreversible antiproliferative fate of death or senescence . thus , failed mitotic catastrophe can promote the unrestrained growth of defective cells , thereby representing a major gateway to tumour development . furthermore , the activation of mitotic catastrophe offers significant therapeutic advantage which has been exploited in the action of conventional and targeted anticancer agents . yet , despite its importance in tumour prevention and treatment , the molecular mechanism of mitotic catastrophe is not well understood . a better understanding of the signals that determine cell fate following failed or defective mitosis will reveal new opportunities to selectively target and enhance the programme for therapeutic benefit and reveal biomarkers to predict patient response . this review is focused on the molecular mechanism of mitotic catastrophe induction and signalling and highlights current strategies to exploit the process in cancer therapy .

1. Introduction 2. Mitosis 3. The Spindle Assembly Checkpoint 4. Mechanism of Mitotic Catastrophe 5. Induction of Mitotic Catastrophe by Mitotic Perturbations 6. Induction of Mitotic Catastrophe following DNA Damage 7. Exploiting Mitotic Catastrophe in Cancer Therapy

although it does not constitute a bona fide cell death mechanism in itself , mitotic catastrophe precedes and uses antiproliferative measures including apoptosis , necrosis , and senescence to prevent the proliferation of defective mitotic cells [ 2 , 3 ] . mitotic catastrophe is characterised by unique nuclear alterations that lead to multinucleation and/or micronucleation and are used as morphological markers for detection . failure of the mitotic catastrophe antiproliferative process leads to persistent genome instability and aneuploidy ( c f ) . furthermore , as a result of the various antiproliferative pathways adopted by mitotic catastrophe it is often accompanied by morphological and biochemical features of apoptosis and necrosis [ 2 , 3 ] . the detection and removal of mitotically defective cells are important steps in the prevention of genome instability . defective or failed mitosis leads to the generation of aneuploid or tetraploid cells , which are a common feature of tumour cells [ 5 , 6 ] . on the other hand , tetraploid cells have twice the normal diploid chromosome content , which can arise due to mitotic slippage , cytokinesis failure , cell fusion , and endoreplication . tetraploid cells also contain twice the normal centrosome content , which promotes multipolar mitosis and whole chromosome missegregation , and provides a mechanism for the transition of cells from a tetraploid state to an aneuploid state . while there has been much debate over the role of aneuploidy and tetraploidy in tumour onset , mounting evidence suggests that tetraploid cells can trigger cellular transformation and tumour formation [ 5 , 6 ] . in these cases transformation was coupled with extensive genome instability with abnormalities in the number and structure of chromosomes , providing evidence that tetraploidy represents an intermediate stage to promote aneuploidy and genome instability . it is clear that aneuploidy alters the path of tumour development , and a variety of factors influence the final outcome including the combination of chromosomes involved , cell type , genetic context , for example , the presence of additional cooperating mutations in key regulatory genes , as well as the microenvironment within different tissue . more recently it was suggested that the rate of chromosome missegregation will determine whether aneuploidy will promote or suppress tumour growth , where low rates of chromosome missegregation can promote tumourigenesis , and high rates lead to cell death and thereby prevent tumour growth . in each scenario , the final outcome will be influenced by the functional status of damage sensing mitotic catastrophe signals as well as the cell survival and death machinery . thus , mitotic catastrophe represents an important part of our genome maintenance machinery and abrogated or compromised signals will contribute to tumour onset . understanding the molecular mechanism that dictates mitotic catastrophe here we provide an update on current knowledge about the mechanism of mitotic catastrophe induction and signalling and highlight approaches to target and exploit the process in cancer treatment . pioneering work over the past four decades has revealed the molecular components that control the cell cycle in eukaryotes . activation of cdk1 , which occurs upon formation of a cdk1/cyclin b complex , regulates entry and progression through mitosis . active cdk1/cyclin b phosphorylates substrates involved in nuclear envelope breakdown , assembly of the mitotic spindle , chromosome condensation , and activation of the spindle assembly checkpoint [ 23 , 24 ] . during metaphase the mitotic chromosomes , which are composed of sister chromatids held together by cohesion , are aligned on the mitotic spindle by stable microtubule attachment through their kinetochores . properly aligned chromosomes are separated during anaphase and move towards opposite ends of the spindle . during cytokinesis , the midbody acts as a platform for components required during abscission of the plasma membrane and eventual daughter cell separation [ 2629 ] . as well as the coordinated activation and inactivation of cdk1 that controls mitotic progression , the fidelity of the process is maintained by an independent and evolutionary conserved checkpoint known as the spindle assembly checkpoint ( sac ) . upon proper attachment , the sac is switched off and cdc20 activates the e3 ubiquitin ligase , anaphase promoting complex ( apc ) , leading to ubiquitination and proteolytic degradation of two substrates , cyclin b , which maintains cdk1 in an active form , and securin , which inhibits separase . following degradation of securin , the liberated separase targets cohesion causing sister chromatid separation , and , anaphase onset . furthermore , apc - mediated degradation of cyclin b leads to inactivation of cdk1 and signals mitotic exit . a single unattached or incorrectly attached chromosome is sufficient to block progression to anaphase by inhibition of apc activity , thereby leading to mitotic arrest . mitotic catastrophe senses mitotic damage and directs the defective cell to one of three possible antiproliferative fates ( figure 2 ) . defective mitotic cells can engage the cell death machinery and undergo death in mitosis , when cyclin b levels remain high . alternatively , defective cells can exit mitosis , known as slippage , and undergo cell death execution during g1 in the subsequent cell cycle . finally , defective cells can exit mitosis and undergo senescence [ 2 , 3 ] . it is clear that mitotic catastrophe is always accompanied by mitotic arrest ; however , the mechanisms that dictate cell fate following mitotic catastrophe remain unclear [ 2 , 3 ] . it was originally proposed that death signals gradually accumulate during mitotic arrest , and therefore the length of mitotic arrest determines cell fate . since then a model has been proposed whereby cell fate is dictated by two independent , yet competing networks ; one involves activation of prodeath signals and the other protects against cyclin b degradation . both pathways work in opposite directions during prolonged mitosis ; that is , cell death signals accumulate and cyclin b levels decline . both pathways have a threshold and the fate of the cell is determined by which threshold is breached first . it is known that cyclin b levels slowly decline during prolonged mitotic arrest even in presence of an active sac ; thus , if levels fall below the threshold that dictates mitotic exit , slippage occurs , whereas if the death threshold is met first the cell will undergo death in mitosis . additional work has focused on determining the molecular events that govern each network and its threshold in order to understand how cells respond to mitotic stress . the activity of the bcl-2 protein family is a key determinant of fate following mitotic catastrophe [ 6771 ] and phosphorylation mediated by cdk1 is an important signal that controls bcl-2 family activity [ 67 , 68 , 72 , 73 ] . thus , it is proposed that during a typical mitosis the transient phosphorylation of mcl-1 by cdk1/cyclin b is not sufficient to drive cell death before cyclin b levels drop sufficiently to inactive cdk1 . this transient effect ensures that normal cells are not subject to a fate of apoptosis during normal mitosis . in contrast , however , the sustained cdk1 activity that occurs during mitotic arrest leads to a significant drop in mcl-1 levels thereby suppressing its antiapoptotic effect and triggering cell death before mitotic exit . phosphorylation of mcl-1 also controls interaction with the fbw7 tumour - suppressor and subsequent degradation by the skp - cullin - f - box ( scf ) complex during mitotic arrest . collectively , these reports highlight a role for posttranslational phosphorylation and ubiquitination in the crosstalk between the mitotic and apoptotic machinery to control cell fate during mitotic arrest . it is suggested that cdk-1-mediated phosphorylation of bim alters its heterodimer formation with bcl-2 , leading to enhanced activation of bak and mitochondrial cell death , consistent with the view that mitochondrial proapoptotic signalling entails the interplay between pro- and antiapoptotic bcl-2 proteins . thus , based on the competing model that was proposed by gascoigne and taylor , the gradual loss of cyclin b during prolonged mitotic arrest will lead to the loss of cdk-1-mediated bim phosphorylation , which will alter its cell death activity during prolonged mitosis and following slippage , either through stability or heterodimer formation . this may partially explain the contradictory reports that mitotic catastrophe - induced death occurs via bim - dependent and bim - independent mechanisms [ 7982 ] . cdk1 also phosphorylates members of the proteolytic caspase family , specifically caspase-2 , caspase-8 , and caspase-9 , leading to inhibition of their apoptotic activity , which is believed to be a cytoprotective measure during normal mitosis [ 8385 ] . furthermore , caspase activity is not required for spindle assembly checkpoint function or mitotic slippage following mitotic catastrophe ; however , the downstream cell death can manifest in a caspase - dependent or caspase - independent manner . cell death can involve mitochondrial perturbations including mitochondrial outer membrane permeabilisation ( momp ) and cytochrome c release induced following bax / bak oligomerisation and pore formation on the outer mitochondrial membrane , leading to caspase - dependent apoptosis . alternatively cell death can occur through the permeability transition pore complex ( ptpc ) , a large complex that bridges the junction between the inner and outer mitochondrial membranes . although details of the mitotic and cell death processes are well characterised , the molecular signals that link these events during mitotic catastrophe remain poorly understood and are the focus of intense investigation . two interesting candidates are mad2 and survivin that are reported to have dual functions in regulating spindle checkpoint and cell death [ 58 , 89 ] . phosphorylation of mitochondrial ptp1b increases its phosphatase activity and sensitises cells to antimitotic agents representing a new molecular link between the mitotic machinery and the mitochondrion during mitotic catastrophe . it was also recently demonstrated that cdk1 can directly phosphorylate and regulate numerous mitochondrial proteins , including subunits of the respiratory chain to regulate respiration in a cell cycle specific manner [ 91 , 92 ] , providing a further link between cdk1 activity and mitochondrial function . during mitosis microtubules form a bipolar spindle array that emanates from the centrosomes located at opposite sides of the cell . mcc binds to and potently inhibits apc by sequestering cdc20 , thereby preventing mitotic exit [ 25 , 9496 ] ( figure 2 ) . biorientated attachment of all sister chromatid pairs to their kinetochore microtubules promotes displacement of the sac proteins , allowing release of cdc20 from the mcc . consistent with that , cancer cells are more susceptible to cell death following mitotic damage in comparison to nontransformed cells , and a number of mitotic targets have been identified . aurora b localises to kinetochores and forms part of the chromosome passenger complex that plays critical roles during chromosome condensation , biorientation , and cytokinesis . thus , the critical role of aurora kinases during mitosis makes them indispensible with faithful mitosis . mps1 forms a core component of the spindle assembly checkpoint ( sac ) and functions in the alignment and orientation of chromosomes during metaphase . the most widely studied is plk1 , which is involved in assembly of the mitotic spindle , maturation of centrosomes , activation of the sac , chromosome segregation , and cytokinesis [ 102 , 106 ] . deregulation of the centrosome cycle leading to supernumerary centrosomes generates multipolar mitosis that promotes genome instability , sac activation , and mitotic catastrophe [ 107 , 108 ] . disruption of eg5 function during mitosis leads to monopolar spindles and activation of the sac . furthermore , the centromere - associated motor protein ( cenp - e ) is a component of the kinetochore corona fibres of mammalian centromeres and is required for chromosome biorientated attachment and proper mitotic checkpoint signalling [ 109111 ] . in addition to disrupting chromosome segregation , inhibition of cytoplasmic division following anaphase onset will generate genome instability and stimulate mitotic catastrophe in the next cell cycle [ 109 , 112 ] ( figure 2 ) . the development of pharmacological agents that induce mitotic catastrophe via disruption of bipolar spindle function or faithful chromosome segregation is discussed in more details later . the dna damage response ( ddr ) is a complex mechanism to sense various types of dna damage and respond appropriately to maintain genomic integrity . following dna damage the phosphatidylinositol 3-kinase - related kinases atm ( ataxia - telangiectasia mutated ) and atr ( atm and rad-3 related ) are activated and coordinate activation of the dna damage checkpoints through the phosphorylation of numerous downstream substrates . at this point , chk2 , which is activated by atm , phosphorylates and suppresses the phosphatase cdc25-a , thereby preventing activation of cyclin e / cdk2 and thus halting the cell cycle . thus , wee1 and cdc25 act as a central switch for mitosis and are regulated by posttranslational alterations thereby enabling rapid switching . thus , the g2 checkpoint is the last opportunity to halt the cycle and repair dna damage in cells that have escaped the g1 and s phase checkpoints . abrogated or compromised g2 checkpoint will allow premature mitotic entry of defective cells that fail to undergo proper chromosome segregation thereby leading to mitotic catastrophe ( figure 2 ) . in support of this , the fusion of interphase and mitotic cells led to mitotic catastrophe which was due to the cyclin b / cdk1 driven - premature entry of cells into mitosis before they had completed s or g2 . furthermore , knockout of the cytoplasmic binding protein 14 - 3 - 3 in colorectal cancer cells resulted in failure to sequester cyclin b1 and prevented g2 arrest following dna damage , culminating in mitotic catastrophe . in contrast , cells that harbour dna damage and undergo death in interphase do not constitute an example of mitotic catastrophe [ 2 , 3 , 113 ] . furthermore , while an abrogated or defective g2 checkpoint is essential for dna damage - induced mitotic catastrophe , the eventual mode of cell death induced is determined by whether p53 is present or absent . for example , dna damage induces two distinct forms of cell death in ovarian carcinoma . the exact mechanism of p53 activation during or after mitotic catastrophe remains to be elucidated ; however , it was shown that phosphorylated h2ax - atm - p53 pathway dictates an apoptotic outcome following mitotic catastrophe . thus it is proposed that the initiation of mitotic catastrophe occurs independently of p53 status and caspase activity ; however , the presence of functional p53 is required for a caspase - mediated apoptotic response . mitotic catastrophe is induced by a variety of agents classified as those that disrupt mitotic progression or directly damage dna ( figure 2 and table 1 ) . for example , taxanes have been used in the treatment of kaposi 's sarcoma , non - small - cell lung cancer , breast cancer , ovarian cancer , and prostate cancer , whereas vinca alkaloids are used to treat haematological malignancies [ 34 , 121 ] . furthermore , the development of drug resistance limits their use , which can be ascribed to drug efflux pumps , overexpression of prosurvival bcl-2 proteins , and mutations in tubulin that abrogates drug binding [ 122 , 123 ] . resistance may also occur as a result of mitotic slippage . thus , research efforts have focused on the development of non - microtubule antimitotic therapeutics , such as those targeted at mitotic kinases and spindle motor proteins , with the hope that that they would overcome some of the drawbacks associated with microtubule targeting agents . moreover , their depletion or inhibition impairs the proliferation of cancer cells , thus , making them an attractive target for cancer treatment [ 35 , 36 ] . mps1 kinase inhibitors have been developed which induce mitotic defects and death in cancer cells , across a variety of preclinical models , either alone or in combination with microtubule inhibitors [ 4951 ] . furthermore , agents that disrupt the centrosome cycle in tumour cells , through centrosome amplification and centrosome declustering promote multipolar mitosis , genome instability , and mitotic catastrophe [ 5557 ] . monastrol is a selective inhibitor of the kinesin-5 motor protein ( ksp , also termed eg5 ) . the small molecule cenp - e inhibitor , gsk923295 , induces defective mitosis and displays antiproliferative effects in vivo and has recently entered clinical trial . cancer cells often harbour a deficient or defective g1 checkpoint due to aberrant p53 signalling , which ultimately leads to increased dna damage at the g2 checkpoint compared to normal cells . thus , cancer cells that are defective in g1 and g2 checkpoints will undergo mitotic catastrophe following dna damage induced by radiation , alkylating agents , and doxorubicin . in addition to chk1 , targeting wee1 kinase activity together with dna damage can effectively induce mitotic catastrophe . furthermore , histone deacetylase ( hdac ) inhibitors promote mitotic catastrophe and cell death and have shown promise in multiple myeloma and glioma treatments [ 6466 ] . despite the promising preclinical data displayed by new generation antimitotic agents , their clinical efficacy has been disappointing in comparison to microtubule targeting agents [ 37 , 128 ] . furthermore , the development of resistance to antimitotic agents represents a major challenge that occurs following mitotic slippage when defective cells adapt and survive , although in some cases mitotic slippage is required for cell death . based on this observation , recent research has focused on strategies to block slippage and mitotic exit in order to maximise mitotic arrest - induced death . small molecule dynamin gtpase inhibitors have shown antiproliferative effects and induce cytokinesis failure and cell death in cancer cells . it is clear that mitotic catastrophe is an important anticancer strategy that is achieved by a variety of mechanisms that target the cell cycle . although these approaches target proteins that are upregulated in cancer cells , thereby providing a therapeutic window to preferentially kill the cancer cells , they are not specific to cancer cells and are likely to be accompanied by some side effects . moreover , loss of myc activity due to inhibition of sumoylation or transcriptional inactivation by omomyc led to mitotic catastrophe and cell death in in vivo models of breast cancer and glioma , respectively . thus , targeting myc activity , using approaches that inhibit sumoylation and/or mimic omomyc action , represents new approaches to selectively induce mitotic catastrophe in cancer cells . furthermore , a better understanding of the postmitotic signals that connect to the cell death and senescence pathways will reveal new approaches to push cells down a defined antiproliferative route and is likely to synergise with current antimitotic drugs to kill cancer cells before adaption and the development of drug resistance . these new approaches may provide more effective strategies to exploit mitotic catastrophe in cancer prevention and treatment .

hydrogenases are enzymes that catalyze reversible reactions with h2 as reactant or product ( eqs . 1a , 1b , 1c ) : 1a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{h}}_2 + { \text{x } } \ , \rightleftharpoons\ , { \text{xh}}_2 , $ $ \end{document}1b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{h}}_2 + { \text{x } } \ , \rightleftharpoons \ , { \text{xh}}^ { - } + { \text { h}}^ + , $ $ \end{document}1c\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{h}}_2 + { \text{x } } \ , \rightleftharpoons { \text{x}}^{2 - } + 2{\text{h}}^ { + } , $ $ \end{document}where x is either an enzyme or a coenzyme . this definition is independent of the hydrogenase s catalytic mechanism which can be ping - pong or ternary complex . it is therefore broader than the definition used historically that hydrogenases catalyze the reversible oxidation of h2 to two electrons and two protons , which excludes hydrogenases with a ternary complex mechanism [ 1 , 2 ] . three types are presently known which are phylogenetically not related [ 3 , 4 ] : [ nife ] hydrogenases found in archaea and bacteria , [ fefe ] hydrogenases found in bacteria and eukarya [ 68 ] and [ fe ] hydrogenases only found in methanogenic archaea [ 9 , 10 ] . [ fefe ] hydrogenases are still also referred to as iron - only hydrogenases , which was the name used before it was shown that [ fe ] hydrogenase also contains iron . [ nife ] hydrogenases and [ fefe ] hydrogenases have many properties in common . they both contain a dinuclear metal center in their active site and at least one iron sulfur cluster . the iron in both the [ nife ] and the [ fefe ] center is complexed by co , cyanide and sulfur ligands , and the iron in [ nife ] hydrogenase and the distal iron in [ fefe ] hydrogenase ( distal to the [ 4fe4s ] cluster ) are in a low - spin fe(ii ) oxidation state [ 1215 ] . both enzymes catalyze the reversible heterolytic cleavage of h2 to two protons and two electrons as evidenced by the reversible reduction of the artificial one - electron acceptor methyl viologen ( mv ) with h2 [ 1 , 16 ] ( eq . 2 ) . they also catalyze a single and a double exchange of d2 with protons of bulk water ( eqs . 3 , 4 ) and the exergonic conversion of para - h2 to ortho - h2 ( eq . 5 ) in the absence of exogenous electron acceptors , indicating that [ nife ] and [ fefe ] hydrogenases have a ping - pong catalytic mechanism . 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{h}}_2\ : + 2\:{\text { mv } } \ : \rightleftharpoons\ : { \text { } } 2\:{\text { mv}}^ { - } \:+ \:2{\text{h}}^ + $ $ \end{document}3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{d}}_2\:+ { \text{h}}_2 { \text{o}}\ : \to \:{\text{hd}}\ : + \:{\text{dho}}\quad ( { \text{electron acceptor independent } } ) $ $ \end{document}4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{d}}_2 \:+ { \text{h}}_2 { \text{o } } \to { \text { h}}_2 + { \text{d}}_2 { \text{o}}\quad ( { \text{electron acceptor independent } } ) $ $ \end{document}5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ para{\text{-h}}_2 { \text { } } \to { \text { } } ortho{\text{-h}}_2 { \text { } } ( { \text{electron acceptor independent } } ) $ $ \end{document } the exchange reactions shown in eqs . 3 and 4 are essentially irreversible because the d concentration in the bulk h2o is very low . mutants of [ nife ] hydrogenase have been made that can catalyze only the reaction shown in eq . 5 or only the reactions shown in eqs . 3 , 4 and 5 , indicating that h2 binding to the enzyme , exchange of protons with bulk water and electron transfer to the electron acceptor are individual steps . [ fe ] hydrogenase differs from [ nife ] and [ fefe ] hydrogenases in that it contains a mononuclear metal center rather than a dinuclear metal center in its active site and is devoid of iron sulfur clusters , which is why the enzyme is also referred to as iron sulfur - cluster - free hydrogenase . [ fe ] hydrogenase catalyzes the reversible reduction of methenyltetrahydromethanopterin ( methenyl - h4mpt ) with h2 to methylenetetrahydromethanopterin ( methylene - h4mpt ) ( eq . 6 ) , a reaction involved in co2 reduction with h2 to methane in many methanogenic archaea [ 9 , 1923 ] . in the reaction a hydride from h2 is transferred into the pro - r position of the c(14a ) methylene group of the reaction product ( fig . 1 ) . the systematic name for [ fe ] hydrogenase is hydrogen - forming methylenetetrahydromethanopterin dehydrogenase , abbreviated hmd . the km and vmax of the enzyme for h2 and d2 were found to be almost identical , indicating that a step other than the activation of h2 is rate - determining in the reaction shown in eq . [ fe ] hydrogenase also catalyzes a stereospecific direct exchange of the pro - r hydrogen of methylene - h4mpt with protons of water ( eq . 7 ) , a methenyl - h4mpt - dependent single and double exchange between d2 and protons of bulk water ( eqs . 8 , 9 ) [ 27 , 28 ] and a methenyl - h4mpt - dependent conversion of para - h2 into ortho - h2 ( eq . 10 ) . [ fe ] hydrogenase does not catalyze the reduction of viologen dyes ( eq . 2 ) or other artificial one - electron or two - electron acceptors neither in the absence nor in the presence of methenyl - h4mpt . the catalytic mechanism of the enzyme is thus clearly ternary complex rather than ping - pong and thus quite different from that of [ nife ] and [ fefe ] hydrogenases . 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{h}}_2 + { \text{methenyl - h}}_4 { \text{mpt}}^ + { \text { } } \rightleftharpoons { \text { methylene - h}}_4 { \text{mpt } } + { \text { h}}^ + \quad\updelta { \text{g}}^{\circ \prime } = -5.5\,{\text{kj}}\,{\text{mol}}^ { { - 1}}$$\end{document}7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ ( 14{\text{a}}rs){\text{-}}[14{\text{a - h}}]{\text{methylene - h}}_4 { \text{mpt } } + { \text{d}}_2 { \text{o } } \to { \text { } } ( 14{\text{a}}r){\text{-}}[14{\text{a - d}}]{\text{methylene - h}}_4 { \text{mpt } } + { \text{hdo } } $ $ \end{document}8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{d}}_2 + { \text{h}}_2 { \text{o } } \to { \text { hd } } + { \text{hdo}}\quad ( { \text{methenyl - h}}_4 { \text{mpt}}^ + { \text{-dependent } } ) $ $ \end{document}9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{d}}_2 + { \text{h}}_2 { \text{o } } \to { \text { h}}_2 + { \text{d}}_2 { \text{o}}\;({\text{methenyl - h}}_4 { \text{mpt}}^ + { \text{-dependent } } ) $ $ \end{document}10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ para{\text{-h}}_2 { \text { } } \to { \text { } } ortho{\text{-h}}_2 \quad ( { \text{methenyl - h}}_4 { \text{mpt}}^ + { \text{-dependent } } ) $ $ \end{document}fig . 1reaction catalyzed by [ fe ] hydrogenase . n , n - methenyltetrahydromethanopterin ( methenyl - h4mpt ) reduction with h2 to n , n - methylenetetrahydromethanopterin ( methylene - h4mpt ) and a proton , whereby a hydride is stereospecifically transferred from h2 into the pro - r side of methylene - h4mpt reaction catalyzed by [ fe ] hydrogenase . n , n - methenyltetrahydromethanopterin ( methenyl - h4mpt ) reduction with h2 to n , n - methylenetetrahydromethanopterin ( methylene - h4mpt ) and a proton , whereby a hydride is stereospecifically transferred from h2 into the pro - r side of methylene - h4mpt of special interest is the dependence of the reactions in eqs . 8 and 9 on the presence of methenyl - h4mpt . in its absence the h2/h exchange activity the residual activity was attributed to small contaminations of the [ fe ] hydrogenase preparations with methenyl - h4mpt present in high concentrations in the methanogens from which [ fe ] hydrogenase was isolated . [ fe ] hydrogenase is a cytoplasmic homodimeric enzyme ( 2 38 kda ) harboring an iron - containing cofactor , which can be extracted from the enzyme after denaturation in the presence of mercaptoethanol and with which active hydrogenase can be reconstituted from heterologously produced apoprotein . the iron in the cofactor , both when bound to the enzyme and in the free state , is complexed by a pyridone ( possibly functionally equivalent to a cyanide ) , two co and a sulfur and is in a low - spin and low - oxidation state [ 10 , 11 , 3032 ] . [ fe ] hydrogenase is thus similar to [ nife ] and [ fefe ] hydrogenases in containing a low - spin iron carbonyl complex in its active site , albeit in a mononuclear rather than in a dinuclear metal center . this is remarkable since the three hydrogenases are not phylogenetically related and since iron carbonyl complexes have until now not been found in the active site of any other metalloenzyme . the presence of an iron carbonyl complex in [ fe ] , [ fefe ] and [ nife ] hydrogenases indicates that the low - spin iron could play a crucial role in h2 activation in all three enzymes . the low - spin iron in [ fe ] hydrogenase could in principle function in the activation of h2 as a lewis acid or a lewis base . as a lewis acid ( electrophile ) it will bind h2 side - on [ ( -h2)fe ] by which the pk of h2 of 35 is significantly lowered [ 3335 ] . depending on the decrease in pk , the thus - activated h2 would exchange more or less rapidly with protons of water and essentially this exchange is not predicted to require the presence of the hydride acceptor methenyl - h4mpt . when the iron functions as a lewis base ( nucleophile ) , exchange of the bound h2 with protons of water is only possible in the presence of the hydride acceptor . these predictions fostered the question whether the residual h2/h exchange activity shown by [ fe ] hydrogenase preparations might not be independent of methenyl - h4mpt after all . because of the importance of this question for the catalytic mechanism , we took up the problem again and measured the residual exchange activity of [ fe ] hydrogenase reconstituted from highly purified recombinant apoenzyme and highly purified iron - containing cofactor . tritium - labeled h2o ( 185 mbq ml ) was from ge healthcare ( munich , germany ) . lithium wire ( 99% ) of 3.2-mm diameter in mineral oil was from sigma - aldrich ( taufkirchen , germany ) . the detection limit of the t2/h2o exchange assay employed is strongly dependent on the tritium - labeled h2 used being as free as possible of t2o and nt3 , which is why the method of t2 generation from tritium - labeled water and elemental lithium is described in greater detail . the reaction of lithium with h2o had to be done under argon rather than n2 since lithium in water can slowly reduce n2 to nh3 , which is tritium - labeled when the reaction is in t2o . any tritium - labeled nh3 in the gas phase will increase the background radioactivity in the exchange assay and thus increase the lower detection limit . t2 was produced from t2o and an excess of metallic lithium under argon [ 18 , 38 ] . glass vials with a total volume of 8.5 ml , closed with a rubber stopper , were degassed via vacuum and refilled with n2 . the lithium wire was then prepared so that it could be added to the vial . approximately 2.5 cm ( 0.15 g ; approximately 20 mol ) of the wire was removed from the oil in which it was stored and placed on a piece of wax - coated paper . an additional drop of oil was added on top of the wire to ensure that the lithium was protected from n2 , o2 and h2o . under a cover of oil , the lithium wire was dissected into little pieces with a width of approximately 1 mm or less using a scalpel . at this point , the 8.5-ml vial , which was under vacuum , was filled with argon via a needle through the rubber stopper such that the rubber stopper could be lifted slightly under a steady flow of argon and no n2 or o2 could enter the vial . each sliver of lithium was then picked up using tweezers and submersed in a vial containing petroleum ether . once the oil had been removed the sliver was quickly transferred into the vial , which was being kept under an argon cover . after all of the lithium pieces had been transferred in such a manner , the vial was again closed and the stopper fastened in place with an aluminum cap . a vacuum was drawn on the vial and then to the evacuated vial 50 l of tritiated h2o ( 2.8 mol ) was added . the vial was placed in a sonic bath for 2 h and then left to stand at room temperature at least 7 days to allow the excess lithium to react with any residual t2o . the [ fe ] hydrogenase apoenzyme from methanocaldococcus jannaschii was heterologously produced in escherichia coli bl21 ( de3 ) harboring the expression vector pet-24b , which carried the hmd gene from m. jannaschii . the transformed bacterium was cultured aerobically in 2 l tryptone phosphate medium . at an optical density at 578 nm of 1.4 hmd transcription was induced by the addition of isopropylthiogalactoside ( 1 mm ) . three hours after induction , the 2 l culture was harvested by centrifugation . the e. coli cells ( 45 g wet mass ) were anaerobically resuspended in 15 ml of 50 mm tris(hydoxymethyl)aminomethane / hcl ph 7.8 supplemented with 1 mm dithiothreitol and were then disrupted anaerobically at 4 c using a french press and a pressure of 130 mpa . cell debris was removed by ultracentrifugation at 116,000 g for 45 min at 4 c . the supernatant was heated for 20 min at 70 c , centrifuged again for the removal of denatured proteins , and then supplemented with ammonium sulfate to a final concentration of 2 m , centrifuged again and then applied to a phenyl - sepharose column ( 2.6 cm 20 cm ) equilibrated with 50 mm 3-(n - morpholino)propanesulfonic acid ( mops)/koh ph 7.0 containing 2 m ammonium sulfate and 1 mm dithiothreitol . [ fe ] hydrogenase apoenzyme was eluted with a linear decreasing gradient of ammonium sulfate ( 5-ml fractions ) . the apoenzyme - containing fractions [ 1.20.8 m ( nh4)2so4 ] were combined and subsequently washed with 50 mm mops / koh ph 7.0 containing 1 mm dithiothreitol and then concentrated to 35 mg protein ml using microconcentrators with a 10-kda cutoff . in one preparation typically 40 mg of [ fe ] [ fe ] hydrogenase apoenzyme was converted completely into [ fe ] hydrogenase holoenzyme by mixing the apoenzyme with a threefold excess of the iron - containing [ fe ] hydrogenase cofactor . the reconstituted enzyme was then washed three times with 120 mm potassium phosphate ph 6.0 containing 1 mm edta . this step was followed by a concentration of the solution to a final protein concentration of 30 mg ml using microconcentrators with a 10-kda cutoff . the reconstituted enzyme catalyzed the dehydrogenation of methylene - h4mpt ( 20 m ) at 25 c and at ph 6.0 with a specific activity of 150 u mg protein . the [ fe ] hydrogenase cofactor was extracted from [ fe ] hydrogenase holoenzyme of m. marburgensis . the holoenzyme was purified from 80 g cells ( wet mass ) grown at 65 c under nickel - limiting conditions , under which the synthesis of [ fe ] hydrogenase in m. marburgensis is enhanced [ 4042 ] . to extract the cofactor 50 mg purified enzyme was incubated in 10 ml h2o containing 60% methanol , 1 mm mercaptoethanol and 1% ammonia at 4 c for 20 h , conditions under which the cofactor was completely released . then the cofactor was separated from the denatured apoprotein by ultrafiltration using amicon ultra-15 ( 10-kda cutoff ) . after concentration by anoxic evaporation at 4 c the cofactor solution was ultrafiltrated again . the iron - containing cofactor was further purified at 18 c by preparative high - performance liquid chromatography ( hplc ; sykam ) on hitrapqff ( ge healthcare ; 0.7 cm 2.5 cm ) , which was equilibrated with 10 mm ammonium carbonate ph 9.0 containing 1 mm mercaptoethanol ( acm buffer ) . the cofactor was eluted using a linear increasing gradient of nacl from 0 to 1 m in acm buffer . all purification steps were done under red light in an anaerobic chamber ( coy ) filled with 95% n2/5% h2 and containing a palladium catalyst for the continuous removal of o2 . the rate of methylene - h4mpt dehydrogenation was determined at ph 6.0 and 25 c by following the formation of methenyl - h4mpt from methylene - h4mpt ( 20 m ) photometrically at 336 nm ( = 21.6 mm cm ) . one unit of hydrogenase activity is equivalent to 1 mol methenyl - h4mpt formed per minute . the rate of t2/h2o exchange was determined at 25 c in 3.5-ml serum vials closed with a rubber stopper . the 1 ml assay mixtures contained 120 mm potassium phosphate ph 6.0 ( the optimum ph of exchange ) , 1 mm edta and methenyl - h4mpt and [ fe ] hydrogenase in the amounts indicated in the legends to the figures . the 2.5 ml n2 gas phase at 1.2 10 pa contained either 14 or 24% tritium - labeled h2 with a specific radioactivity of 2.4 or 3.3 kbq mol . before the start of the reaction with [ fe ] hydrogenase the gas phase was equilibrated with the liquid phase by 15 min of shaking at 1,100 rpm . then a 0.1 ml sample was taken ( for the determination of the background ) and subsequently the enzyme was added . upon further shaking 0.1 ml samples were withdrawn and placed into empty 20-ml plastic scintillation vials to which 5 ml scintillation cocktail quicksafe a ( zinsser analytic ) was added . then the samples were counted for tritium radioactivity in beckman ls6500 scintillation system . from the specific radioactivity of the tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase added to the assay one unit of exchange activity is equivalent to 1 mol h2 exchanged into water per minute . it was previously shown that the methenyl - h4mpt - dependent exchange between d2 and h2o and that between h2 and d2o catalyzed by [ fe ] hydrogenase proceed with almost the same specific activity , indicating the absence of major isotope effects . the detection limit of the t2/h2o exchange assay employed is strongly dependent on the tritium - labeled h2 used being as free as possible of t2o and nt3 , which is why the method of t2 generation from tritium - labeled water and elemental lithium is described in greater detail . the reaction of lithium with h2o had to be done under argon rather than n2 since lithium in water can slowly reduce n2 to nh3 , which is tritium - labeled when the reaction is in t2o . any tritium - labeled nh3 in the gas phase will increase the background radioactivity in the exchange assay and thus increase the lower detection limit . t2 was produced from t2o and an excess of metallic lithium under argon [ 18 , 38 ] . glass vials with a total volume of 8.5 ml , closed with a rubber stopper , were degassed via vacuum and refilled with n2 . the lithium wire was then prepared so that it could be added to the vial . approximately 2.5 cm ( 0.15 g ; approximately 20 mol ) of the wire was removed from the oil in which it was stored and placed on a piece of wax - coated paper . an additional drop of oil was added on top of the wire to ensure that the lithium was protected from n2 , o2 and h2o . under a cover of oil , the lithium wire was dissected into little pieces with a width of approximately 1 mm or less using a scalpel . at this point , the 8.5-ml vial , which was under vacuum , was filled with argon via a needle through the rubber stopper such that the rubber stopper could be lifted slightly under a steady flow of argon and no n2 or o2 could enter the vial . each sliver of lithium was then picked up using tweezers and submersed in a vial containing petroleum ether . once the oil had been removed the sliver was quickly transferred into the vial , which was being kept under an argon cover . after all of the lithium pieces had been transferred in such a manner , the vial was again closed and the stopper fastened in place with an aluminum cap . a vacuum was drawn on the vial and then to the evacuated vial 50 l of tritiated h2o ( 2.8 mol ) was added . the vial was placed in a sonic bath for 2 h and then left to stand at room temperature at least 7 days to allow the excess lithium to react with any residual t2o . the [ fe ] hydrogenase apoenzyme from methanocaldococcus jannaschii was heterologously produced in escherichia coli bl21 ( de3 ) harboring the expression vector pet-24b , which carried the hmd gene from m. jannaschii . the transformed bacterium was cultured aerobically in 2 l tryptone phosphate medium . at an optical density at 578 nm of 1.4 hmd transcription was induced by the addition of isopropylthiogalactoside ( 1 mm ) . three hours after induction , the 2 l culture was harvested by centrifugation . the e. coli cells ( 45 g wet mass ) were anaerobically resuspended in 15 ml of 50 mm tris(hydoxymethyl)aminomethane / hcl ph 7.8 supplemented with 1 mm dithiothreitol and were then disrupted anaerobically at 4 c using a french press and a pressure of 130 mpa . cell debris was removed by ultracentrifugation at 116,000 g for 45 min at 4 c . the supernatant was heated for 20 min at 70 c , centrifuged again for the removal of denatured proteins , and then supplemented with ammonium sulfate to a final concentration of 2 m , centrifuged again and then applied to a phenyl - sepharose column ( 2.6 cm 20 cm ) equilibrated with 50 mm 3-(n - morpholino)propanesulfonic acid ( mops)/koh ph 7.0 containing 2 m ammonium sulfate and 1 mm dithiothreitol . [ fe ] hydrogenase apoenzyme was eluted with a linear decreasing gradient of ammonium sulfate ( 5-ml fractions ) . the apoenzyme - containing fractions [ 1.20.8 m ( nh4)2so4 ] were combined and subsequently washed with 50 mm mops / koh ph 7.0 containing 1 mm dithiothreitol and then concentrated to 35 mg protein ml using microconcentrators with a 10-kda cutoff . in one preparation typically 40 mg of [ fe ] [ fe ] hydrogenase apoenzyme was converted completely into [ fe ] hydrogenase holoenzyme by mixing the apoenzyme with a threefold excess of the iron - containing [ fe ] hydrogenase cofactor . the reconstituted enzyme was then washed three times with 120 mm potassium phosphate ph 6.0 containing 1 mm edta . this step was followed by a concentration of the solution to a final protein concentration of 30 mg ml using microconcentrators with a 10-kda cutoff . the reconstituted enzyme catalyzed the dehydrogenation of methylene - h4mpt ( 20 m ) at 25 c and at ph 6.0 with a specific activity of 150 u mg protein . the [ fe ] hydrogenase cofactor was extracted from [ fe ] hydrogenase holoenzyme of m. marburgensis . the holoenzyme was purified from 80 g cells ( wet mass ) grown at 65 c under nickel - limiting conditions , under which the synthesis of [ fe ] hydrogenase in m. marburgensis is enhanced [ 4042 ] . to extract the cofactor 50 mg purified enzyme was incubated in 10 ml h2o containing 60% methanol , 1 mm mercaptoethanol and 1% ammonia at 4 c for 20 h , conditions under which the cofactor was completely released . then the cofactor was separated from the denatured apoprotein by ultrafiltration using amicon ultra-15 ( 10-kda cutoff ) . after concentration by anoxic evaporation at 4 c the cofactor solution was ultrafiltrated again . the iron - containing cofactor was further purified at 18 c by preparative high - performance liquid chromatography ( hplc ; sykam ) on hitrapqff ( ge healthcare ; 0.7 cm 2.5 cm ) , which was equilibrated with 10 mm ammonium carbonate ph 9.0 containing 1 mm mercaptoethanol ( acm buffer ) . the cofactor was eluted using a linear increasing gradient of nacl from 0 to 1 m in acm buffer . all purification steps were done under red light in an anaerobic chamber ( coy ) filled with 95% n2/5% h2 and containing a palladium catalyst for the continuous removal of o2 . the rate of methylene - h4mpt dehydrogenation was determined at ph 6.0 and 25 c by following the formation of methenyl - h4mpt from methylene - h4mpt ( 20 m ) photometrically at 336 nm ( = 21.6 mm cm ) . one unit of hydrogenase activity is equivalent to 1 mol methenyl - h4mpt formed per minute . the rate of t2/h2o exchange was determined at 25 c in 3.5-ml serum vials closed with a rubber stopper . the 1 ml assay mixtures contained 120 mm potassium phosphate ph 6.0 ( the optimum ph of exchange ) , 1 mm edta and methenyl - h4mpt and [ fe ] hydrogenase in the amounts indicated in the legends to the figures . the 2.5 ml n2 gas phase at 1.2 10 pa contained either 14 or 24% tritium - labeled h2 with a specific radioactivity of 2.4 or 3.3 kbq mol . before the start of the reaction with [ fe ] hydrogenase the gas phase was equilibrated with the liquid phase by 15 min of shaking at 1,100 rpm . then a 0.1 ml sample was taken ( for the determination of the background ) and subsequently the enzyme was added . upon further shaking 0.1 ml samples were withdrawn and placed into empty 20-ml plastic scintillation vials to which 5 ml scintillation cocktail quicksafe a ( zinsser analytic ) was added . then the samples were counted for tritium radioactivity in beckman ls6500 scintillation system . from the specific radioactivity of the tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase added to the assay one unit of exchange activity is equivalent to 1 mol h2 exchanged into water per minute . it was previously shown that the methenyl - h4mpt - dependent exchange between d2 and h2o and that between h2 and d2o catalyzed by [ fe ] hydrogenase proceed with almost the same specific activity , indicating the absence of major isotope effects . the h2/h exchange activity of [ fe ] hydrogenase was measured by following the incorporation of tritium from tritium - labeled h2 ( 2.4 kbq mol ) into 1 ml h2o . this method is much more sensitive than following the formation of hd and d2 from h2 in d2o or of hd and h2 from d2 in h2o by mass spectrometry . at the times indicated , 0.1 ml samples were withdrawn and counted by liquid scintillation counting . via this method an increase in radioactivity in the 0.1 ml sample by 10 bq ( 4.1 nmol tritium - labeled h2 exchanged into 0.1 ml h2o ) within 60 min could easily be seen ( fig . 2 , triangles ) . also an increase by 5 bq ( 2.05 nmol ) in 60 min was still well observable ( fig . 2 , circles ) . an increase of less than 1.2 bq ( 0.5 nmol ) in 60 min is the lower detection limit , which is equivalent to an exchange activity of the [ fe ] hydrogenase in the 1 ml assay of 0.1 mu . this indicates that at a [ fe ] hydrogenase concentration of 1 mg ml assay , the specific exchange activity would have to be higher than 0.1 mu mg to be observable . 2detection limit of the method employed for the determination of the t2/h2o exchange activity of [ fe ] hydrogenase . the vials contained 1 ml standard assay mixture : 120 mm potassium phosphate ph 6.0 , 1 mm edta and either no enzyme ( open squares ) or 0.5 mg ( circles ) , 1.0 mg ( triangles ) or 10 mg ( filled squares ) purified [ fe ] hydrogenase from methanothermobacter marburgensis . the 2.5 ml gas phase consisted of 24% tritium - labeled h2 ( 2.4 kbq mol ) and 76% n2 at 1.2 10 pa . at the times indicated , 0.1 ml liquid samples were withdrawn and analyzed for tritium radioactivity . from the specific radioactivity of tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase in the 1 ml assay is calculated to be 0.35 mu ( circles ) , 0.7 mu ( triangles ) and 8 mu ( filled squares ) . one unit is equivalent to 1 mol h2 exchanged into water per minute detection limit of the method employed for the determination of the t2/h2o exchange activity of [ fe ] hydrogenase . the vials contained 1 ml standard assay mixture : 120 mm potassium phosphate ph 6.0 , 1 mm edta and either no enzyme ( open squares ) or 0.5 mg ( circles ) , 1.0 mg ( triangles ) or 10 mg ( filled squares ) purified [ fe ] hydrogenase from methanothermobacter marburgensis . the 2.5 ml gas phase consisted of 24% tritium - labeled h2 ( 2.4 kbq mol ) and 76% n2 at 1.2 10 pa . at the times indicated , 0.1 ml liquid samples were withdrawn and analyzed for tritium radioactivity . from the specific radioactivity of tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase in the 1 ml assay is calculated to be 0.35 mu ( circles ) , 0.7 mu ( triangles ) and 8 mu ( filled squares ) . one unit is equivalent to 1 mol h2 exchanged into water per minute the following experiments were performed with [ fe ] hydrogenase reconstituted from recombinant [ fe ] hydrogenase apoenzyme from m. jannaschii and iron - containing [ fe ] hydrogenase cofactor from m. marburgensis . the apoenzyme from m. jannaschii rather than that from m. marburgensis was chosen because it did not form inclusion bodies when heterologously overproduced in e. coli . the apoenzyme was purified via heat denaturation of most of the e. coli proteins ( m. jannaschii is a hyperthermophile ) followed by chromatography on phenyl - sepharose . sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the preparations contain only one polypeptide of apparent molecular mass of approximately 40 kda ( not shown ) . the iron - containing cofactor was extracted from [ fe ] hydrogenase purified from m. marburgensis . reconstitution of the enzyme of m. jannaschii with this cofactor was possible since the [ fe ] hydrogenases from different methanogens all appear to have the same iron - containing cofactor . under the alkaline extraction conditions employed ( 1% nh3 in h2o ; ph 11 ) any contaminating methenyl - h4mpt would have been destroyed . other contaminants and the destroyed methenyl - h4mpt were separated from the iron - containing cofactor by hplc . [ fe ] hydrogenase holoenzyme was reconstituted by the addition of 3 times the molar amount of the iron - containing cofactor to the apoprotein and subsequently excess cofactor was removed by ultrafiltration . the t2/h2o exchange activity of the reconstituted [ fe ] hydrogenase was determined at room temperature since at this temperature the enzyme was active in the exchange assay for more than 60 min ( fig . 2 ) . at 65 c the reconstituted enzyme was rapidly inactivated despite the fact that the optimum temperature for growth of m. jannaschii is 85 c . the reconstituted enzyme catalyzed the conversion of methylene - h4mpt ( 20 m ) to methenyl - h4mpt and h2 with a specific activity of 150 u mg at ph 6.0 and room temperature . the apparent km for methylene - h4mpt was 10 m and the apparent vmax was 220 u mg . at 65 c the specific activity was above 1,000 u mg ( results not shown ) . reconstituted [ fe ] hydrogenase catalyzed the t2/h2o exchange reaction in the presence of methenyl - h4mpt with a specific activity of 10 u mg under our standard assay conditions ( 5 m methenyl - h4mpt , 24% h2 in the gas phase equilibrated with the liquid phase by shaking at 1,100 rpm , ph 6.0 , room temperature ) . the reaction proceeded linearly with time for more than 20 min ( fig . 3a ) and the rate was proportional to the protein concentration up to 40 g ml ( fig . the specific exchange activity decreased because the rate of h2 diffusion from the gas phase into the liquid phase became rate limiting as indicated by the finding that at shaking rates lower than 1,100 rpm the specific exchange activity decreased at lower protein concentrations ( not shown ) . when methenyl - h4mpt was omitted from the assays , no t2/h2o exchange was observable ( fig . 3t2/h2o exchange activity of [ fe ] hydrogenase in the presence and absence of methenyl - h4mpt . a time dependence at different protein concentrations ; b protein dependence . the assay conditions were essentially the same as described in the legend to fig . hydrogenase from m. marburgensis , the assays contained the indicated microgram amounts of reconstituted [ fe ] hydrogenase from methanocaldococcus jannaschii ( jhmd ) . where indicated , the assays contained 5 m methenyl - h4mpt . one unit is equivalent to 1 mol h2 exchanged into water per minute t2/h2o exchange activity of [ fe ] hydrogenase in the presence and absence of methenyl - h4mpt . a time dependence at different protein concentrations ; b protein dependence . the assay conditions were essentially the same as described in the legend to fig . hydrogenase from m. marburgensis , the assays contained the indicated microgram amounts of reconstituted [ fe ] hydrogenase from methanocaldococcus jannaschii ( jhmd ) . where indicated one unit is equivalent to 1 mol h2 exchanged into water per minute in fig . 4 the dependence of the t2/h2o exchange rate on the methenyl - h4mpt concentration is shown at two different h2 concentrations . reciprocal plots of the rates versus the methenyl - h4mpt concentration yielded two straight lines intersecting on the abscissa to the left of the ordinate at a ka of 4 m . at zero methenyl - h4mpt concentration the rate was essentially zero . at infinite methenyl - h4mpt concentration the apparent vmax was 7 u mg for 14% tritium - labeled h2 and 12 u mg for 24% tritium - labeled h2 in the gas phase ( fig . when the h2 concentration in the gas phase was increased above 24% the exchange activity increased hyperbolically with the h2 concentration , half - maximal activity being reached at a h2 concentration of approximately 60% in the gas phase ( approximately 0.5 mm h2 in the liquid phase at 25 c ) . the extrapolated maximal specific activity at infinitely high h2 and methenyl - h4mpt concentrations was 40 u mg ( vmax ) ( results not shown ) . vmax for the exchange reaction ( 40 u mg ) is thus 18% of vmax of methylene - h4mpt dehydrogenation to methenyl - h4mpt and h2 ( 220 u mg ) under the same experimental conditions . the kinetic properties of the reconstituted [ fe]hydrogenase from m. jannaschii are very similar to those of the holoenzyme purified from m. marburgensis . 4dependence of the t2/h2o exchange activity of [ fe ] hydrogenase on the methenyl - h4mpt concentration at two different h2 concentrations . the 1 ml assay contained 120 mm potassium phosphate ph 6.0 , 1 mm edta , 2.0 g reconstituted [ fe ] hydrogenase from m. jannaschii and methenyl - h4mpt at the concentrations indicated . the gas phase was either 24% tritium - labeled h2/76% n2 or 14% tritium - labeled h2/78% n2 . the specific radioactivity of the tritium - labeled h2 was 3.3 kbq mol . one unit is equivalent to 1 mol h2 exchanged into water per minute dependence of the t2/h2o exchange activity of [ fe ] hydrogenase on the methenyl - h4mpt concentration at two different h2 concentrations . the 1 ml assay contained 120 mm potassium phosphate ph 6.0 , 1 mm edta , 2.0 g reconstituted [ fe ] hydrogenase from m. jannaschii and methenyl - h4mpt at the concentrations indicated . the gas phase was either 24% tritium - labeled h2/76% n2 or 14% tritium - labeled h2/78% n2 . the specific radioactivity of the tritium - labeled h2 was 3.3 kbq mol . one unit is equivalent to 1 mol h2 exchanged into water per minute to substantiate the finding that the t2/h2o exchange activity of [ fe ] hydrogenase is absolutely dependent on methenyl - h4mpt , the [ fe ] hydrogenase concentration was increased to 1 mg ml ( 0.025 mm ) ( fig . , there was no measurable t2/h2o exchange in the absence of added methenyl - h4mpt : the radioactivity measured for the 0.1 ml samples was 1.3 0.4 bq throughout the incubation time of more than 2 h. the same radioactivity was found when bovine serum albumin ( 1 mg ml ) instead of [ fe ] hydrogenase was present in the assays ( not shown ) . the relatively high but constant background radioactivity of 1.3 0.4 bq determined by scintillation counting is mainly due to traces of t2o in the t2 gas , which was generated from t2o and li(0 ) . the results show that [ fe ] hydrogenase has a relatively high affinity for methenyl - h4mpt ( ka = 4 m ; keq = 1/ka = 0.25 10 m ) ( fig . 4 ) and that the enzyme catalyzes t2/h2o exchange only in the presence of methenyl - h4mpt even when determined at very high [ fe ] hydrogenase concentrations ( fig . 3b ) . the high affinity of the enzyme for methenyl - h4mpt indicates that some methenyl - h4mpt , if present , will partially copurify with [ fe ] hydrogenase . this can explain why [ fe ] hydrogenase holoenzyme purified from methanogenic archaea , which contain high h4mpt and methenyl - h4mpt concentrations ( more than 1 mm ) , exhibit some exchange activity in the absence of added methenyl - h4mpt ( approximately 1 compared with 10 u mg in the presence of methenyl - h4mpt ) . we therefore heterologously produced the apoenzyme in e. coli which does not contain h4mpt and reconstituted the holoenzyme from the apoenzyme and highly purified iron - containing cofactor . in the absence of added methenyl - h4mpt the reconstituted enzyme did not catalyze the exchange reaction within the detection limit of the method employed , which was near 0.1 mu mg ( fig . 2 ) . the apoprotein and iron - containing [ fe ] hydrogenase cofactor , when tested alone in the absence or presence of methenyl - h4mpt , did not catalyze the exchange reaction , indicating that for t2/h2o exchange the apoprotein , the iron - containing cofactor and methenyl - h4mpt are required . it has been shown that c(14a ) of methenyl - h4mpt , when bound to [ fe ] hydrogenase , has carbocation character [ 4446 ] . carbocations can bind h2 either side - on or end - on , which is followed by a heterolytic cleavage of h2 , the hydride reacting with the carbocation and the proton with a base [ 36 , 47 , 48 , 49 ] . for methenyl - h4mpt the activation barrier for the reduction reaction has been calculated to be reasonable only when in the transition state h2 binds end - on to the carbocation and a base is positioned relative to h2 such that it can directly accept the proton [ 5052 ] . the base was assumed to be the amine group of a lysine or the carboxyl group of an aspartate or a glutamate and the protonated base to be in proton exchange with bulk water [ 36 , 47 ] . at that time iron was not considered as a base since the enzyme was then still thought to be metal free [ 23 , 36 ] ( for metal - free hydrogen activation see ) . but the supposed base could principally also be an iron(0 ) complex or an iron(ii ) complex forming an iron(ii ) hydride or an iron(iv ) hydride [ 5456 ] , respectively , upon protonation . the proposed mechanism predicts that any h2/h exchange catalyzed by [ fe ] hydrogenase should be absolutely dependent on the presence of methenyl - h4mpt , which is what we found . the mechanism described above fails to explain why the base has to be an iron and can not be a proton - accepting group of the protein . it is also not favored by the finding that the exchange reaction is inhibited by co and by cyanide . co inhibition is competitive to h2 , which strongly suggests that h2 binds to an open coordination site rather than to a free electron pair . iron carbonyl complexes with an open coordination site have been shown to form side - on ( -h2)fe complexes that are in proton exchange with bulk water [ 5759 ] . since in the absence of methenyl - h4mpt [ fe ] hydrogenase does not catalyze such an exchange , it has to be assumed that in the absence of methenyl - h4mpt h2 binds to the iron in [ fe ] hydrogenase only weakly , resulting in an exchange activity too low to be detected by the method employed . the requirement of methenyl - h4mpt for h2 binding to the enzyme is also indicated by the finding that the ir spectrum and the circular dichroism spectrum ( unpublished result ) of [ fe ] hydrogenase change significantly only when both h2 and methenyl - h4mpt are present . if the iron in [ fe ] hydrogenase functions as lewis acid , as suggested in the paragraph above , then how can the methenyl - h4mpt dependency of the h2/h2o exchange be explained ? there are three possibilities which come to mind : methenyl - h4mpt induces a conformational change within the active site of the enzyme leading to an activation of the iron which catalyzes the h2/h2o exchange without the carbocation center of methenyl - h4mpt being involved.methenyl-h4mpt binds to the enzyme such that its carbocationic c(14a ) is juxtapositioned to the iron such as to allow the interaction of h2 with both the iron and the carbocation . this interaction would lead to a polarization of h2 to an extent in which a bridging hydride and a bridging proton are formed between the two nucleophiles ( shown for d2 binding in fig . 5 ) ( the carbocation is assumed to resemble a second transition metal center as do carbenes ) . the cationic ( -h)2 complex thus formed is predicted to be a relatively strong acid and therefore to be in proton exchange with bulk water.both the methenyl - h4mpt - induced conformational change and the juxta position of the carbocationic c(14a ) of methenyl - h4mpt are required for h2 activation , which is a combination of possibilities 1 and 2.fig . 5mechanism that can explain the parallel formation of hd and h2 from d2 and h2o as catalyzed by [ fe ] hydrogenase in the presence of methenyl - h4mpt ( fig . the carbocationic c(14a ) of methenyl - h4mpt bound to the enzyme is shown as c. the carbocation is assumed to resemble a second transition metal center as do carbenes . the catalytic cycle starts with the formation of the ( -d2)fe complex , which is in electronic equilibrium with the cationic ( -d)2 complex . this is followed by an exchange of the ( -d)2 complex with protons of bulk water and either by a rotation of hd in the ( -hd)fe complex followed by a second exchange or by the dissociation of the complex with the release of hd . the parallel formation of hd and h2 at equal rates ( fig . 6a ) can be explained assuming that koff = kexchange and that all other steps in the catalytic cycle are not rate limiting methenyl - h4mpt induces a conformational change within the active site of the enzyme leading to an activation of the iron which catalyzes the h2/h2o exchange without the carbocation center of methenyl - h4mpt being involved . methenyl - h4mpt binds to the enzyme such that its carbocationic c(14a ) is juxtapositioned to the iron such as to allow the interaction of h2 with both the iron and the carbocation . this interaction would lead to a polarization of h2 to an extent in which a bridging hydride and a bridging proton are formed between the two nucleophiles ( shown for d2 binding in fig . 5 ) ( the carbocation is assumed to resemble a second transition metal center as do carbenes ) . the cationic ( -h)2 complex thus formed is predicted to be a relatively strong acid and therefore to be in proton exchange with bulk water . both the methenyl - h4mpt - induced conformational change and the juxta position of the carbocationic c(14a ) of methenyl - h4mpt are required for h2 activation , which is a combination of possibilities 1 and 2 . mechanism that can explain the parallel formation of hd and h2 from d2 and h2o as catalyzed by [ fe ] hydrogenase in the presence of methenyl - h4mpt ( fig the carbocationic c(14a ) of methenyl - h4mpt bound to the enzyme is shown as c. the carbocation is assumed to resemble a second transition metal center as do carbenes . the catalytic cycle starts with the formation of the ( -d2)fe complex , which is in electronic equilibrium with the cationic ( -d)2 complex . this is followed by an exchange of the ( -d)2 complex with protons of bulk water and either by a rotation of hd in the ( -hd)fe complex followed by a second exchange or by the dissociation of the complex with the release of hd . the parallel formation of hd and h2 at equal rates ( fig . 6a ) can be explained assuming that koff = kexchange and that all other steps in the catalytic cycle are not rate limiting a larger conformational change of [ fe ] hydrogenase upon methenyl - h4mpt binding is indicated by the finding that [ fe ] hydrogenase crystals crack upon soaking with methenyl - h4mpt . also , it was found that in the presence of methenyl - h4mpt , [ fe ] hydrogenase does not crystallize under the conditions under which the enzyme crystallizes in the absence of methenyl - h4mpt ( unpublished results ) . in fig . 6a , the kinetics of exchange between d2 and h2o as catalyzed by [ fe ] hydrogenase in the presence of methenyl - h4mpt are shown . hd ( single exchange ) and h2 ( double exchange ) are formed at almost equal initial rates . the apparent vmax and km of the enzyme for d2 , hd and h2 were shown to be very similar [ 25 , 28 ] . therefore , the almost identical initial single - exchange and double - exchange rates indicate that free hd is not an intermediate in h2 formation from d2 and h2o . 6kinetics of h2 and hd formation from d2 and h2o catalyzed a by [ fe ] hydrogenase from m. marburgensis and b by the h2-signaling [ nife ] hydrogenase from rhodobacter capsulatus . a the 7 ml assay mixtures at 40 c contained 120 mm potassium phosphate ph 6.0 , 7 m methenyl - h4mpt and 150 m d2 ( 20% saturation ) . the reactions were started by the addition of 0.13 u purified [ fe ] hydrogenase from m. marburgensis . b the 1.5 ml assay mixtures at 30 c contained 50 mm citrate phosphate ph 7.0 and cells of r. capsulatus mutants containing only the h2-signaling hydrogenase . the suspension was gassed with 100% d2 before the vessel was closed and the formation of hd and that of h2 were monitored by mass spectrometry . ( a the data were taken from with permission ; b the data were taken from with permission ) kinetics of h2 and hd formation from d2 and h2o catalyzed a by [ fe ] hydrogenase from m. marburgensis and b by the h2-signaling [ nife ] hydrogenase from rhodobacter capsulatus . a the 7 ml assay mixtures at 40 c contained 120 mm potassium phosphate ph 6.0 , 7 m methenyl - h4mpt and 150 m d2 ( 20% saturation ) . the reactions were started by the addition of 0.13 u purified [ fe ] hydrogenase from m. marburgensis . b the 1.5 ml assay mixtures at 30 c contained 50 mm citrate phosphate ph 7.0 and cells of r. capsulatus mutants containing only the h2-signaling hydrogenase . the suspension was gassed with 100% d2 before the vessel was closed and the formation of hd and that of h2 were monitored by mass spectrometry . ( a the data were taken from with permission ; b the data were taken from with permission ) the parallel formation of h2 and hd from d2 and h2o ( fig . 6a ) is explainable via the exchange mechanism outlined in fig . 5 in which it is assumed that the iron and the c(14a ) of methenyl - h4mpt have to be juxtapositioned such that the h2 can interact with both the iron and the c(14a ) ( possibilities 2 or 3 discussed above ) . the mechanism implies that upon binding of d2 side - on to [ fe ] hydrogenase in the presence of methenyl - h4mpt an ( -d2)fe complex is formed which is in rapid equilibrium with a cationic ( -d)2 complex . only the bridging d , the one not covalently bound to c(14a ) , exchanges with a proton of water , yielding a ( -h)(-d ) complex in equilibrium with a ( -hd)fe complex . from the alternatively , after rotation of hd in the ( -hd)fe complex by 180 a ( -h)(-d ) complex is re - formed in which the bridging h is covalently bound to c(14a ) of methenyl - h4mpt and in which the bridging d is acidic and therefore exchanges with protons of bulk water . for the observed double exchange the rotation is required since the bridging hydride ends up only in the pro - r position of the methylene group of methylene - h4mpt . the second exchange yields a ( -h)2 complex in equilibrium with a ( -h2)fe complex , from which h2 can dissociate off ( fig . 5 ) . with respect to the kinetics it is assumed that the two proton exchange rates ( kexchange ) are equal and that also the two off - dissociation rates ( koff ) ( include diffusion rates out of the enzyme s active - site pocket ) are equal and that both the exchange rates and the off - dissociation rates are slow relative to the rates of all other steps , including the rotation step involved in the proposed catalytic cycle . with these assumptions the exchange mechanism proposed in fig . 5 predicts that hd and h2 are parallely formed from d2 and h2o at equal rates when kexchange koff . if kexchange < koff then more hd than h2 is formed and if kexchange > koff then more h2 than hd is generated . the cationic ( -h)2 complex proposed to be formed upon binding of methenyl - h4mpt and h2 to [ fe ] hydrogenase ( shown for d2 binding in fig . 5 ) is most likely also formed upon binding of methylene - h4mpt to the enzyme , which can explain the direct exchange of the pro - r hydrogen of methylene - h4mpt with protons of bulk water ( eq . 7 ) . in fig . 6b the kinetics of single and double exchange of d2 with h2o catalyzed by h2-signaling [ nife ] hydrogenase from rhodobacter capsulatus are shown . 6a for the [ fe ] hydrogenase from m. marburgensis but are also similar to the exchange kinetics for reactions catalyzed by other [ nife ] and [ fefe ] hydrogenases , the only differences being that the relative rates of the single - exchange and the double - exchange reaction vary from enzyme to enzyme ( table 1 ) . this finding substantiates the notion that the low - spin iron carbonyl in the active sites of the three types of hydrogenases could have the same function , namely , to help polarize the h2 such that a hydride can be transferred to an acceptor , which is the c(14a ) of methenyl - h4mpt in the case of [ fe ] hydrogenase , the proximal iron of the diiron center in the case of [ fefe ] hydrogenases and the nickel in the nife center in case of [ nife ] hydrogenases . table 1h2 and hd formation from d2 and h2o catalyzed by purified [ fe ] , [ fefe ] , [ nife ] and [ nifese ] hydrogenasestypeorganismtopologyinitial rates of hd and h2 formationreferences[fe]methanothermobacter marburgensiscytoplasmichd h2[fefe]desulfovibrio vulgarisperiplasmichd > h2[fefe]desulfovibrio desulfricansperiplasmichd > h2[nife]desulfovibrio gigasperiplasmichd > h2[nife]desulfovibrio fructosovoransperiplasmichd > h2[63 , 64][nifese]desulfovibrio baculatusperiplasmichd < h2[61 , 64 , 65][nifese]desulfovibrio baculatuscytoplasmichd < h2[nifese]desulfovibrio baculatusmembrane - boundhd < h2[nifese]desulfovibrio salexigensperiplasmichd < h2[nife]thiocapsa roseopersicinaperiplasmichd > h2[nife]rhodobacter capsulatusmembrane - boundhd < h2[nife]rhodobacter capsulatuscytoplasmichd h2[64 , 67][nife]methanosarcina barkericytoplasmichd > h2[nife]ralstonia eutrophacytoplasmich2 hd[nife]azotobacter vinelandiimembrane - boundh2 hdcell suspensions h2 and hd formation from d2 and h2o catalyzed by purified [ fe ] , [ fefe ] , [ nife ] and [ nifese ] hydrogenases

[ fe ] hydrogenase ( iron sulfur - cluster - free hydrogenase ) catalyzes the reversible reduction of methenyltetrahydromethanopterin ( methenyl - h4mpt+ ) with h2 to methylene - h4mpt , a reaction involved in methanogenesis from h2 and co2 in many methanogenic archaea . the enzyme harbors an iron - containing cofactor , in which a low - spin iron is complexed by a pyridone , two co and a cysteine sulfur . [ fe ] hydrogenase is thus similar to [ nife ] and [ fefe ] hydrogenases , in which a low - spin iron carbonyl complex , albeit in a dinuclear metal center , is also involved in h2 activation . like the [ nife ] and [ fefe ] hydrogenases , [ fe ] hydrogenase catalyzes an active exchange of h2 with protons of water ; however , this activity is dependent on the presence of the hydride - accepting methenyl - h4mpt+ . in its absence the exchange activity is only 0.01% of that in its presence . the residual activity has been attributed to the presence of traces of methenyl - h4mpt+ in the enzyme preparations , but it could also reflect a weak binding of h2 to the iron in the absence of methenyl - h4mpt+ . to test this we reinvestigated the exchange activity with [ fe ] hydrogenase reconstituted from apoprotein heterologously produced in escherichia coli and highly purified iron - containing cofactor and found that in the absence of added methenyl - h4mpt+ the exchange activity was below the detection limit of the tritium method employed ( 0.1 nmol min1 mg1 ) . the finding reiterates that for h2 activation by [ fe ] hydrogenase the presence of the hydride - accepting methenyl - h4mpt+ is essentially required . this differentiates [ fe ] hydrogenase from [ fefe ] and [ nife ] hydrogenases , which actively catalyze h2/h2o exchange in the absence of exogenous electron acceptors .

Introduction Materials and methods Generation of tritium-labeled H Purification of the [Fe]hydrogenase apoenzyme Purification of the iron-containing [Fe]hydrogenase cofactor Determination of [Fe]hydrogenase activities Results Discussion

three types are presently known which are phylogenetically not related [ 3 , 4 ] : [ nife ] hydrogenases found in archaea and bacteria , [ fefe ] hydrogenases found in bacteria and eukarya [ 68 ] and [ fe ] hydrogenases only found in methanogenic archaea [ 9 , 10 ] . [ fefe ] hydrogenases are still also referred to as iron - only hydrogenases , which was the name used before it was shown that [ fe ] hydrogenase also contains iron . the iron in both the [ nife ] and the [ fefe ] center is complexed by co , cyanide and sulfur ligands , and the iron in [ nife ] hydrogenase and the distal iron in [ fefe ] hydrogenase ( distal to the [ 4fe4s ] cluster ) are in a low - spin fe(ii ) oxidation state [ 1215 ] . 5 ) in the absence of exogenous electron acceptors , indicating that [ nife ] and [ fefe ] hydrogenases have a ping - pong catalytic mechanism . [ fe ] hydrogenase differs from [ nife ] and [ fefe ] hydrogenases in that it contains a mononuclear metal center rather than a dinuclear metal center in its active site and is devoid of iron sulfur clusters , which is why the enzyme is also referred to as iron sulfur - cluster - free hydrogenase . [ fe ] hydrogenase catalyzes the reversible reduction of methenyltetrahydromethanopterin ( methenyl - h4mpt ) with h2 to methylenetetrahydromethanopterin ( methylene - h4mpt ) ( eq . 6 ) , a reaction involved in co2 reduction with h2 to methane in many methanogenic archaea [ 9 , 1923 ] . [ fe ] hydrogenase also catalyzes a stereospecific direct exchange of the pro - r hydrogen of methylene - h4mpt with protons of water ( eq . the catalytic mechanism of the enzyme is thus clearly ternary complex rather than ping - pong and thus quite different from that of [ nife ] and [ fefe ] hydrogenases . n , n - methenyltetrahydromethanopterin ( methenyl - h4mpt ) reduction with h2 to n , n - methylenetetrahydromethanopterin ( methylene - h4mpt ) and a proton , whereby a hydride is stereospecifically transferred from h2 into the pro - r side of methylene - h4mpt reaction catalyzed by [ fe ] hydrogenase . n , n - methenyltetrahydromethanopterin ( methenyl - h4mpt ) reduction with h2 to n , n - methylenetetrahydromethanopterin ( methylene - h4mpt ) and a proton , whereby a hydride is stereospecifically transferred from h2 into the pro - r side of methylene - h4mpt of special interest is the dependence of the reactions in eqs . in its absence the h2/h exchange activity the residual activity was attributed to small contaminations of the [ fe ] hydrogenase preparations with methenyl - h4mpt present in high concentrations in the methanogens from which [ fe ] hydrogenase was isolated . [ fe ] hydrogenase is a cytoplasmic homodimeric enzyme ( 2 38 kda ) harboring an iron - containing cofactor , which can be extracted from the enzyme after denaturation in the presence of mercaptoethanol and with which active hydrogenase can be reconstituted from heterologously produced apoprotein . the iron in the cofactor , both when bound to the enzyme and in the free state , is complexed by a pyridone ( possibly functionally equivalent to a cyanide ) , two co and a sulfur and is in a low - spin and low - oxidation state [ 10 , 11 , 3032 ] . [ fe ] hydrogenase is thus similar to [ nife ] and [ fefe ] hydrogenases in containing a low - spin iron carbonyl complex in its active site , albeit in a mononuclear rather than in a dinuclear metal center . the presence of an iron carbonyl complex in [ fe ] , [ fefe ] and [ nife ] hydrogenases indicates that the low - spin iron could play a crucial role in h2 activation in all three enzymes . the low - spin iron in [ fe ] hydrogenase could in principle function in the activation of h2 as a lewis acid or a lewis base . depending on the decrease in pk , the thus - activated h2 would exchange more or less rapidly with protons of water and essentially this exchange is not predicted to require the presence of the hydride acceptor methenyl - h4mpt . when the iron functions as a lewis base ( nucleophile ) , exchange of the bound h2 with protons of water is only possible in the presence of the hydride acceptor . these predictions fostered the question whether the residual h2/h exchange activity shown by [ fe ] hydrogenase preparations might not be independent of methenyl - h4mpt after all . because of the importance of this question for the catalytic mechanism , we took up the problem again and measured the residual exchange activity of [ fe ] hydrogenase reconstituted from highly purified recombinant apoenzyme and highly purified iron - containing cofactor . the [ fe ] hydrogenase apoenzyme from methanocaldococcus jannaschii was heterologously produced in escherichia coli bl21 ( de3 ) harboring the expression vector pet-24b , which carried the hmd gene from m. jannaschii . from the specific radioactivity of the tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase added to the assay one unit of exchange activity is equivalent to 1 mol h2 exchanged into water per minute . it was previously shown that the methenyl - h4mpt - dependent exchange between d2 and h2o and that between h2 and d2o catalyzed by [ fe ] hydrogenase proceed with almost the same specific activity , indicating the absence of major isotope effects . the [ fe ] hydrogenase apoenzyme from methanocaldococcus jannaschii was heterologously produced in escherichia coli bl21 ( de3 ) harboring the expression vector pet-24b , which carried the hmd gene from m. jannaschii . from the specific radioactivity of the tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase added to the assay one unit of exchange activity is equivalent to 1 mol h2 exchanged into water per minute . it was previously shown that the methenyl - h4mpt - dependent exchange between d2 and h2o and that between h2 and d2o catalyzed by [ fe ] hydrogenase proceed with almost the same specific activity , indicating the absence of major isotope effects . an increase of less than 1.2 bq ( 0.5 nmol ) in 60 min is the lower detection limit , which is equivalent to an exchange activity of the [ fe ] hydrogenase in the 1 ml assay of 0.1 mu . from the specific radioactivity of tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase in the 1 ml assay is calculated to be 0.35 mu ( circles ) , 0.7 mu ( triangles ) and 8 mu ( filled squares ) . from the specific radioactivity of tritium - labeled h2 and the radioactivity above the background incorporated into water per 60 min , the exchange activity of the [ fe ] hydrogenase in the 1 ml assay is calculated to be 0.35 mu ( circles ) , 0.7 mu ( triangles ) and 8 mu ( filled squares ) . reconstitution of the enzyme of m. jannaschii with this cofactor was possible since the [ fe ] hydrogenases from different methanogens all appear to have the same iron - containing cofactor . [ fe ] hydrogenase holoenzyme was reconstituted by the addition of 3 times the molar amount of the iron - containing cofactor to the apoprotein and subsequently excess cofactor was removed by ultrafiltration . reconstituted [ fe ] hydrogenase catalyzed the t2/h2o exchange reaction in the presence of methenyl - h4mpt with a specific activity of 10 u mg under our standard assay conditions ( 5 m methenyl - h4mpt , 24% h2 in the gas phase equilibrated with the liquid phase by shaking at 1,100 rpm , ph 6.0 , room temperature ) . 3t2/h2o exchange activity of [ fe ] hydrogenase in the presence and absence of methenyl - h4mpt . one unit is equivalent to 1 mol h2 exchanged into water per minute t2/h2o exchange activity of [ fe ] hydrogenase in the presence and absence of methenyl - h4mpt . one unit is equivalent to 1 mol h2 exchanged into water per minute to substantiate the finding that the t2/h2o exchange activity of [ fe ] hydrogenase is absolutely dependent on methenyl - h4mpt , the [ fe ] hydrogenase concentration was increased to 1 mg ml ( 0.025 mm ) ( fig . , there was no measurable t2/h2o exchange in the absence of added methenyl - h4mpt : the radioactivity measured for the 0.1 ml samples was 1.3 0.4 bq throughout the incubation time of more than 2 h. the same radioactivity was found when bovine serum albumin ( 1 mg ml ) instead of [ fe ] hydrogenase was present in the assays ( not shown ) . 4 ) and that the enzyme catalyzes t2/h2o exchange only in the presence of methenyl - h4mpt even when determined at very high [ fe ] hydrogenase concentrations ( fig . this can explain why [ fe ] hydrogenase holoenzyme purified from methanogenic archaea , which contain high h4mpt and methenyl - h4mpt concentrations ( more than 1 mm ) , exhibit some exchange activity in the absence of added methenyl - h4mpt ( approximately 1 compared with 10 u mg in the presence of methenyl - h4mpt ) . in the absence of added methenyl - h4mpt the reconstituted enzyme did not catalyze the exchange reaction within the detection limit of the method employed , which was near 0.1 mu mg ( fig . the apoprotein and iron - containing [ fe ] hydrogenase cofactor , when tested alone in the absence or presence of methenyl - h4mpt , did not catalyze the exchange reaction , indicating that for t2/h2o exchange the apoprotein , the iron - containing cofactor and methenyl - h4mpt are required . it has been shown that c(14a ) of methenyl - h4mpt , when bound to [ fe ] hydrogenase , has carbocation character [ 4446 ] . the proposed mechanism predicts that any h2/h exchange catalyzed by [ fe ] hydrogenase should be absolutely dependent on the presence of methenyl - h4mpt , which is what we found . since in the absence of methenyl - h4mpt [ fe ] hydrogenase does not catalyze such an exchange , it has to be assumed that in the absence of methenyl - h4mpt h2 binds to the iron in [ fe ] hydrogenase only weakly , resulting in an exchange activity too low to be detected by the method employed . the requirement of methenyl - h4mpt for h2 binding to the enzyme is also indicated by the finding that the ir spectrum and the circular dichroism spectrum ( unpublished result ) of [ fe ] hydrogenase change significantly only when both h2 and methenyl - h4mpt are present . if the iron in [ fe ] hydrogenase functions as lewis acid , as suggested in the paragraph above , then how can the methenyl - h4mpt dependency of the h2/h2o exchange be explained ? there are three possibilities which come to mind : methenyl - h4mpt induces a conformational change within the active site of the enzyme leading to an activation of the iron which catalyzes the h2/h2o exchange without the carbocation center of methenyl - h4mpt being involved.methenyl-h4mpt binds to the enzyme such that its carbocationic c(14a ) is juxtapositioned to the iron such as to allow the interaction of h2 with both the iron and the carbocation . 5mechanism that can explain the parallel formation of hd and h2 from d2 and h2o as catalyzed by [ fe ] hydrogenase in the presence of methenyl - h4mpt ( fig . 6a ) can be explained assuming that koff = kexchange and that all other steps in the catalytic cycle are not rate limiting methenyl - h4mpt induces a conformational change within the active site of the enzyme leading to an activation of the iron which catalyzes the h2/h2o exchange without the carbocation center of methenyl - h4mpt being involved . mechanism that can explain the parallel formation of hd and h2 from d2 and h2o as catalyzed by [ fe ] hydrogenase in the presence of methenyl - h4mpt ( fig the carbocationic c(14a ) of methenyl - h4mpt bound to the enzyme is shown as c. the carbocation is assumed to resemble a second transition metal center as do carbenes . also , it was found that in the presence of methenyl - h4mpt , [ fe ] hydrogenase does not crystallize under the conditions under which the enzyme crystallizes in the absence of methenyl - h4mpt ( unpublished results ) . 6a , the kinetics of exchange between d2 and h2o as catalyzed by [ fe ] hydrogenase in the presence of methenyl - h4mpt are shown . 6kinetics of h2 and hd formation from d2 and h2o catalyzed a by [ fe ] hydrogenase from m. marburgensis and b by the h2-signaling [ nife ] hydrogenase from rhodobacter capsulatus . ( a the data were taken from with permission ; b the data were taken from with permission ) kinetics of h2 and hd formation from d2 and h2o catalyzed a by [ fe ] hydrogenase from m. marburgensis and b by the h2-signaling [ nife ] hydrogenase from rhodobacter capsulatus . the mechanism implies that upon binding of d2 side - on to [ fe ] hydrogenase in the presence of methenyl - h4mpt an ( -d2)fe complex is formed which is in rapid equilibrium with a cationic ( -d)2 complex . the cationic ( -h)2 complex proposed to be formed upon binding of methenyl - h4mpt and h2 to [ fe ] hydrogenase ( shown for d2 binding in fig . 5 ) is most likely also formed upon binding of methylene - h4mpt to the enzyme , which can explain the direct exchange of the pro - r hydrogen of methylene - h4mpt with protons of bulk water ( eq . 6a for the [ fe ] hydrogenase from m. marburgensis but are also similar to the exchange kinetics for reactions catalyzed by other [ nife ] and [ fefe ] hydrogenases , the only differences being that the relative rates of the single - exchange and the double - exchange reaction vary from enzyme to enzyme ( table 1 ) . this finding substantiates the notion that the low - spin iron carbonyl in the active sites of the three types of hydrogenases could have the same function , namely , to help polarize the h2 such that a hydride can be transferred to an acceptor , which is the c(14a ) of methenyl - h4mpt in the case of [ fe ] hydrogenase , the proximal iron of the diiron center in the case of [ fefe ] hydrogenases and the nickel in the nife center in case of [ nife ] hydrogenases . table 1h2 and hd formation from d2 and h2o catalyzed by purified [ fe ] , [ fefe ] , [ nife ] and [ nifese ] hydrogenasestypeorganismtopologyinitial rates of hd and h2 formationreferences[fe]methanothermobacter marburgensiscytoplasmichd h2[fefe]desulfovibrio vulgarisperiplasmichd > h2[fefe]desulfovibrio desulfricansperiplasmichd > h2[nife]desulfovibrio gigasperiplasmichd > h2[nife]desulfovibrio fructosovoransperiplasmichd > h2[63 , 64][nifese]desulfovibrio baculatusperiplasmichd < h2[61 , 64 , 65][nifese]desulfovibrio baculatuscytoplasmichd < h2[nifese]desulfovibrio baculatusmembrane - boundhd < h2[nifese]desulfovibrio salexigensperiplasmichd < h2[nife]thiocapsa roseopersicinaperiplasmichd > h2[nife]rhodobacter capsulatusmembrane - boundhd < h2[nife]rhodobacter capsulatuscytoplasmichd h2[64 , 67][nife]methanosarcina barkericytoplasmichd > h2[nife]ralstonia eutrophacytoplasmich2 hd[nife]azotobacter vinelandiimembrane - boundh2 hdcell suspensions h2 and hd formation from d2 and h2o catalyzed by purified [ fe ] , [ fefe ] , [ nife ] and [ nifese ] hydrogenases

during the past 20 years , proteoglycans ( pgs ) have emerged as critical modulators of most major cellular processes , including cell proliferation , adhesion , and migration , and have been involved in many pathological conditions , such as inflammation , cancer , or infection [ 15 ] . these complex glycoproteins , which consist of a core protein bearing polysaccharide chains of glycosaminoglycans ( gags ) , are abundantly found at the surface of cells and in extracellular matrices ( ecms ) . from there , they benefit from an ideal positioning to interact , either through their protein core or their gag chains , with soluble signaling effectors ( growth factors , cytokines , chemokines ) [ 6 , 7 ] , structural components of the ecm ( fibronectin , collagens ) and membrane - associated proteins ( receptors , integrins ) [ 811 ] , or intracellular molecules ( pkc , syntenin , cask / lin-2 ) [ 12 , 13 ] . however , pg activities are not restricted to the control of local contacts and molecular interactions at the cell surface vicinity . shedding of cell - surface pgs ( particularly hspg syndecans and glypicans ) , enabling release of soluble gag - associated ectodomains , is known to play a key role in many physiopathological conditions , such as wound healing , inflammation , tumor progression , and infection [ 1416 ] . paracrine activities can also be exerted by free , soluble pgs , such as endocan , a small , soluble dermatan sulfate proteoglycan ( dspg ) expressed by endothelial cells and naturally found in the blood circulation of healthy subjects [ 1720 ] . the history of endocan originates from the discovery in 1996 of a soluble protein produced by endothelial cells : endothelial cell - specific molecule-1 ( esm-1 ) . posttranslational modification studies later led to the identification of a ds chain on esm-1 protein core , which was consequently included to the pg family , under the new name of endocan . since then , endocan has rapidly emerged as a very distinctive pg , in many aspects : ( i ) endocan is the one of very few circulating pg sensu stricto , ( ii ) in contrast with most ecm pgs , which are usually large , modular proteins that harbor several gag chains , endocan comprises a relatively small polypeptide associated to one single polysaccharide chain , and ( iii ) characterization of endocan ds chain revealed uncommon structural features and saccharide composition . interestingly , increasing experimental evidence has reported that endocan is overexpressed in cancer , sepsis , obesity , or inflammatory conditions [ 20 , 21 , 2328 ] . furthermore , blood levels of freely circulating pg have been associated with poor prognosis in several types of cancers ( reviewed in ) . the aim of this paper is to review the present knowledge about endocan expression , structure , and activity , with a special focus on the recent developments that have been highlighting that endocan , as an endothelial proteoglycan not only is a biomarker of neoangiogenesis but also appears today as a signature of the tumor progression when it is expressed by the tumor cells themselves . endocan is the product of one single gene , esm , located in the proximal region of chromosome 5 long arm ( 5q11.2 ) . the esm gene is organized in 3 exons separated by 2 introns , encoding an 552 bp open reading frame . transcriptional control is ensured by a 3888 bp promoter at the 5 flanking region of esm , which features a typical tata box and a number of putative transcriptional binding sites , including ets , hhex , and cre - like motifs [ 29 , 30 ] . noteworthy , ets and hhex motifs have been involved in the regulation of genes encoding several angiogenic and ecm remodeling factors , such as vegf receptors-1 and -2 , tie-1 and -2 , neuropilin-1 , or ve - cadherin [ 24 , 29 , 30 ] . endocan mrnas were first identified from cultures of human umbilical vein endothelial cells ( huvecs ) , but expression of endocan was subsequently reported in a large panel of cultured human endothelial cells , such as endothelial cells from the dermal microvasculature , the coronary and pulmonary arteries , or capillaries from adipose tissues [ 29 , 31 ] . surprisingly , endocan could not be detected in highly vascularized organs , such as brain , heart , pancreas , or liver [ 21 , 31 ] , suggesting that endocan expression may be characteristic of activated rather than resting endothelial cells . in support to this , a recent study reported endocan expression during the endothelial - mesenchymal transition process , suggesting a role during arterial wall remodeling . in vivo , endocan is detected with an average concentration of ~1 ng / ml in the serum healthy patients however , as detailed below , levels significantly increase in patients with septic shock or cancers such as lung , kidney , or colon cancers . tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , transforming growth factor-1 ( tgf-1 ) , fibroblast growth factor-2 ( fgf-2 ) , and vascular endothelial growth factor ( vegf ) have been shown to induce endocan expression in vitro [ 3540 ] , while interferon- ( ifn- ) inhibits tnf- induced upregulation of endocan . one of the first studies on this topic came from daly and collaborators , who reported that endocan ( as angiopoetin 2 ) is a downstream target of the endogenous transcription factor fkhr that regulates many genes associated to vascular destabilization / remodeling . using pharmacological compounds , abid and collaborators investigated intracellular pathways involved in vegf - induced secretion of endocan in cultured endothelial cells . vegf induction of endocan mrna was blocked by bim ( pkc inhibitor ) but not pd98059 ( mek1/2 inhibitor ) . in contrast , preincubation of huvec with ly294002 ( pi3 k inhibitor ) or with pma ( pkc activator ) resulted in increased basal levels of endocan mrna . vegf - mediated induction of endocan was therefore found to be positively and negatively regulated by pkc / nfb and pi3 k / akt / fkhrl1 signaling pathways , respectively . this was further supported by rennel and collaborators , who also reported that vegf - a induction of endocan expression in cultured endothelial cells was regulated via the pi3 k pathway . furthermore , they showed that pi3 k inhibitor ly294002 alone caused an increase in endocan transcripts to levels higher than those induced by vegf . noteworthy , recent studies have suggested that endocan expression may regulate cell survival in cancer cells via the nfkb pathway [ 43 , 44 ] . this variant , resulting from alternative splicing of exon 2 , is not secreted and not glycosylated ( deletion of the gag attachment site ) [ 18 , 31 ] . endocan is one of the rare circulating pgs that have been identified , along with bikunin and macrophage colony stimulating factor-1 ( mcsf-1 ) , which are both cspgs . structurally , endocan is composed of a 165 amino acid core protein bearing one single ds chain ( figure 1 ) . the protein moiety features an n - terminal cysteine - rich region ( w - k ) that includes an egf - like domain ( y - m ) , a phenylalanine - rich region ( f - y ) , and a c - terminal region ( s - r ) that comprises the sg dipeptide , where attachment of the ds chain occurs . surprisingly , endocan shares no obvious homologies with other extracellular members of the pg family . endocan does not include leucin - rich repeats , which are the signature of small leucine - rich protein ( slrp ) pgs , nor c - type lectin - like domains that are typically found in hyalectans . egf - like domains are also present in hyalectan pgs as well as in hspgs , such as perlecan or agrin . in perlecan , such domains have been suggested to play a role in protein oligomerisation . however , no precise function has been associated yet with the single egf - like domain found in endocan . in contrast , 1528% of sequence identity can be found between the cystein - rich n - terminal region of endocan and members of the insulin - like growth factor binding protein ( igfbp ) family . however , unlike these proteins , endocan does not bind to igf [ 21 , 47 ] . in addition , endocan sequence shows some homology with human 2 integrin , fibrillin-1 , laminin-2 , and procollagen 2 . finally , a 74% sequence homology was found with pg25 , a protein isolated from rat pineal gland . pg25 has therefore been categorized as rat endocan , thereby representing the first endocan ortholog to be identified at the protein level . expression of endocan orthologs has also been identified by southern blot in mouse , rabbit , dog , cow , and monkey , with a high degree of gene conservation [ 21 , 29 , 48 ] . the saccharide moiety of human endocan is represented by as single chain of dermatan sulfate ( ds ) . ds is a member of the gag family , which include glucosaminoglycan ha , heparin and hs , and galactosaminoglycans cs and ds [ 49 , 50 ] . gags are complex , linear polysaccharides , constituted of a repeating disaccharide unit comprising an n - substituted hexosamine and an uronic acid . in galactosaminoglycans , the hexosamine is an n - acetylgalactosamine , which is associated with either glucuronic acid ( glca ) for cs or glca and a variable proportion of its c5-epimer iduronic acid ( idoa ) for ds . in addition , this saccharide backbone is modified further by addition of o - sulfate groups : at c-2 of the uronic acid and at c-4/c6 of the galactosamine . resulting sulfation patterns have led to a classification of cs disaccharide units as 0 units standing for glca - galnac , a units for glca - galnac,4s , b units for glca,2s - galnac,4s , c units for glca - galnac,6s , d - units for glca,2s - galnac,6s , and e - units for glca - galnac,4,6s ( figure 2 ) . for ds comprising a idoa instead of glca , i0 , ia , ib , ic i d , and ie units have been defined accordingly . both saccharide backbone and sulfation pattern provide the structural basis of cs / ds large interactive properties . idoa residue confers functional specificities to ds [ 5153 ] , most probably by enhancing flexibility of saccharide motifs . furthermore , increasing evidence has shown that specific cs / ds sulfation patterns were involved in precise biological functions and that variations of these sulfation patterns can be associated with physiological changes and pathological developments [ 5557 ] . in contrast with most extracellular pg , one single gag chain is found attached to endocan protein core . early studies reported partial susceptibility to chondroitinase b , thereby identifying endocan chain as of the ds type . since then , its structural features and binding properties have been extensively studied [ 17 , 22 ] . the ds chain endocan expressed by endothelial cells is ~15 kda in size ( e.g. , ~32 disaccharides ) , which is relatively shorter than cs / ds chains found on pgs . this unique gag chain includes a significant proportion of idoa residue ( 31% ) that are distributed along the polysaccharide chain , either as alternating glca / idoa - containing disaccharide sequences or in short clusters of 2 - 3 disaccharides . most interestingly , when compared to cs / ds from various sources [ 55 , 57 ] , disaccharide analysis of endocan ds revealed a higher proportion of nonsulfated disaccharides ( 1012% of hexa - galnac ) and of the disulfated b , d , and e units that are rather rare in mammals . as highly sulfated domains are usually involved in gag / protein interactions , it is speculated that the presence of such disaccharide may be critical for endocan biological properties . as many pgs , biological properties of endocan depend on ligand interactions involving either its protein core or its ds chain . in vitro , endocan protein core binds to lymphocytes and monocytes through a high affinity interaction with the integrin cd11a endocan inhibits lfa-1/icam-1 interaction , thereby affecting leukocyte adhesion and migration in tissues [ 19 , 58 ] . endocan ds chain has been shown to bind and activate hepatocyte growth factor ( hgf ) in vitro [ 17 , 22 ] . hgf is a multifunctional growth factor that can bind to both hs and ds gags and promotes , through interaction with its specific receptor met , motility and proliferation of many different cell types cells [ 59 , 60 ] . importantly , endocan variant lacking the ds chain failed to induce hgf activation , thus confirming the role played by the gag chain in this process . furthermore , biosensor analysis reported a kd of ~36 nm for hgf / endocan interaction , which is well in the 0.220 nm range of previously published affinity values for hgf binding to free hs and ds chains [ 51 , 61 , 62 ] . high affinity interaction of gags with hgf is critical for its activation of the met receptor , which plays major roles in many physiopathological processes , including development , wound healing , and tumor progression [ 6365 ] . through its ability to promote hgf activity , endocan in addition , endocan has been shown to promote the mitogenic and migratory activities of vegf - a and -c in vitro . finally , ds is known to bind to a multitude of signaling and adhesion molecules , including l- and p - selectins , matrix proteins such as fibronectin , chemokines / cytokines rantes , sdf-1 , il-8 , mcp-1 , ifn- , and pf-4 , and growth factors midkine , pleiotrophin fgf-2 , and -7 [ 24 , 57 ] , leaving open the question of a possible interaction of these ligands with endocan . this process gives rise to a new blood supply so that an emerging tumor shifts from a localized stage to an aggressive behavior [ 67 , 68 ] . pertinent biomarkers of angiogenesis are urgently needed to detect this shift in order to apply the best personalized treatment to the patient . the major growth factors and cytokines involved in vascular growth have been failing as biomarkers of the progression in tumor development . endocan , as a pg secreted by endothelial cells when activated , has appeared over the years as a promising tissue - based and also blood - based marker and we will detail later the most recent literature evidences . the release of endocan by cultured endothelial cells has been largely described to be strongly upregulated in presence of proangiogenic molecules such as vegf , fgf-2 , and vegf - c that are critical mediators involved in angiogenesis ( i.e. , formation of blood vessel ) , lymphangiogenesis ( i.e. , formation of lymphatic vessel ) , and cancer progression ( spreading and metastasis ) [ 23 , 28 , 35 , 36 , 39 , 66 ] . in addition , endocan has been shown to directly promote the mitogenic and migratory activities of vegf - a and -c on cultured endothelial cells . interestingly , sirna silencing of endocan expression inhibits vegf - a/-c induced proliferation of lymphatic endothelial cells . furthermore , addition of recombinant endocan protein to endocan sirna - transfected endothelial cells restores the stimulatory effect of vegf - a on migration , hence suggesting a role of endocan in lymphangiogenesis . endocan expression is also induced in cultured endothelial cells undergoing tube formation as an assay to assess angiogenesis and vasculogenesis in vitro . in animal models , endocan gene is one of the main genes involved in the angiogenic switch occurring during retinal neovascularization induced by hypoxia . a link has been recently established between endocan and the tip cells , the specialized subset of endothelial cells known to mediate vessel growth in neoangiogenesis [ 70 , 71 ] . these tip cells are the sensor cells at the end ( the tip ) of the growing new vessels , which lead the building of the tumor vascular network required for tumor progression . the stalk cells ( constituting the vessel to be ) are not or less expressing endocan , which may suggest that endocan will decrease with vascular regression . taken together , all these experimental data highlight endocan as a marker of endothelial cell activation during the growth of new vessels required for tumor progression . data obtained from cancer tissue samples strengthen all that experimental evidence of endocan as a relevant biomarker for clinics . by immunohistochemistry , endocan is visualized in the cytoplasm of endothelial cells of the newly growing tumor vessels ( figure 3 ) . in the literature , a marked cytoplasmic expression of vascular endocan has been then described inside the tumor vessels in different types of highly vascularized tumors such as cancer of lung [ 20 , 23 ] , brain [ 35 , 36 ] , colon [ 35 , 44 , 72 ] , liver [ 73 , 74 ] , kidney , the pituitary , stomach [ 35 , 76 ] , prostate , and testis . endocan detection may be today the most pertinent tool to discriminate the good ( the resting vessel in normal tissue ) , the bad ( the emerging vessel dedicated tumor angiogenesis ) , and the ugly ( the proliferating multilayered capillary as seen in highly aggressive tumor of the brain ) . indeed , endocan is detected in the vessels primarily activated by vegf [ 23 , 74 ] . glioblastoma , which is the most aggressive tumor of the brain in adults , is a vegf - driven tumor characterized by extensive angiogenesis and proliferating multilayered capillaries . in glioblastoma , endocan expression is always associated with the abnormal vasculature reflecting neoangiogenesis [ 35 , 36 ] , as shown in figure 3(a ) . in contrast , endocan is never detected in low - grade gliomas ( grade i and grade ii ) and in the cerebral cortex distant from the tumors [ 35 , 36 ] . another cancer of particular interest is the clear cell renal carcinoma ( ccrc ) that usually shows a huge neoangiogenesis driven by the vhl ( von hippel lindau ) mutation , and that is currently mainly treated by antiangiogenic drugs . endocan is reported to be clearly expressed in all the vessels of the ccrc , inside the tumor sample , but is never detected in the vessels of other types of kidney tumors . furthermore , in the same tumor section , endocan as a biomarker of activated endothelial cells helps to distinguish the resting from the emerging capillaries . microscopically , the quantification of the density of capillaries per mm ( also called microvascular density : mvd ) in a cancer sample is not by itself relevant to show the angiogenic process if all vessels ( normal and pathologicals ) are taken into account . interestingly , the endocan immunolabeling ( endocan - mvd ) was shown to correlate more strongly with aggressiveness ( and shortened survival ) for the first time in liver carcinoma than an mvd measured using a pan - endothelial surface marker such as cd34 , as described for the first time in liver carcinoma . since then , other studies in colon , liver , and pituitary tumors have described that mvd stained by endocan is correlated with microscopic venous invasion , vegf expression , recurrence , and/or bad prognosis [ 43 , 73 , 75 , 77 ] . the immunodetection of endocan in tissues may help to reveal an aggressive behavior and/or recurrence according to the type of tumors . for instance , endocan immunoreactivity within endothelial cells clearly correlates with a shorter survival in glioblastoma patients . similarly , endocan expression by endothelial cells in hepatocarcinoma is associated with unfavorable prognostic features . in pituitary tumors , a strong association between endocan immunoreactivity of the vessels and endocan immunodetection in activated colorectal cancer endothelial cells has also been reported as an independent prognostic factor for disease recurrence and worse survival outcome . since endocan expression reflects endothelial activation occurring during neoangiogenesis of tumors , dosage of the secreted protein in the blood circulation could also provide a valuable read out of tumor progression and of patient response to drugs . levels of circulating endocan are found to be from 3- to 10-fold higher in the sera of patients with renal carcinoma compared to healthy subjects . accordingly , increased blood levels of endocan are detected in patients with lung cancers , with the highest levels of endocan in patients with poor prognosis [ 20 , 23 ] . endocan is also a potential serum marker for early detection of colorectal cancer and also of liver carcinoma . these different studies have all highlighted the potential of endocan as a blood - based biomarker to assess endothelial activation in angiogenesis - driven tumorigenesis . indeed , endocan as a biomarker could also be of great interest for the selection of patients susceptible to angiogenesis - directed therapies or for identifying the probability of resistance , the major cause of failure of cancer treatment . interestingly , treatment of cultured endothelial cells by antiangiogenic drugs ( such as anti - vegf antibodies or small drugs ) abolishes the vegf - induced secretion of endocan [ 23 , 33 , 42 ] . for instance , endocan expression was shown to decrease under treatment with specific vegf receptor-2 kinase inhibitors or the addition of sutent ( a multityrosine kinase inhibitor ) directly into the culture medium completely abolished the secretion of endocan induced by vegf from huvec cells . furthermore , a combined blockade of vegfr-2 and vegfr-3 by specific antibodies completely prevented vegf - c - mediated induction of endocan expression in endothelial cells . other pgs have been proposed as potential biomarkers of tumor progression , such as the proteolytically shed ectodomains of syndecan-1 and glypican-3 . for instance , the level of shed syndecan-1 is increased in sera of patients with lung cancer , myeloma , or liver cancer and was associated with bad prognosis [ 7981 ] . however , their use as diagnostic tools remains controversial . as such , further investigations will be required to fully assess the pertinence of endocan as biomarker . no other molecule is currently available to assess endothelial activation and/or dysfunction during tumor progression and endocan could represent a pertinent blood based marker that helps detect neoangiogenesis and response of treatment . study of endocan in cancer samples took another twist , when expression of the pg by the tumor cells themselves has been reported by immunohistochemistry . surprisingly , endocan is always present in the cytoplasm of the tumor cells of high grade gliomas also called glioblastoma ( figure 4(a ) ) . furthermore , endocan tumor immunolabeling was always associated with the typical hypoxic palisades of poorly differentiated tumor cells in glioblastoma as shown in figure 4(b ) . another recent example of endocan expression in tumor cells came from the pituitary where endocan was detected in both endothelial and tumor cells as shown in figures 4(c ) and 4(d ) . the question was then raised about the role of endocan expression in tumor cells themselves . going through the literature , an early study had already presented endocan as a protumorigenic molecule when expressed in tumor epithelial cells . when colon tumor ht29 cells ( that are nave for endocan ) were manipulated to express endocan , they became more tumorigenic in a mouse xenotransplantation model . interestingly , when overexpressed in tumor cells , only the clones expressing the dspg ( i.e. , fully glycanated endocan ) enhance tumorigenesis , thereby underlining the role of the ds chain in the protumorigenic activity of endocan [ 20 , 82 ] . in addition , increased endocan expression in tumor cells was associated with enhanced invasiveness and metastasis in prostate cancer cells . endocan was upregulated by 26.5-fold in rat prostate mat - lylu cell lines with highly metastatic phenotype compared to nonmetastatic cells derived from dunning r3327 cells . further investigations will be needed to clarify the role(s ) of endocan expression in the tumor cells , but the first hints arose when endocan was described in cultured tumor cell lines as one of the genes associated with two of the major events in tumor progression : the angiogenic switch and the vascular mimicry of tumor cells . first , the angiogenic switch has been described as the key step that determines the development from a dormant and localized tumor into an aggressive tumor able to disseminate and spread over the body . the esm gene encoding endocan appears as a signature of the angiogenic switch from dormant to aggressive phenotype of breast , brain , osteosarcoma , and liposarcoma tumor cell lines [ 84 , 85 ] . interestingly , levels of endocan expression correlated with tumor aggressiveness , with elevated levels of endocan reported in the aggressive clones generating angiogenic fast - growing tumors in xenografted animal models , and low levels of endocan in clones leading to dormant tumors [ 84 , 85 ] . increase of endocan gene expression was notably high ( 30-fold increase ) in glioblastoma and liposarcoma models . interestingly , the tumor cell lines release in vitro endocan as a dspg [ 36 , 84 ] and the secretion of endocan by these tumor cells was shown to be upon regulation of proinflammatory molecules such as tnf- and of proangiogenic growth factors such as fgf-2 as previously described for endothelial cultured cells . the expression of endocan in tumor cells could also be explained by the mechanism described as the vasculogenic mimicry . vasculogenic mimicry is defined as the unique capability of highly aggressive tumor cells but not of poorly aggressive cells to express endothelium - associated genes , to form patterned , tubular networks in three - dimensional culture , which could convey blood plasma and red blood cells and therefore mimics the vasculogenic networks formed by endothelial cells . when involved in vasculogenic mimicry , skin melanoma tumor cells have been shown to strongly express endothelial cell - associated genes including endocan [ 87 , 88 ] . indeed , hendrix and collaborators were the first to describe endocan to be one of the most upregulated genes in vitro ( 44-fold increase ) in highly aggressive skin and uveal melanoma cells compared to poorly aggressive melanoma cells [ 87 , 88 ] . the vasculogenic mimicry of tumor cells may then facilitate tumor perfusion and consequently the tumor metastasis . this process has been already observed in various types of cancers from prostate , bladder , breast , ovary , and brain [ 89 , 90 ] . the tumor cell plasticity seems to trigger vascular mimicry and the aggressive tumor cells may revert to an undifferentiated stem cell - like phenotype , with a pluripotent gene expression pattern . in glioblastoma , the ability of these cancer stem - like cells to directly contribute to the tumor vasculature by endothelial cell differentiation has largely been described [ 89 , 9193 ] . endocan is always expressed in tumor cells in glioblastoma , particularly in the palisade area and was never observed in low grade tumors [ 36 , 84 , 85 ] . the potential of endocan as a marker of vascular mimicry , of tumor plasticity , or its potential role in cancer stem - like cells for tumor progression still needs to be further investigated . endocan is therefore a unique circulating pg that appears today as a molecule of versatile interest in the study of tumor progression ( figure 5 ) . the dermatan sulfate chain seems critical to bear the roles of endocan in vascular and tumor growth . there is now an urgent need to assess , in a larger trial , the clinical value of endocan as a tissue- and blood - based biomarker in cancers where neoangiogenesis is the main driver of tumor progression . as no suitable biomarker has been yet validated in clinics to identify the patients clearly susceptible to benefit of angiogenesis - directed therapies , clinical studies should be set up to evaluate endocan as a predictive biomarker for antiangiogenic therapies . furthermore , the recent identification of endocan expression by tumor cells represents a novel and major development in the study of this pg . indeed , the large gene expression studies from microarrays that originally revealed the potential of endocan as a gene of bad prognosis signature in various types of cancers did not take into account the part of the endothelial versus the tumor expression of endocan . this particular expression of endocan in tumor cells will need further investigation to better understand the prognostic value of endocan expression in tumor versus endothelial compartment .

as most proteoglycans exert their biological activities in the pericellular region , circulating endocan has appeared since its discovery as an atypical dermatan sulfate proteoglycan , with distinctive structural and functional properties . endocan is naturally expressed by endothelial cells , highly regulated in presence of proinflammatory and proangiogenic molecules , binds to matrix proteins , growth factors , integrin , and cells , and may be then considered as an accurate marker of endothelial activation . consequently , endocan expression has been associated with a growing number of pathological conditions where endothelium gets challenged and notably in highly vascularized cancers . in this context , endocan has indeed been rapidly emerging as a promising tissue- and blood - based marker of the vascular growth and neoangiogenesis during cancer progression . furthermore , very recent studies have reported an expression of endocan by the tumor cells themselves . this highlights endocan as a multifaceted molecule with a great interest for researchers and clinicians to better understand tumor development , from the bench to the clinics . with promising perspectives of clinical applications , endocan thus appears as an exciting model for on going and future developments of proteoglycan - based approaches in cancer diagnostics and/or therapy .

1. Introduction 2. Expression of Endocan: A Marker of Endothelial Activation? 3. Endocan: An Atypical Structure for an Atypical Proteoglycan 4. Binding Properties of Endocan 5. Vascular Endocan as a Biomarker of Neoangiogenesis in Cancer 6. Endocan Is Also Expressed by Tumor Cells: A Signature of Tumor Progression? 7. Conclusion

from there , they benefit from an ideal positioning to interact , either through their protein core or their gag chains , with soluble signaling effectors ( growth factors , cytokines , chemokines ) [ 6 , 7 ] , structural components of the ecm ( fibronectin , collagens ) and membrane - associated proteins ( receptors , integrins ) [ 811 ] , or intracellular molecules ( pkc , syntenin , cask / lin-2 ) [ 12 , 13 ] . paracrine activities can also be exerted by free , soluble pgs , such as endocan , a small , soluble dermatan sulfate proteoglycan ( dspg ) expressed by endothelial cells and naturally found in the blood circulation of healthy subjects [ 1720 ] . the history of endocan originates from the discovery in 1996 of a soluble protein produced by endothelial cells : endothelial cell - specific molecule-1 ( esm-1 ) . posttranslational modification studies later led to the identification of a ds chain on esm-1 protein core , which was consequently included to the pg family , under the new name of endocan . since then , endocan has rapidly emerged as a very distinctive pg , in many aspects : ( i ) endocan is the one of very few circulating pg sensu stricto , ( ii ) in contrast with most ecm pgs , which are usually large , modular proteins that harbor several gag chains , endocan comprises a relatively small polypeptide associated to one single polysaccharide chain , and ( iii ) characterization of endocan ds chain revealed uncommon structural features and saccharide composition . interestingly , increasing experimental evidence has reported that endocan is overexpressed in cancer , sepsis , obesity , or inflammatory conditions [ 20 , 21 , 2328 ] . furthermore , blood levels of freely circulating pg have been associated with poor prognosis in several types of cancers ( reviewed in ) . the aim of this paper is to review the present knowledge about endocan expression , structure , and activity , with a special focus on the recent developments that have been highlighting that endocan , as an endothelial proteoglycan not only is a biomarker of neoangiogenesis but also appears today as a signature of the tumor progression when it is expressed by the tumor cells themselves . transcriptional control is ensured by a 3888 bp promoter at the 5 flanking region of esm , which features a typical tata box and a number of putative transcriptional binding sites , including ets , hhex , and cre - like motifs [ 29 , 30 ] . noteworthy , ets and hhex motifs have been involved in the regulation of genes encoding several angiogenic and ecm remodeling factors , such as vegf receptors-1 and -2 , tie-1 and -2 , neuropilin-1 , or ve - cadherin [ 24 , 29 , 30 ] . endocan mrnas were first identified from cultures of human umbilical vein endothelial cells ( huvecs ) , but expression of endocan was subsequently reported in a large panel of cultured human endothelial cells , such as endothelial cells from the dermal microvasculature , the coronary and pulmonary arteries , or capillaries from adipose tissues [ 29 , 31 ] . surprisingly , endocan could not be detected in highly vascularized organs , such as brain , heart , pancreas , or liver [ 21 , 31 ] , suggesting that endocan expression may be characteristic of activated rather than resting endothelial cells . in vivo , endocan is detected with an average concentration of ~1 ng / ml in the serum healthy patients however , as detailed below , levels significantly increase in patients with septic shock or cancers such as lung , kidney , or colon cancers . tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , transforming growth factor-1 ( tgf-1 ) , fibroblast growth factor-2 ( fgf-2 ) , and vascular endothelial growth factor ( vegf ) have been shown to induce endocan expression in vitro [ 3540 ] , while interferon- ( ifn- ) inhibits tnf- induced upregulation of endocan . using pharmacological compounds , abid and collaborators investigated intracellular pathways involved in vegf - induced secretion of endocan in cultured endothelial cells . this was further supported by rennel and collaborators , who also reported that vegf - a induction of endocan expression in cultured endothelial cells was regulated via the pi3 k pathway . noteworthy , recent studies have suggested that endocan expression may regulate cell survival in cancer cells via the nfkb pathway [ 43 , 44 ] . endocan is one of the rare circulating pgs that have been identified , along with bikunin and macrophage colony stimulating factor-1 ( mcsf-1 ) , which are both cspgs . structurally , endocan is composed of a 165 amino acid core protein bearing one single ds chain ( figure 1 ) . surprisingly , endocan shares no obvious homologies with other extracellular members of the pg family . in contrast , 1528% of sequence identity can be found between the cystein - rich n - terminal region of endocan and members of the insulin - like growth factor binding protein ( igfbp ) family . in addition , endocan sequence shows some homology with human 2 integrin , fibrillin-1 , laminin-2 , and procollagen 2 . expression of endocan orthologs has also been identified by southern blot in mouse , rabbit , dog , cow , and monkey , with a high degree of gene conservation [ 21 , 29 , 48 ] . the saccharide moiety of human endocan is represented by as single chain of dermatan sulfate ( ds ) . ds is a member of the gag family , which include glucosaminoglycan ha , heparin and hs , and galactosaminoglycans cs and ds [ 49 , 50 ] . furthermore , increasing evidence has shown that specific cs / ds sulfation patterns were involved in precise biological functions and that variations of these sulfation patterns can be associated with physiological changes and pathological developments [ 5557 ] . the ds chain endocan expressed by endothelial cells is ~15 kda in size ( e.g. most interestingly , when compared to cs / ds from various sources [ 55 , 57 ] , disaccharide analysis of endocan ds revealed a higher proportion of nonsulfated disaccharides ( 1012% of hexa - galnac ) and of the disulfated b , d , and e units that are rather rare in mammals . importantly , endocan variant lacking the ds chain failed to induce hgf activation , thus confirming the role played by the gag chain in this process . furthermore , biosensor analysis reported a kd of ~36 nm for hgf / endocan interaction , which is well in the 0.220 nm range of previously published affinity values for hgf binding to free hs and ds chains [ 51 , 61 , 62 ] . high affinity interaction of gags with hgf is critical for its activation of the met receptor , which plays major roles in many physiopathological processes , including development , wound healing , and tumor progression [ 6365 ] . through its ability to promote hgf activity , endocan in addition , endocan has been shown to promote the mitogenic and migratory activities of vegf - a and -c in vitro . finally , ds is known to bind to a multitude of signaling and adhesion molecules , including l- and p - selectins , matrix proteins such as fibronectin , chemokines / cytokines rantes , sdf-1 , il-8 , mcp-1 , ifn- , and pf-4 , and growth factors midkine , pleiotrophin fgf-2 , and -7 [ 24 , 57 ] , leaving open the question of a possible interaction of these ligands with endocan . the major growth factors and cytokines involved in vascular growth have been failing as biomarkers of the progression in tumor development . endocan , as a pg secreted by endothelial cells when activated , has appeared over the years as a promising tissue - based and also blood - based marker and we will detail later the most recent literature evidences . the release of endocan by cultured endothelial cells has been largely described to be strongly upregulated in presence of proangiogenic molecules such as vegf , fgf-2 , and vegf - c that are critical mediators involved in angiogenesis ( i.e. in addition , endocan has been shown to directly promote the mitogenic and migratory activities of vegf - a and -c on cultured endothelial cells . interestingly , sirna silencing of endocan expression inhibits vegf - a/-c induced proliferation of lymphatic endothelial cells . furthermore , addition of recombinant endocan protein to endocan sirna - transfected endothelial cells restores the stimulatory effect of vegf - a on migration , hence suggesting a role of endocan in lymphangiogenesis . endocan expression is also induced in cultured endothelial cells undergoing tube formation as an assay to assess angiogenesis and vasculogenesis in vitro . in animal models , endocan gene is one of the main genes involved in the angiogenic switch occurring during retinal neovascularization induced by hypoxia . a link has been recently established between endocan and the tip cells , the specialized subset of endothelial cells known to mediate vessel growth in neoangiogenesis [ 70 , 71 ] . these tip cells are the sensor cells at the end ( the tip ) of the growing new vessels , which lead the building of the tumor vascular network required for tumor progression . taken together , all these experimental data highlight endocan as a marker of endothelial cell activation during the growth of new vessels required for tumor progression . data obtained from cancer tissue samples strengthen all that experimental evidence of endocan as a relevant biomarker for clinics . by immunohistochemistry , endocan is visualized in the cytoplasm of endothelial cells of the newly growing tumor vessels ( figure 3 ) . in the literature , a marked cytoplasmic expression of vascular endocan has been then described inside the tumor vessels in different types of highly vascularized tumors such as cancer of lung [ 20 , 23 ] , brain [ 35 , 36 ] , colon [ 35 , 44 , 72 ] , liver [ 73 , 74 ] , kidney , the pituitary , stomach [ 35 , 76 ] , prostate , and testis . endocan detection may be today the most pertinent tool to discriminate the good ( the resting vessel in normal tissue ) , the bad ( the emerging vessel dedicated tumor angiogenesis ) , and the ugly ( the proliferating multilayered capillary as seen in highly aggressive tumor of the brain ) . indeed , endocan is detected in the vessels primarily activated by vegf [ 23 , 74 ] . in glioblastoma , endocan expression is always associated with the abnormal vasculature reflecting neoangiogenesis [ 35 , 36 ] , as shown in figure 3(a ) . in contrast , endocan is never detected in low - grade gliomas ( grade i and grade ii ) and in the cerebral cortex distant from the tumors [ 35 , 36 ] . another cancer of particular interest is the clear cell renal carcinoma ( ccrc ) that usually shows a huge neoangiogenesis driven by the vhl ( von hippel lindau ) mutation , and that is currently mainly treated by antiangiogenic drugs . endocan is reported to be clearly expressed in all the vessels of the ccrc , inside the tumor sample , but is never detected in the vessels of other types of kidney tumors . furthermore , in the same tumor section , endocan as a biomarker of activated endothelial cells helps to distinguish the resting from the emerging capillaries . for instance , endocan immunoreactivity within endothelial cells clearly correlates with a shorter survival in glioblastoma patients . similarly , endocan expression by endothelial cells in hepatocarcinoma is associated with unfavorable prognostic features . in pituitary tumors , a strong association between endocan immunoreactivity of the vessels and endocan immunodetection in activated colorectal cancer endothelial cells has also been reported as an independent prognostic factor for disease recurrence and worse survival outcome . since endocan expression reflects endothelial activation occurring during neoangiogenesis of tumors , dosage of the secreted protein in the blood circulation could also provide a valuable read out of tumor progression and of patient response to drugs . these different studies have all highlighted the potential of endocan as a blood - based biomarker to assess endothelial activation in angiogenesis - driven tumorigenesis . indeed , endocan as a biomarker could also be of great interest for the selection of patients susceptible to angiogenesis - directed therapies or for identifying the probability of resistance , the major cause of failure of cancer treatment . for instance , endocan expression was shown to decrease under treatment with specific vegf receptor-2 kinase inhibitors or the addition of sutent ( a multityrosine kinase inhibitor ) directly into the culture medium completely abolished the secretion of endocan induced by vegf from huvec cells . furthermore , a combined blockade of vegfr-2 and vegfr-3 by specific antibodies completely prevented vegf - c - mediated induction of endocan expression in endothelial cells . as such , further investigations will be required to fully assess the pertinence of endocan as biomarker . no other molecule is currently available to assess endothelial activation and/or dysfunction during tumor progression and endocan could represent a pertinent blood based marker that helps detect neoangiogenesis and response of treatment . study of endocan in cancer samples took another twist , when expression of the pg by the tumor cells themselves has been reported by immunohistochemistry . surprisingly , endocan is always present in the cytoplasm of the tumor cells of high grade gliomas also called glioblastoma ( figure 4(a ) ) . furthermore , endocan tumor immunolabeling was always associated with the typical hypoxic palisades of poorly differentiated tumor cells in glioblastoma as shown in figure 4(b ) . another recent example of endocan expression in tumor cells came from the pituitary where endocan was detected in both endothelial and tumor cells as shown in figures 4(c ) and 4(d ) . the question was then raised about the role of endocan expression in tumor cells themselves . going through the literature , an early study had already presented endocan as a protumorigenic molecule when expressed in tumor epithelial cells . , fully glycanated endocan ) enhance tumorigenesis , thereby underlining the role of the ds chain in the protumorigenic activity of endocan [ 20 , 82 ] . in addition , increased endocan expression in tumor cells was associated with enhanced invasiveness and metastasis in prostate cancer cells . further investigations will be needed to clarify the role(s ) of endocan expression in the tumor cells , but the first hints arose when endocan was described in cultured tumor cell lines as one of the genes associated with two of the major events in tumor progression : the angiogenic switch and the vascular mimicry of tumor cells . the esm gene encoding endocan appears as a signature of the angiogenic switch from dormant to aggressive phenotype of breast , brain , osteosarcoma , and liposarcoma tumor cell lines [ 84 , 85 ] . interestingly , levels of endocan expression correlated with tumor aggressiveness , with elevated levels of endocan reported in the aggressive clones generating angiogenic fast - growing tumors in xenografted animal models , and low levels of endocan in clones leading to dormant tumors [ 84 , 85 ] . interestingly , the tumor cell lines release in vitro endocan as a dspg [ 36 , 84 ] and the secretion of endocan by these tumor cells was shown to be upon regulation of proinflammatory molecules such as tnf- and of proangiogenic growth factors such as fgf-2 as previously described for endothelial cultured cells . the expression of endocan in tumor cells could also be explained by the mechanism described as the vasculogenic mimicry . vasculogenic mimicry is defined as the unique capability of highly aggressive tumor cells but not of poorly aggressive cells to express endothelium - associated genes , to form patterned , tubular networks in three - dimensional culture , which could convey blood plasma and red blood cells and therefore mimics the vasculogenic networks formed by endothelial cells . the vasculogenic mimicry of tumor cells may then facilitate tumor perfusion and consequently the tumor metastasis . this process has been already observed in various types of cancers from prostate , bladder , breast , ovary , and brain [ 89 , 90 ] . the tumor cell plasticity seems to trigger vascular mimicry and the aggressive tumor cells may revert to an undifferentiated stem cell - like phenotype , with a pluripotent gene expression pattern . in glioblastoma , the ability of these cancer stem - like cells to directly contribute to the tumor vasculature by endothelial cell differentiation has largely been described [ 89 , 9193 ] . endocan is always expressed in tumor cells in glioblastoma , particularly in the palisade area and was never observed in low grade tumors [ 36 , 84 , 85 ] . the potential of endocan as a marker of vascular mimicry , of tumor plasticity , or its potential role in cancer stem - like cells for tumor progression still needs to be further investigated . endocan is therefore a unique circulating pg that appears today as a molecule of versatile interest in the study of tumor progression ( figure 5 ) . the dermatan sulfate chain seems critical to bear the roles of endocan in vascular and tumor growth . there is now an urgent need to assess , in a larger trial , the clinical value of endocan as a tissue- and blood - based biomarker in cancers where neoangiogenesis is the main driver of tumor progression . as no suitable biomarker has been yet validated in clinics to identify the patients clearly susceptible to benefit of angiogenesis - directed therapies , clinical studies should be set up to evaluate endocan as a predictive biomarker for antiangiogenic therapies . furthermore , the recent identification of endocan expression by tumor cells represents a novel and major development in the study of this pg . indeed , the large gene expression studies from microarrays that originally revealed the potential of endocan as a gene of bad prognosis signature in various types of cancers did not take into account the part of the endothelial versus the tumor expression of endocan . this particular expression of endocan in tumor cells will need further investigation to better understand the prognostic value of endocan expression in tumor versus endothelial compartment .

this study was conducted in the department of orthodontics and dentofacial orthopedics , vinayaka missions shankarachariyar dental college , salem , tamil nadu . the in vitro tests were conducted at the sree chitra tirunal institute for medical science and technology , thiruvanthapuram kerala . the basic study was designed to compare the coefficient of kinetic friction of stainless steel , self - ligating stainless steel , and titanium brackets ( ormco , columbia , united states ) , using stainless steel ( ortho - organizers , carlsbad , ca , united states ) and tma archwires ( ormco , columbia , united states ) . the property investigated was the frictional characteristic using 0.017 0.025 and 0.019 0.025 rectangular archwires in 0.018 and 0.022-inch slot , respectively , ligated with elastic modules . the canine brackets used were coded as follows : titanium bracket 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 1].self - ligating stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 2].stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 3].stainless steel wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ortho - organizers ) [ figure 4].tma wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ormco ) [ figure 5 ] . titanium bracket 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 1 ] . self - ligating stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 2 ] . stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 3 ] . stainless steel wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ortho - organizers ) [ figure 4 ] . tma wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ormco ) [ figure 5 ] . self ligating stainless steel brackets stainless steel brackets stainless steel arch wires /titanium molybdenum arch wires the evaluation of frictional resistance between brackets and the archwire was carried out as per the protocol described by tidy . the four premolar brackets on either side were bonded to a rigid perspex sheet at 8-mm intervals with a 16-mm space for a movable bracket at the center . both 0.018-inch slot and 0.022-inch slot brackets were set up in this way using in turn archwires of 0.017 0.025 inch and 0.019 0.025 inch , respectively , for sliding the canine bracket to simulate canine retraction . the archwires used were stainless steel and tma of 0.017 0.025 inch and 0.019 0.025 , were secured using elastomeric ligatures ( ortho - organizers ) [ figure 6 ] . the movable bracket was fitted with a 10-mm power arm from which weights of 100 g were hung to represent the single equivalent force acting at the center of resistance of the tooth root . the length of the power arm was chosen to represent the distance from the slot to the center of resistance of a typical canine tooth . all the tests were conducted under dry conditions with instron universal testing machine model no . 3365 [ figure 7 ] , with a cross head moving downward at a speed of 5 mm / min . the movable bracket was suspended from the load cell of the testing machine , while the perspex sheet was mounted on the cross head below [ figure 8 ] . the load cell readings represented the clinical force of retraction that would be applied to the tooth , part of which would be lost in friction , while the remainder was transmitted to the tooth root . the difference between the load cell reading and the load on the power arm thus represented the friction . brackets of both the sides of the upper arch were mounted on separate perspex sheets . the 0.017 0.025 inch stainless steel and tma archwires were secured to the 0.018 slot bracket and 0.0.019 0.025 inch stainless steel and tma archwires were secured to the 0.022 slot bracket with elastomeric modules that were done to standardize the ligature force . a trial run was performed on the power arm without any load to check whether the elastomeric module was binding onto the archwire prior to each test . then , each side was tested with 100 g of weight suspended from the power arm . the load needed to move the canine bracket across the central span was recorded separately as kinetic friction from the graphic plotter . the coefficient of kinetic friction was calculated from this value with the help of the following formula : where p is the frictional resistance , f the equivalent force acting at a distance , w the bracket distance , h is 10 mm , and is coefficient of friction . the readings for 0.018 slot and 0.022 slot using 0.017 0.025 inch and 0.019 0.025 inch archwires , respectively , for sliding mechanics were taken with three variant brackets ( the stainless steel , self - ligating stainless steel , and titanium brackets ) using two different archwires , the stainless steel and tma ( titanium ) archwires , and were averaged . osb_ssstainless steel brackets using tma wire osb_tmtitanium brackets using stainless steel wire otb_sstitanium brackets using tma wire otb_tmself - ligating stainless steel using stainless steel wire otb_tm self - ligating stainless steel using stainless steel wire oslb_ss self - ligating stainless steel using tma wire stainless steel brackets using stainless steel wire osb_ssstainless steel brackets using tma wire osb_tmtitanium brackets using stainless steel wire otb_sstitanium brackets using tma wire otb_tmself - ligating stainless steel using stainless steel wire oslb_ssself - ligating stainless steel using tma wire oslb_tm stainless steel brackets using stainless steel wire osb_ss stainless steel brackets using tma wire osb_tm titanium brackets using stainless steel wire otb_ss titanium brackets using tma wire otb_tm self - ligating stainless steel using stainless steel wire oslb_ss self - ligating stainless steel using tma wire analysis of variance ( anova ) test was conducted for mean score analysis to compare between three types of brackets and two types of wire combinations . if the p value is greater than 0.05 , it means the hypothesis is accepted with higher range frictional resistance . null hypothesis giving values for different bracket and wire combinations and the post hoc test signified the marginal differences subjecting the variance into three groups . the paired student 's t - test compared mean scores of frictional resistance for titanium self - ligating stainless steel and stainless steel brackets of 0.018-inch and 0.022-inch slots using stainless steel and tma wires , hence signifying the results to quantify the study . the archwires are fitted in the bracket and sliding mechanics require either the bracket or the archwires to slide through the bracket slot . of all the different materials tested so far , stainless steel brackets are preferred for their low frictional force values . however , concerns have been expressed about the nickel content in the stainless steel causing hypersensitivity and corrosion in the oral environment . in addition , there are instances where mri or ct imaging may have been distorted because of the stainless steel alloys . to overcome these difficulties , these brackets have excellent corrosion resistance and are biocompatible , but have not been subjected to studies related to the frictional resistance force an orthodontist may encounter during sliding mechanics . to reduce friction clinically , some practitioners prefer the use of round wire or they reduce rectangular wire in the buccal segments to a more rounded cross section . round wires generally produce less friction than rectangular wire when engaged in brackets out of alignment because of their greater flexibility . friction as a result of two - point contact is largely independent of wire stiffness or cross section and so is likely to be little affected by the choice of round or rectangular wire . very few studies have been conducted and discussed in the literature about corrosion and sensitivity to the nickel present and stainless steel brackets . titanium has heralded as a material totally compatible in the oral environment and superior in structural integrity compared to stainless steel . the study was designed to compare the frictional properties of stainless steel , titanium , and self - ligating brackets in 0.018 and 0.022 slots , in 0.017 0.025 and 0.019 0.025 stainless steel and tma archwires with second - order angulations , ligated with elastic modules to the brackets slot . in this study , the apparatus consisted of a simulated arch fixed appliance with archwire in a vertical position . four premolar pre - adjusted edgewise brackets were bonded to rigid perspex sheet at 8-mm interval and a space of 16 mm was left at the center of a movable canine bracket . the movable canine bracket was fitted with a 10-mm power arm from which weights of 100 g were hung to represent single equivalent force acting at the center of resistance of the tooth root . all tests were carried out under standard conditions with an instron universal testing machine , with the cross - head speed at 5 mm / min . however , the force exerted on the bracket wire combination is less variable in elastomeric ligation , compared to stainless steel ligation ( bazokidou 1997 ) . standardization of ligation force necessitated the use of elastomeric ligatures , as the frictional forces are proportional to the ligational force . the anova and paired student 's t - test were done to evaluate statistically significant differences between the mean coefficients of kinetic friction for different bracket systems . this study shows that the self - ligating stainless steel brackets have the least kinetic friction compared to titanium and stainless steel brackets in 0.018- and 0.022-inch slots . in keeping with several previous studies , we found that both the slots , 0.018 and 0.022 , with bracket and arch wire , considering the advantages of the self - ligating brackets , showing lesser frictional resistance , they could be used in day - to - day practice . the result shows that titanium archwires generated significantly less frictional resistance than the stainless steel wires . on the other hand , the laboratory measurements may be more affected by the other wire properties such as stiffness . the speculation is supported by this study that frictional resistance is delivered when wires are pulled through the bracket slot under resisting 100 g of weight . the substantial differences in friction between titanium , stainless steel , and self - ligating brackets , using stainless steel and tma archwires , indicate differences in the coefficient of friction applicable for these materials sliding along the slot . to reduce the friction clinically , some practitioners prefer the use of round wire or they reduce rectangular wire in the buccal segments to a more rounded cross section . round wires generally produce less friction than rectangular wire when engaged into the brackets that are out of alignment because of their greater flexibility . friction as a result of two - point contact is largely independent of wire stiffness or cross section and so is likely to be little affected by the choice of round or rectangular wire . it is therefore concluded that after insertion of the archwire , friction with round wire may be initially less than the rectangular wire . however , as the brackets align and any torque becomes passive , the difference will become small . in this respect , the common practice is leaving a rectangular wire in position for a month before sliding forces are used . it is becoming apparent that stainless steel and elastomeric ligatures will eventually be as outdated like the full banding technique one day . considering the advantages of the self- ligating brackets , showing lesser frictional resistance , they could be used in day - to - day practice . the archwires are fitted in the bracket and sliding mechanics require either the bracket or the archwires to slide through the bracket slot . of all the different materials tested so far , stainless steel brackets are preferred for their low frictional force values . however , concerns have been expressed about the nickel content in the stainless steel causing hypersensitivity and corrosion in the oral environment . in addition , there are instances where mri or ct imaging may have been distorted because of the stainless steel alloys . to overcome these difficulties , these brackets have excellent corrosion resistance and are biocompatible , but have not been subjected to studies related to the frictional resistance force an orthodontist may encounter during sliding mechanics . friction is largely independent of wire stiffness or cross section and so is likely to be little affected by the choice of round or rectangular wire . andreasen and quevodo ( 1970 ) pioneered an in - depth frictional study to evaluate the frictional forces in the 0.022 0.028 inches edgewise bracket system . articolo et al ( 2000 ) stated that three mechanical cause of resistance to sliding of a bracket along an arch - wire are classical friction , binding and notching . drescher et al ( 1989 ) in their study found the following factors to affect friction in tooth - guided arch wire mechanics ( in decreasing order ) : retarding force ( biologic resistance ) , surface roughness of wire , wire size ( vertical dimension ) , and bracket width and elastic properties of the wire . bednar et al ( 1991 ) found that the elastomeric - ligated ceramic brackets demonstrated the greatest friction when compared with other bracket / ligation technique combinations . david et al states that the opposing frictional forces at the interface of the bracket , archwire , and ligature can influence the sliding arch - guided system generally referred to as sliding mechanics . dickson et al experimentation demonstrated that the conventional ceramic bracket had highest static frictional characteristics . downing et al evaluated the effect of artificial saliva on the static and kinetic forces of stainless steel and polycrystalline ceramic brackets in combination with stainless , nickel titanium and beta titanium arch wire materials , under a constant ligature force . the stainless steel brackets produced more friction than polycrystalline brackets , but these results were not statistically significant except against beta titanium arch wires . baker et al investigated on frictional changes in the force values caused by saliva substitution on stainless steel arch wire bracket combination . their studies clearly shows that when compared to a dry medium less force is required to move teeth in a saliva medium . stannard et al did a comparative study of friction of wires under dry and wet conditions . they found artificial saliva to increase coefficients of friction for stainless steel , m titanium and nickel - titanium when compared to dry conditions . very few studies have been conducted and discussed in the literature about corrosion and sensitivity to the nickel present and stainless steel brackets . titanium has heralded as a material totally compatible in the oral environment and superior in structural integrity compared to stainless steel . the study was designed to compare the frictional properties of stainless steel , titanium , and self - ligating brackets in 0.018 and 0.022 slots , in 0.017 0.025 and 0.019 0.025 stainless steel and tma archwires with second - order angulations , ligated with elastic modules to the brackets slot . in this study , the apparatus consisted of a simulated arch fixed appliance with archwire in a vertical position . the substantial differences in friction between titanium , stainless steel , self - ligating brackets , using stainless steel and tma archwire , indicate differences in the coefficient of friction applicable for these materials sliding along the slot . to reduce friction clinically , some practitioners prefer the use of round wire , or they reduce rectangular wire in the buccal segments to a more rounded cross section . round wires , of course , eliminate friction caused by active torque . round wires generally produce less friction than rectangular wire when engaged into the brackets that are out of alignment because of their greater flexibility . friction as a result of two - point contact is largely independent of wire stiffness or cross section , and so is likely to be little affected by the choice of round or rectangular wire . it is therefore concluded that after insertion of the archwire , friction with round wire may be initially less than the rectangular wire . however , as the brackets align and any torque becomes passive , the difference will become small . in this respect , the common practice is leaving a rectangular wire in position for a month before sliding forces are used . it is becoming apparent that stainless steel and elastomeric ligatures will eventually be outdated like the full banding technique one day . considering the advantages of the self - ligating brackets that they show lesser frictional resistance , the frictional resistance of 0.022 inch and 0.018 inch slot were measured using instron universal testing machine and recorded [ table 1 ] . value recorded from the instron universal testing machine diagrammatic representation of six bracket and wire combinations of 0.018-inch and 0.022-inch slots the independent samples t - test procedure is used to compare the mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is no significant difference between the mean scores of stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . t - test group statistics the results of levene 's test analysis are given in table 3 . h0 : there is significant difference between the mean scores of self - ligating stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . t - test group statistics the independent samples t - test procedure is used to compare mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is significant difference between the mean scores of titanium bracket with stainless steel wire and tma wire regarding the level of satisfaction . graphs 2 and 3 represents mean frictional resistance of 0.22 slot and 0.18 slot brackets with displacement respectively . t - test group statistics 0.22 slot brackets with displacement 0.18 slot brackets with displacement the table displays the descriptive statistics of the sample size , mean , standard deviation , and standard error . the table also [ tables 510 ] shows the t - statistics , which is calculated as the ratio of the difference between sample means divided by the standard error of the difference . the column hence , there is no significant / there is a significant difference in the mean scores of the two groups with respect to the level of frictional resistance . t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics the mean score analysis ( anova ) test procedure [ tables 1116 ] is used to compare the mean scores of level of frictional resistance for more than two groups . the procedure assumes that the variances of the groups are equal and it was tested with levene 's test statistics . the significant difference between the mean scores of level of frictional resistance is tested with respect to the various titanium , self - ligating stainless steel , and stainless steel brackets , using stainless steel and tma archwires . test for mean scores of level of frictional resistance and six groups of brackets and wire combinations . the mean scores of level of frictional resistance and six groups of 0.018-inch and 0.022-inch slot brackets were tested with anova test procedures and the results of the analysis are given in table 11 . h0 : there is no significant difference between the mean scores of frictional resistance among the six groups regarding the level of coefficient of friction . once we have determined that differences exist among the means , post hoc rank tests can determine which means differ . tukey 's tests identified the homogeneous subsets of means that are not different from each other and the results are given in tables 1316 . we used tukey 's test to identify the homogeneous subsets of means which are not different from each other . the self - ligating stainless steel brackets and titanium brackets come under one group of subset 1 and stainless steel brackets are in subset 2 . the independent samples t - test procedure is used to compare the mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is no significant difference between the mean scores of stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . t - test group statistics the results of levene 's test analysis are given in table 3 . h0 : there is significant difference between the mean scores of self - ligating stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . t - test group statistics the independent samples t - test procedure is used to compare mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is significant difference between the mean scores of titanium bracket with stainless steel wire and tma wire regarding the level of satisfaction . graphs 2 and 3 represents mean frictional resistance of 0.22 slot and 0.18 slot brackets with displacement respectively . t - test group statistics 0.22 slot brackets with displacement 0.18 slot brackets with displacement the table displays the descriptive statistics of the sample size , mean , standard deviation , and standard error . the table also [ tables 510 ] shows the t - statistics , which is calculated as the ratio of the difference between sample means divided by the standard error of the difference . the column hence , there is no significant / there is a significant difference in the mean scores of the two groups with respect to the level of frictional resistance . t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics the mean score analysis ( anova ) test procedure [ tables 1116 ] is used to compare the mean scores of level of frictional resistance for more than two groups . the procedure assumes that the variances of the groups are equal and it was tested with levene 's test statistics . the significant difference between the mean scores of level of frictional resistance is tested with respect to the various titanium , self - ligating stainless steel , and stainless steel brackets , using stainless steel and tma archwires . test for mean scores of level of frictional resistance and six groups of brackets and wire combinations . the mean scores of level of frictional resistance and six groups of 0.018-inch and 0.022-inch slot brackets were tested with anova test procedures and the results of the analysis are given in table 11 . h0 : there is no significant difference between the mean scores of frictional resistance among the six groups regarding the level of coefficient of friction . once we have determined that differences exist among the means , post hoc rank tests can determine which means differ . tukey 's tests identified the homogeneous subsets of means that are not different from each other and the results are given in tables 1316 . we used tukey 's test to identify the homogeneous subsets of means which are not different from each other . the self - ligating stainless steel brackets and titanium brackets come under one group of subset 1 and stainless steel brackets are in subset 2 . thus , the aim of the study was to compare the frictional resistance of titanium , self - ligating stainless steel , and conventional stainless steel brackets using stainless steel and tma archwires , with the help of instron universal testing machine , one - way anova , student 's t test , and post hoc multiple range test at level of < 0.05 showed statistically significant difference in the mean values of all the groups . results demonstrated the following : the titanium , self - ligating stainless steel , and stainless steel brackets of 0.018-inch and 0.022-inch slots had no significant variations in frictional resistance.the self - ligating bracket with tma archwires showed relatively less frictional resistance compared with the other groups.the titanium bracket with tma archwires showed relatively less frictional resistance compared with the stainless steel brackets . the titanium , self - ligating stainless steel , and stainless steel brackets of 0.018-inch and 0.022-inch slots had no significant variations in frictional resistance . the self - ligating bracket with tma archwires showed relatively less frictional resistance compared with the other groups . the titanium bracket with tma archwires showed relatively less frictional resistance compared with the stainless steel brackets .

aim : the aim of the study was to compare the frictional resistance of titanium , self - ligating stainless steel , and conventional stainless steel brackets , using stainless steel and titanium molybdenum alloy ( tma ) archwires.materials and methods : we compared the frictional resistance in 0.018 slot and 0.022 slot of the three brackets titanium , self - ligating stainless steel , and conventional stainless steel using stainless steel archwires and tma archwires . an in vitro study of simulated canine retraction was undertaken to evaluate the difference in frictional resistance between titanium , self - ligating stainless steel , and stainless steel brackets , using stainless steel and tma archwires.results and conclusion : we compared the frictional resistance of titanium , self - ligating stainless steel , and conventional stainless steel brackets , using stainless steel and tma archwires , with the help of instron universal testing machine . one - way analysis of variance ( anova ) , student 's t test , and post hoc multiple range test at level of < 0.05 showed statistically significant difference in the mean values of all groups . results demonstrated that the titanium , self - ligating stainless steel , and stainless steel brackets of 0.018-inch and 0.022-inch slot had no significant variations in frictional rsistance . the self - ligating bracket with tma archwires showed relatively less frictional resistance compared with the other groups . the titanium bracket with tma archwires showed relatively less frictional resistance compared with the stainless steel brackets .

Materials and Methods Codes used for the study Statistical Analysis Discussion Results Independent samples Frictional resistance in the groups of 0.022-inch slot ANOVA Post hoc tests homogeneous subsets SS_0.018 Conclusion

this study was conducted in the department of orthodontics and dentofacial orthopedics , vinayaka missions shankarachariyar dental college , salem , tamil nadu . the basic study was designed to compare the coefficient of kinetic friction of stainless steel , self - ligating stainless steel , and titanium brackets ( ormco , columbia , united states ) , using stainless steel ( ortho - organizers , carlsbad , ca , united states ) and tma archwires ( ormco , columbia , united states ) . the property investigated was the frictional characteristic using 0.017 0.025 and 0.019 0.025 rectangular archwires in 0.018 and 0.022-inch slot , respectively , ligated with elastic modules . the canine brackets used were coded as follows : titanium bracket 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 1].self - ligating stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 2].stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 3].stainless steel wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ortho - organizers ) [ figure 4].tma wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ormco ) [ figure 5 ] . titanium bracket 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 1 ] . self - ligating stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 2 ] . stainless steel brackets 20 nos in each slot ( ormco ; 0.018-inch slot and 0.022-inch slot ) [ figure 3 ] . stainless steel wires 0.017 inch 0.025 inch and 0.019 inch 0.025 inch ( ortho - organizers ) [ figure 4 ] . self ligating stainless steel brackets stainless steel brackets stainless steel arch wires /titanium molybdenum arch wires the evaluation of frictional resistance between brackets and the archwire was carried out as per the protocol described by tidy . both 0.018-inch slot and 0.022-inch slot brackets were set up in this way using in turn archwires of 0.017 0.025 inch and 0.019 0.025 inch , respectively , for sliding the canine bracket to simulate canine retraction . the archwires used were stainless steel and tma of 0.017 0.025 inch and 0.019 0.025 , were secured using elastomeric ligatures ( ortho - organizers ) [ figure 6 ] . the movable bracket was fitted with a 10-mm power arm from which weights of 100 g were hung to represent the single equivalent force acting at the center of resistance of the tooth root . the length of the power arm was chosen to represent the distance from the slot to the center of resistance of a typical canine tooth . all the tests were conducted under dry conditions with instron universal testing machine model no . the movable bracket was suspended from the load cell of the testing machine , while the perspex sheet was mounted on the cross head below [ figure 8 ] . brackets of both the sides of the upper arch were mounted on separate perspex sheets . the 0.017 0.025 inch stainless steel and tma archwires were secured to the 0.018 slot bracket and 0.0.019 0.025 inch stainless steel and tma archwires were secured to the 0.022 slot bracket with elastomeric modules that were done to standardize the ligature force . the coefficient of kinetic friction was calculated from this value with the help of the following formula : where p is the frictional resistance , f the equivalent force acting at a distance , w the bracket distance , h is 10 mm , and is coefficient of friction . the readings for 0.018 slot and 0.022 slot using 0.017 0.025 inch and 0.019 0.025 inch archwires , respectively , for sliding mechanics were taken with three variant brackets ( the stainless steel , self - ligating stainless steel , and titanium brackets ) using two different archwires , the stainless steel and tma ( titanium ) archwires , and were averaged . osb_ssstainless steel brackets using tma wire osb_tmtitanium brackets using stainless steel wire otb_sstitanium brackets using tma wire otb_tmself - ligating stainless steel using stainless steel wire otb_tm self - ligating stainless steel using stainless steel wire oslb_ss self - ligating stainless steel using tma wire stainless steel brackets using stainless steel wire osb_ssstainless steel brackets using tma wire osb_tmtitanium brackets using stainless steel wire otb_sstitanium brackets using tma wire otb_tmself - ligating stainless steel using stainless steel wire oslb_ssself - ligating stainless steel using tma wire oslb_tm stainless steel brackets using stainless steel wire osb_ss stainless steel brackets using tma wire osb_tm titanium brackets using stainless steel wire otb_ss titanium brackets using tma wire otb_tm self - ligating stainless steel using stainless steel wire oslb_ss self - ligating stainless steel using tma wire analysis of variance ( anova ) test was conducted for mean score analysis to compare between three types of brackets and two types of wire combinations . if the p value is greater than 0.05 , it means the hypothesis is accepted with higher range frictional resistance . the paired student 's t - test compared mean scores of frictional resistance for titanium self - ligating stainless steel and stainless steel brackets of 0.018-inch and 0.022-inch slots using stainless steel and tma wires , hence signifying the results to quantify the study . of all the different materials tested so far , stainless steel brackets are preferred for their low frictional force values . however , concerns have been expressed about the nickel content in the stainless steel causing hypersensitivity and corrosion in the oral environment . in addition , there are instances where mri or ct imaging may have been distorted because of the stainless steel alloys . to overcome these difficulties , these brackets have excellent corrosion resistance and are biocompatible , but have not been subjected to studies related to the frictional resistance force an orthodontist may encounter during sliding mechanics . very few studies have been conducted and discussed in the literature about corrosion and sensitivity to the nickel present and stainless steel brackets . titanium has heralded as a material totally compatible in the oral environment and superior in structural integrity compared to stainless steel . the study was designed to compare the frictional properties of stainless steel , titanium , and self - ligating brackets in 0.018 and 0.022 slots , in 0.017 0.025 and 0.019 0.025 stainless steel and tma archwires with second - order angulations , ligated with elastic modules to the brackets slot . the movable canine bracket was fitted with a 10-mm power arm from which weights of 100 g were hung to represent single equivalent force acting at the center of resistance of the tooth root . all tests were carried out under standard conditions with an instron universal testing machine , with the cross - head speed at 5 mm / min . the anova and paired student 's t - test were done to evaluate statistically significant differences between the mean coefficients of kinetic friction for different bracket systems . this study shows that the self - ligating stainless steel brackets have the least kinetic friction compared to titanium and stainless steel brackets in 0.018- and 0.022-inch slots . in keeping with several previous studies , we found that both the slots , 0.018 and 0.022 , with bracket and arch wire , considering the advantages of the self - ligating brackets , showing lesser frictional resistance , they could be used in day - to - day practice . the result shows that titanium archwires generated significantly less frictional resistance than the stainless steel wires . the substantial differences in friction between titanium , stainless steel , and self - ligating brackets , using stainless steel and tma archwires , indicate differences in the coefficient of friction applicable for these materials sliding along the slot . it is becoming apparent that stainless steel and elastomeric ligatures will eventually be as outdated like the full banding technique one day . considering the advantages of the self- ligating brackets , showing lesser frictional resistance , they could be used in day - to - day practice . of all the different materials tested so far , stainless steel brackets are preferred for their low frictional force values . however , concerns have been expressed about the nickel content in the stainless steel causing hypersensitivity and corrosion in the oral environment . in addition , there are instances where mri or ct imaging may have been distorted because of the stainless steel alloys . to overcome these difficulties , these brackets have excellent corrosion resistance and are biocompatible , but have not been subjected to studies related to the frictional resistance force an orthodontist may encounter during sliding mechanics . andreasen and quevodo ( 1970 ) pioneered an in - depth frictional study to evaluate the frictional forces in the 0.022 0.028 inches edgewise bracket system . drescher et al ( 1989 ) in their study found the following factors to affect friction in tooth - guided arch wire mechanics ( in decreasing order ) : retarding force ( biologic resistance ) , surface roughness of wire , wire size ( vertical dimension ) , and bracket width and elastic properties of the wire . bednar et al ( 1991 ) found that the elastomeric - ligated ceramic brackets demonstrated the greatest friction when compared with other bracket / ligation technique combinations . david et al states that the opposing frictional forces at the interface of the bracket , archwire , and ligature can influence the sliding arch - guided system generally referred to as sliding mechanics . dickson et al experimentation demonstrated that the conventional ceramic bracket had highest static frictional characteristics . downing et al evaluated the effect of artificial saliva on the static and kinetic forces of stainless steel and polycrystalline ceramic brackets in combination with stainless , nickel titanium and beta titanium arch wire materials , under a constant ligature force . the stainless steel brackets produced more friction than polycrystalline brackets , but these results were not statistically significant except against beta titanium arch wires . baker et al investigated on frictional changes in the force values caused by saliva substitution on stainless steel arch wire bracket combination . they found artificial saliva to increase coefficients of friction for stainless steel , m titanium and nickel - titanium when compared to dry conditions . very few studies have been conducted and discussed in the literature about corrosion and sensitivity to the nickel present and stainless steel brackets . titanium has heralded as a material totally compatible in the oral environment and superior in structural integrity compared to stainless steel . the study was designed to compare the frictional properties of stainless steel , titanium , and self - ligating brackets in 0.018 and 0.022 slots , in 0.017 0.025 and 0.019 0.025 stainless steel and tma archwires with second - order angulations , ligated with elastic modules to the brackets slot . the substantial differences in friction between titanium , stainless steel , self - ligating brackets , using stainless steel and tma archwire , indicate differences in the coefficient of friction applicable for these materials sliding along the slot . it is becoming apparent that stainless steel and elastomeric ligatures will eventually be outdated like the full banding technique one day . considering the advantages of the self - ligating brackets that they show lesser frictional resistance , the frictional resistance of 0.022 inch and 0.018 inch slot were measured using instron universal testing machine and recorded [ table 1 ] . value recorded from the instron universal testing machine diagrammatic representation of six bracket and wire combinations of 0.018-inch and 0.022-inch slots the independent samples t - test procedure is used to compare the mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is no significant difference between the mean scores of stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . h0 : there is significant difference between the mean scores of self - ligating stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . t - test group statistics the independent samples t - test procedure is used to compare mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is significant difference between the mean scores of titanium bracket with stainless steel wire and tma wire regarding the level of satisfaction . graphs 2 and 3 represents mean frictional resistance of 0.22 slot and 0.18 slot brackets with displacement respectively . t - test group statistics 0.22 slot brackets with displacement 0.18 slot brackets with displacement the table displays the descriptive statistics of the sample size , mean , standard deviation , and standard error . the table also [ tables 510 ] shows the t - statistics , which is calculated as the ratio of the difference between sample means divided by the standard error of the difference . the column hence , there is no significant / there is a significant difference in the mean scores of the two groups with respect to the level of frictional resistance . t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics the mean score analysis ( anova ) test procedure [ tables 1116 ] is used to compare the mean scores of level of frictional resistance for more than two groups . the procedure assumes that the variances of the groups are equal and it was tested with levene 's test statistics . the significant difference between the mean scores of level of frictional resistance is tested with respect to the various titanium , self - ligating stainless steel , and stainless steel brackets , using stainless steel and tma archwires . test for mean scores of level of frictional resistance and six groups of brackets and wire combinations . the mean scores of level of frictional resistance and six groups of 0.018-inch and 0.022-inch slot brackets were tested with anova test procedures and the results of the analysis are given in table 11 . h0 : there is no significant difference between the mean scores of frictional resistance among the six groups regarding the level of coefficient of friction . the self - ligating stainless steel brackets and titanium brackets come under one group of subset 1 and stainless steel brackets are in subset 2 . the independent samples t - test procedure is used to compare the mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is no significant difference between the mean scores of stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . h0 : there is significant difference between the mean scores of self - ligating stainless steel bracket with stainless steel wire and tma wire regarding the level of satisfaction . t - test group statistics the independent samples t - test procedure is used to compare mean scores of level of frictional resistance for the titanium , self - ligating stainless steel , and stainless steel brackets of slots 0.018 inch and 0.022 inch . the procedure assumes that the variances of the two groups are equal and it was tested with levene 's test statistics . h0 : there is significant difference between the mean scores of titanium bracket with stainless steel wire and tma wire regarding the level of satisfaction . graphs 2 and 3 represents mean frictional resistance of 0.22 slot and 0.18 slot brackets with displacement respectively . t - test group statistics 0.22 slot brackets with displacement 0.18 slot brackets with displacement the table displays the descriptive statistics of the sample size , mean , standard deviation , and standard error . the table also [ tables 510 ] shows the t - statistics , which is calculated as the ratio of the difference between sample means divided by the standard error of the difference . the column hence , there is no significant / there is a significant difference in the mean scores of the two groups with respect to the level of frictional resistance . t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics t - test group statistics the mean score analysis ( anova ) test procedure [ tables 1116 ] is used to compare the mean scores of level of frictional resistance for more than two groups . the procedure assumes that the variances of the groups are equal and it was tested with levene 's test statistics . the significant difference between the mean scores of level of frictional resistance is tested with respect to the various titanium , self - ligating stainless steel , and stainless steel brackets , using stainless steel and tma archwires . test for mean scores of level of frictional resistance and six groups of brackets and wire combinations . the mean scores of level of frictional resistance and six groups of 0.018-inch and 0.022-inch slot brackets were tested with anova test procedures and the results of the analysis are given in table 11 . h0 : there is no significant difference between the mean scores of frictional resistance among the six groups regarding the level of coefficient of friction . the self - ligating stainless steel brackets and titanium brackets come under one group of subset 1 and stainless steel brackets are in subset 2 . thus , the aim of the study was to compare the frictional resistance of titanium , self - ligating stainless steel , and conventional stainless steel brackets using stainless steel and tma archwires , with the help of instron universal testing machine , one - way anova , student 's t test , and post hoc multiple range test at level of < 0.05 showed statistically significant difference in the mean values of all the groups . results demonstrated the following : the titanium , self - ligating stainless steel , and stainless steel brackets of 0.018-inch and 0.022-inch slots had no significant variations in frictional resistance.the self - ligating bracket with tma archwires showed relatively less frictional resistance compared with the other groups.the titanium bracket with tma archwires showed relatively less frictional resistance compared with the stainless steel brackets . the titanium , self - ligating stainless steel , and stainless steel brackets of 0.018-inch and 0.022-inch slots had no significant variations in frictional resistance . the self - ligating bracket with tma archwires showed relatively less frictional resistance compared with the other groups . the titanium bracket with tma archwires showed relatively less frictional resistance compared with the stainless steel brackets .

quantum chemical methods , as implemented in many software packages , allow for routine calculations of various properties of molecules . among them , thermodynamic and kinetic data are crucial for the understanding and prediction of chemical processes . a reliable prediction of such properties requires tackling two main issues : first , it is necessary to calculate accurate molecular energies at different geometries defining the energetic minima ( stable states ) and saddle points ( transition states , ts ) on the potential energy surface ( pes ) . modern ab initio methods are well - adapted and efficient in addressing these issues . a second issue arises when one wishes to compute temperature - dependent properties ( entropies , heat capacities , reaction rates ) for which densities of states ( dos ) and/or partition functions need to be computed . this is usually achieved using the simple rigid rotor harmonic oscillator ( rrho ) approximation . indeed , most of the internal vibrations are of small amplitude and are very well - described under this approximation . however , it has been shown that these approximations fail when large amplitude motions are involved , and a rigorous treatment is often required if an accurate prediction of statistical properties is required . this is particularly the case when a torsional motion is present or when a loose ts is involved in a dissociation / recombination reaction . numerous studies have been dedicated to the development of statistical methods in the context of one or the other application , while , to our knowledge , none has been presented for the general case . in cases of torsional motions , the proposed treatments are usually derived from the one - dimensional hindered rotor ( 1dhr ) model , originally proposed by pitzer et al . recent studies show the importance of a rigorous treatment , where quantum effects , multidimensionality , as well as coupling with other internal or external motions can have a non - negligible influence . in the context of dissociation reactions involving a loose complex , two- to five - dimensional large amplitude motions need to be described . analytical expressions for the kinetic energy of two rigid counterparts have already been derived in previous work , and it has been shown that this formalism gives a very good approximation of the reaction rate , as long as the used pes is determined with a high level of accuracy . since the kinetic energy operator has an analytic form , the main and most critical issue involved in studies of large amplitude motion is the determination of the pes . even though the first - principle calculations have proven their ability to yield accurate electronic energies , their computational cost does not generally allow for on - the - fly calculations , and therefore , an appropriate interpolation method has to be used . three approaches can be distinguished : ( i ) local ( or weighted ) interpolation , ( ii ) global ( or nonweighted ) interpolation by a set of mathematical functions , and ( iii ) global interpolation by a parametrized physical model . the first two solutions are general in principle but tedious to set up in practice when the dimension of the problem increases . the practical issues involved , along with the large amount of ab initio energy computations required , are important limitations to their applicability . the third solution is a global interpolation by a parametrized physical model of the interaction energy and is widely referred to as a force field. this particular formalism is extensively used to study the dynamics of large atomic systems . explicit introduction of the physical contributions to the interaction energy allows for a considerable decrease in the amount of information required to set up the model . however , because this simplification can introduce large model errors , it is of primary importance to estimate the confidence expected on a final quantity of interest ( qoi ) . only very recently has this issue been addressed by cailliez and pernot . by using a bayesian approach , they have calculated the parameter uncertainties associated with a van der waals type pes and have evaluated their influence on the uncertainties for the thermodynamic data . a major conclusion of their work is that the pes model error is indeed a critical feature when one wants to predict thermodynamic properties . the purpose of this paper is two - fold : first , we present a general procedure based on a bayesian framework to infer a system - specific force field . this procedure allows the computation of probability density functions ( pdfs ) of the model parameters using a set of ab initio data and also allows their propagatation to the desired qoi ( here , partition function or reaction rate ) . the other concern of this paper is the presentation of a general approach for characterizing the statistical properties of a large amplitude motion ( lam ) . the restriction of previous methods to specific types of motion , largely due to an ad hoc derivation of the kinetic energy of the system , is overcome by introducing a new numerical method based on a functional z - matrix , in which the dof ( dofs ) are written as functions of generalized coordinates . this approach provides several advantages : ( i ) the dynamical variables can be of any kind ( bond length , bending or dihedral angle , reaction path - like coordinate , etc . ) , ( ii ) the geometry relaxation as well as the constraints are easily expressed in the z - matrix formalism , and ( iii ) the numerical implementation is straightforward and robust . other issues , such as quantum effects and couplings between lams with the overall rotation and the internal ho , are approximately taken into account using selected methods from the literature . to illustrate the advantages and accuracies of the proposed methodology , two calculation tests are presented : ( i ) the partition function of 1,3-butadiene , and ( ii ) the high - pressure limit of the ch3 + h ch4 reaction rate . the presented methods are parts of the c++ library opensoams , currently under development . the particular steps that are emphasized in this work are ( i ) the inference of a pes model ( parametrized by the vector ) from a set of ab initio energies ( pes box ) , ( ii ) the implementation of a z - matrix coordinate system for the computation of the kinetic function ( kinetic box ) , and ( iii ) the lagrangian analysis of the lam and pes uncertainty propagation to the qoi ( stat . & uq box ) . in the canonical ensemble , the partition function is the central quantity for computing thermodynamic data and rate constants . in order to compute the partition function , we propose to split the total hamiltonian of a system containing a lam into the following four contributions : ( i ) a large amplitude motion contribution , from which overall rotation and translation are rigorously removed , ( ii ) the small amplitude vibrations , eventually considered loosely coupled to the lam , ( iii ) the overall rotation , loosely coupled to the lam , and ( iv ) the overall translation , rigorously independent of all the other modes . the total partition function of the system is then trivially obtained by simple multiplication of all the contributions:1where t is the temperature and qlm , qhocoupled , qrotcoupled and qtrans are the partition function of the contributions ( i ) to ( iv ) , respectively . in this section , we will address the calculation of qlm , qhocoupled , and qrotcoupled in detail , and the partition function of the overall translation has been discussed extensively elsewhere . acronyms and abbreviations : potential energy surface ( pes ) ; uncertainty quantification ( uq ) ; probability density function ( pdf ) ; thermodynamic ( td ) ; parameters ( param . ) ; translation ( transl . ) ; rotation ( rot . ) ; statistic ( stat . ) . let a lam of a molecular system be described by the positions rat of the atomic nuclei in a system of generalized coordinates q. the lagrangian of the system is written2where v(q ) is the electronic pes and t(q , q ) the kinetic energy , given by the relation:3the matrix a is called the kinetic matrix , its elements are defined by4 the theorem of aston and eidinoff allows a computation of the lam partition function through a configurational integral involving the determinant of the kinetic matrix and the pes:5where kb is the boltzmann constant , t the temperature , n the dimension of q , and the symmetry number of the motion ( see the work of fernndez - ramos et al . for a thorough discussion ) . the term k(q ) = |a(q)| , called the kinetic function here , completely describes the kinetic part of the lam from a statistical point of view . this integral is restricted within configurational space and allows a simple numerical procedure for its evaluation for routine applications . the partition function defines the statistical properties of the lam in the canonical ensemble ; specifically , the probability density that the system occupies a particular position q in the classical formulation is given by6 in the case of one - dimensional motion , the computation of the partition function is improved by using quantum statistics . an effective temperature - dependent kinetic constant is introduced by the following relation:7the fourier grid hamiltonian algorithm is then employed to compute the eingenvalues ieff(t ) of the effective hamiltonian defined by8 the partition function is finally obtained by a direct count of the eigenvalues ieff(t):9 the calibration of a model can be viewed as the update of model parameters in order to get a better respresentation of observations . this model can then be used to make predictions of a quantity of interest for specific scenarios . when calibrating a physico - mathematical model of the pes with respect to ab initio data , uncertainties are associated with the physical model uncertainties , arising from inadequacies of the physical model ( due to underlying assumptions and simplifications ) . as a result , there are model parameter uncertainties , arising from uncertain values of the model parameters . these uncertainties are simultaneously quantified here through the solution of statistical inverse problems based on a bayesian approach , as illustrated by the block pes in figure 1 . a stochastic model m is specified by a set of uncertain parameters a , to which an additional uncertain parameter , variance ( total2 ) , is included as a measure of the total model error . each stochastic model is then specified by the set = a total2 . one can use the data d to compute the posterior pdf p(|d , m ) as defined by bayes theorem:10where c is a normalization constant that makes the probability volume under the posterior pdf equal to unity , p(d| m ) is the likelihood function , and p(|m ) is the prior pdf for ( always chosen to be uniform in this paper ) . the likelihood function expresses the probability of observing d based on the predictive pdf for the system output given by the set of parameters in the model m. to compute the likelihood function , the assumption of statistically independent error is used . we denote by dj the j data point and by yj the model output computed for the same scenario as dj . we also consider an additive error based on the assumption that the error is independent from the value dj:11 in this study we assume that there is no data error , e.g. , that the ab initio values dj calculated at a particular level of theory are perfectly converged with respect to all the numerical parameters of the quantum method used . the error is modeled in this paper as a gaussian deviate with zero mean and variance total2 . based on all these assumptions , the variance total2 is treated as an unknown and thus needs to be calibrated as well . because no data error is assumed , the calibrated variance is a measure of the model adequacy , and thus total2 will be referred to simply as m2 . ( hereafter , we simply say validated ) is subjective ; it requires a metric to compare the predicted quantities produced by the calibration and the data used for the calibration . if the data agree within the acceptable tolerance limit , the model ( denoted as mval ) is then directly used for the forward problem : the pdf model parameters are propagated to the quantity of interest . one of the most important objectives of performing the above analysis is to make robust predictions about the qoi from a data set of the system of interest . based on a candidate m , all the probabilistic information for the prediction of a vector of quantities of interest q is contained in the posterior predictive pdf for m given by the theorem of total probability:13the above equation obtains the prediction p(q|d , m ) of a vector of quantities of interest q r by summing up the prediction p(q|,d , m ) of each model specified by weighted by its posterior probability p(|d , m)d. the evaluation of the multidimensional integrals of eq 13 can not usually be done analytically . a common numerical approach often used is based on simulating samples , k = 1 , 2 , ... , k , ( called posterior samples ) from the posterior pdf p(|d , m ) . the posterior pdf p(|d , m ) can be approximated by using these samples:14we use in this paper the adaptive multilevel stochastic simulation algorithm presented by cheung and beck to generate the posterior samples . the integral in eq 13 is then approximated by15where q is a sample simulated from p(q|,d , m ) . if q is a deterministic function of q ( i.e. , q = q( ) ) , then q = q( ) . estimates for important statistical moments of q conditioned on m and d can be obtained using the samples q , k = 1 , ... , k. for instance , the posterior mean is calculated as follows:16 the 95% confidence interval ( ci ) , usually taken to represent the confidence expected on a predicted outcome , is the interval i defined by:17 in this paper , the mean and the 95% ci on the predicted posterior of q are noted by < q > and [ q ] , respectively . the library queso is used to solve the inverse problem and to compute the posteriors p(|d , m ) . the current numerical methodology is very efficient and feasible for various engineering applications ( e.g. refs ( 4549 ) ) . the computation of the kinetic function |a(q)| defined by eq 4 is achieved using the internal z - matrix coordinates . as illustrated in the kinetic box in figure 1 , the objective is to compute the absolute displacement of the atoms with respect to variations in the generalized coordinates from the atomic relative positions . the contribution of the overall rotation and translation is separable through eq 1 , and the kinetic function is calculated so that the total angular ( j ) and linear ( p ) momenta associated to any variation dq are null . let the internal coordinates of the z - matrix be noted by the vector z of dimension nz , where nz = 3n 5 for a linear molecule or 3n 6 otherwise ( n being the number of atoms ) . the configurations of an n - dimensional motion parametrized by the generalized coordinates q will then be described by nz functions zi(q ) . the connectivity scheme of the z - matrix along with the functions zi(q ) is referred to as a functional z - matrix . the ( relative ) positions of the atoms obtained using the z - matrix definition are denoted by zat(q ) , and their construction rule is straightforward : the first three atoms are defining the orientation of the structure , and the origin of the cartesian coordinates coincides with the center of gravity of the system . given an initial structure rat(q ) directly constructed from the z - matrix ( e.g. , rat(q ) = zat(q ) ) , the rat/qi terms in eq 4 are calculated by determining the structure rat(q + dq ) generated by any displacement dq and associated to j = p = 0. while the condition p = 0 is satisfied by construction in the z - matrix ( because the center of mass of the system is fixed at the origin ) , this is not the case for the condition j = 0. by consequence , the structure rat(q + dq ) is related to zat(q + dq ) by an overall rotation around its center of gravity:18where mrot is a rotational matrix defined by the condition:19 the matrix mrot is optimized by an iterative procedure , generalizing the one presented in our previous paper which was restricted to a fixed axis of rotation . the iterative method is initiated by setting mroti = 0 = i d . at each step i , the angular momentum j is calculated using the current mroti according to20 the matrix mroti is then corrected at the next iteration by21where mrotji , d is the matrix of rotation around the axis j ( current overall angular momentum ) with the counter rotational angle d defined by22where d is an elementary angle ( taken in this paper as 10 radian ) , j is the angular momentum associated with a rotation of d around j of the structure mrotizat(q0 + dq ) . this procedure leads to a progressive annihilation of ji associated with a decrease of the kinetic function . the procedure is stopped in this paper when the kinetic function is converged within a relative factor of 10 . the presence of the large amplitude motion leads to some coupling with the overall rotation , and the usual rr approximation needs to be overcome . the partition function is computed by considering a loose coupling between the lam and the overall rotation , as proposed by vansteenkiste et al . the partition function of the flexible rotor qfr(t ) is based on the rigid rotor ( rr ) expression , calculated at each configuration point of the lam and averaged using the probability density defined in relation ( eq 6):23where qrr(t , q ) is the partition function of a rr of the structure zat(q ) . as for the overall rotation , the presence of the lam may lead to some coupling with small internal vibrations . wong et al . and vansteenkiste et al . proposed to perform an integration similar to eq 23 by using a ho approximation of the small amplitude vibration at each configuration point . this procedure leads to an excellent approximation of the partition function , however it requires the costly evaluation of the hessian over all the configurational space . here , we propose to restrict the integration to the stable configurations involved in the lam . the partition function of the coupled small amplitude vibration under this assumption is given by24where the sum is carried out on all the stable configurations of the lam , qi is the associated configurational point , qho(t , qi ) is the partition function of the small amplitude vibration in the ho approximation of the structure zat(qi ) , and p(qi , t ) is the weighting factor defined by25 the geometry of the molecule at the minimum of the pes is illustrated in figure 2 along with the atom numbering used hereafter . the molecule contains a torsional motion defined by the relative rotation of the two h2ch parts around the central c5c2 bond . previous studies have shown that it involves a highly asymmetric internal rotor associated with a non - negligible geometry relaxation effect as well as a non - negligible coupling between the torsion and the other dof . illustration of the geometry of 1,3-butadiene at the global minimum of the pes . the large circles represent the carbon atoms , and the small circles represent the hydrogen atoms . ground - state electronic energies are calculated using the ab initio methods ub3lyp/6 - 31g(d ) ( hereafter referred to as dft ) for qualitative calculations , and rhf - ccsd(t)/aug - cc - pvdz ( hereafter referred as cc ) for accurate quantitative calculations . geometry optimizations and a normal - mode analysis are performed at the ub3lyp/6 - 31g(d ) level of theory . the torsional pes is obtained by computing the relaxed geometries with respect to the dihedral c1c2c5c7 using a 10 step . we use a local quadratic interpolation to obtain ab initio values of the energy at other torsional angles . the table 1 presents the functional z - matrix used to compute the kinetic function . the functions fi( ) ( i = 1 , ... , 8) define the relaxation of the dof with respect to the generalized coordinate . the dofs contributing to less than 1% of the kinetic function have been considered constant . the functions fi( ) are obtained by fourier series fit to the optimized internal coordinates and are presented in appendix a. the kinetic function calculated using our numerical approach is presented in figure 3 ( solid line ) and compared to those reported by vansteenkiste et al . and wong et al . a very good agreement is observed between the three studies , the difference from the eckart method at 180 being most likely attributed to the different level of theory used in the study of wong et al . for geometry optimization ( mp2 ) . we also present in figure 3 the kinetic function computed when the relaxation of the geometry with respect to is not taken into account ( fi( ) = 0 i , dashed line ) . as it can be seen , the kinetic function is strongly affected and gives values in close agreement with those presented by wong et al . calculated using the pitzer method . because the pitzer values are also based on relaxed geometries , this demonstrates that the pitzer method actually fails to take into account the dynamic influence of the relaxation on the kinetic energy . the definition of the relaxation functions fi( ) is given in appendix a. lengths are in angstroms and angles in degrees . comparison between the kinetic function calculated in this work ( lines ) and the kinetic function obtained using the ehr model and the eckart and the pitzer methods ( presented by wong et al . ) . solid line : flexible torsion ( fi( ) 0 ) ; dashed line : rigid rotation ( fi( ) = 0 ) . the model used to describe the pes of the torsional motion accounts for two physical contributions : ( i ) the contribution of the intrinsic torsional energy of the c5c2 sp chemical bond , and ( ii ) the contribution of a repulsive part , representing the steric repulsion between the atoms h9 and h4 during the torsion . using the definition of the repulsive part of a lennard - jones - type potential , and a modified cosine function to account for the sp torsion energy , the pes is defined using the three parameters a0 , a1 , and a2 by26where dvdw is taken equal to 2.4 ( sum of the van der waals radii of the hydrogen atoms ) , and ga0( ) = 1 a0(1 the function ga0 adds dof to model the bonding contribution of the sp torsional energy , and one can verify that ga0( ) = ga0( ) as well as ga0(180 ) = ga0(180 ) the stochastic pes model m contains four parameters to calibrate : the three involved in eq 26 ( a0,a1,a2 ) , and the variance ( m2 ) . 95% ci of the posterior pes using the data set dndft with n = 5 , 10 , and 20 ( dotted , dashed , and solid lines , respectively ) , and represents the data set d19dft . in order to study how the ab initio data inform the pes parameters , they are denoted by dnx where x { dft , cc } stands for the level used for the electronic energy calculations , and n is the number of values included ( in uniform repartition in [ 0;180 ] ) . the prior pdfs , the 95% cis based on the posterior pdfs , and the posterior means are presented in table 2 . units : a0 , no unit ; and a1 , a2 , and m , kcal / mol . it is shown in rows 38 that the 95% ci is considerably narrowed around the mean value for each parameter each time the data set size increases . also , the posterior mean value < ai > is already accurately predicted when only five points are used . the mean of the variance shows a different behavior : its value keeps decreasing when the amount of data increases . it reaches 1.25 10 kcal / mol ( e.g. , < m > = 3.5 10 kcal / mol ) when 20 points are used . the 95% cis [ v]ndft ( n = 5 , 10 , 20 ) are presented in figure 4 and compared to the d19dft data set . the figure confirms the quality of the proposed model and is able to reproduce almost perfectly the ab initio results when a sufficient amount of data points is used . it is worth mentioning that the uncertainty on the posterior pes ( maximum here at 90 and 180 ) highlights the most relevant volume of the configurational space in which data are needed to improve the calibration process . we now look at the posterior pdfs of model parameters obtained using the data set dncc ( n = 2 , 3 , 5 ) presented in the four last rows of table 2 . in this case , we calibrate model parameters except for m2 , which is set to be 1.25 10 kcal / mol . in other words , we assume that the model error obtained by the dft level of theory is close to the realistic estimation of the true model error . as can be seen in table 2 , this assumption is reasonable since [ v]5cc is already very narrow ( and actually converged ) for all the parameters even when five points are used . it is also shown that using two points ( at 0 and 180 ) is not enough to infer a0 and a2 ( posterior pdfs approximately equal to the prior pdfs ) , while a third point at 90 allows a considerable reduction of the uncertainty on a2 . these results are illustrated in figure 5 , which presents the uncertainty domain of the posterior pes for these three data sets . comparing [ v]5cca posteriori with the d19cc data set demonstrates that the dft level of theory is fully able to estimate the absolute model error for this case as the data points are almost perfectly encapsulated in [ v]5cc ( assuming that the ccsdt / aug - cc - pvdz level of theory gives the exact pes ) . 95% ci of the posterior pes using the data set dncc with n = 2 , 3 , and 5 ( dotted , dashed , and solid lines , respectively ) . the , , and represent the corresponding data set , respectively , and represents the data set d19cc . 95% ci of the posterior torsional partition function using the data set dndft with n = 5 , 10 , and 20 ( dotted , dashed , and solid lines , respectively ) , and mean of the posterior torsional partition function using d5dft . the 95% ci of the posterior partition function [ qlam ] using the dn = 5,10,20dft data sets is presented in figure 6 . the results are normalized to the partition function obtained using an ho approximation . the mean value of the posterior partition function using d5dft is also presented . as for the pes results , the 95% ci becomes very narrow as soon as 10 points are used , and the mean value < q > 5dft is already almost converged . < 400 k ) is important because it demonstrates that the method presented here to treat the 1d lam has a sufficient capability to perfectly reproduce the harmonic approximation results in the low - temperature limit . the total partition function of 1,3-butadiene is obtained by multiplication of the lam partition function by the fr and fo partition functions . to compute the fho partition function , we consider the system constituted by two stable complexes , characterized by the normal - mode frequencies at = 0 and 145 ( the normal - mode frequencies are presented in appendix b ) . it should be noted that the pes uncertainty also affects the fr and fo partition functions through eqs 6 and 25 . the 95% ci of the posterior partition function of 1,3-butadiene using the d5cc ( with m2 kept fixed at 1.25 10 kcal / mol ) and d20dft data sets are presented in figure 7 and compared to the results presented by wong and raman ( pert model ) and those presented by vansteenkiste et al . [ the results are normalized by the partition function obtained using a rrho approximation . we first compare our prediction using d5cc with the low - temperature results presented by wong and raman using their pert method . the comparison is meaningful because the same level of theory has been used to compute the pes and also because the pert method used represents most likely the best model available in the literature . however , this fully coupled quantum method is computationally expensive , which explains why no results for temperatures higher than 500 k have been presented . although our predictions slightly overestimate the partition function , the behavior of the curves is very similar , and the results stay very close to each other . the small overestimation comes from either the simplification introduced by our treatment and/or from differences in the rrho reference partition function . the same dft method used to compute the pes is shared by the two studies ; however , vansteenkiste et al . consider a larger basis set in their work ( 6 - 311g(d , p ) ) . the two pess are nevertheless similar , as reflected by the small difference of the torsional barrier height ( 0.1 kcal / mol ) . as can be seen in the figure , the behavior of the partition function curves is very similar , essentially differing by a small translation factor . this factor seems to be an artifact involved in the ehr method , as the partition function does not converge to 1 at the limit of low temperatures . comparison between the 95% ci of the posterior partition function of the butadiene using d20dft ( dashed line ) and d5cc ( solid line , m2 = 1.2510 ) ; , pert method ; and , ehr method . the model 1d lam method proposed in this paper is able to account for 1d torsional motion with an accuracy comparable to the best methods presented until now . it is worth recalling that only 2 normal - mode analyses have been realized here , while they have been conducted all over the configurational space in the studies of vansteenkiste et al . and wong and raman . also , this efficient formalism , in terms of computational time , further allows it to be used in conjunction with an uncertainty quantification algorithm . the reaction rate of the ch3 + h recombination is computed at the high - pressure limit using variational transition - state theory ( vtst ) with variational reaction coordinate ( vrc ) and spherical dividing surfaces ( ds ) in the canonical ensemble . in the present case , the ds is parametrized using one pivot point p attached to the ch3 part , around which the approaching h * atom is allowed to rotate . the optimal ds ( noted ds * ) , defining the ts , is optimized for every temperature in a way that it is associated to a minimal partition function:27where s is the reaction coordinate ( separation ph * ) , and qdsp,s(t ) is the partition function of the ds defined by s and p. the high - pressure reaction rate is calculated using the standard tst assumptions and is given by28where is planck s constant , qch3 and qh the partition functions of the methyl and hydrogen radicals , respectively , and v * is the electronic barrier height associated with the ch3 + h ds*. over the small amplitude motion involved in the system , the ch3 umbrella motion changes quite substantially and may need to be included in a fo partition function . however , klippenstein et al . have shown that its influence is negligible with at most a 2% increase of the reaction rate for temperatures below 2400 k. in this work , the small amplitude motions are then supposed uncoupled to the reactional motion , and the reaction rate expression is simplified to29where qds*2d is the partition function of the h * motion on the ds * , qds*fr the effective overall rotation partition function of the ds , and qch3trans&rot the partition function of the overall translational and rotational motion of ch3 . ground - state electronic energies are calculated using the cr - cc(2,3)/aug - cc - pvdz level of theory . the cr - cc(2,3 ) method is an improvement over the ccsd(t ) approach to overcome its deficiencies in describing systems involving biradical character . geometry optimizations are carried out at the ub3lyp/6 - 31g(d ) level of theory . the kinetic energy of the ds is obtained by defining the functional z - matrix associated with the h * 2d motion . because of the c3 symmetry of ch3 , the ds is parametrized by two parameters . the first parameter is the distance x between the pivot point p , lying in the c3 axis of symmetry of ch3 , and the carbon atom , taken as the origin . we denote from now on p = x. the second parameter is the distance s between the approaching h * atom and the pivot point . the functional z - matrix which describes the ds the c3 symmetry is imposed to the system , and the relaxation of the ch3 part is taken into account to some extent by using the functions a(|*| ) ( interpolation between the optimized values on the mep ) . illustration of a typical bifaceted dividing surface of the ch3 + h ch4 reaction . it has been shown that a limitation of the spherical dividing surfaces is that they overaccount artificial contributions to the reaction rate when multiple reaction paths are present . to avoid this overestimation , a multifaceted dividing surface is used , composed by the envelope of spherical dss centered around a reactive channel - specific pivot point . considering the equivalence of the two association channels for the h * addition on ch3 , a typical dividing surface for the reaction is illustrated in figure 8 . to compute the ds partition function , the integration domain defined in eq 5 has to be restricted to30where q0min = arcsin(x / s ) is the solid angle from x defining the intersection of the two spherical ds . the overall symmetry number of the irreducible integration domain is then 12 . since this number takes into account the two reaction paths , no symmetry number has to be considered in the overall rotational partition function of both ch3 and the ds . the pes of the ch3/h * interaction accounts for three contributions : ( i ) the stretching energy of the c h * bond ( noted vstr ) , ( ii ) the bending energy of the hich * dof ( noted vbend ) , and ( iii ) the steric repulsion energy between the hi and h*. the stretching energy vstr is written as a morse - like function of |*| with an origin of energy taken at the products state:31where de = 107.75 kcal / mol is the dissociation energy , req = 1.09 is the equilibrium bond length , and a0 and a1 are the first two parameters of the pes model . the bending energy is modeled by a simple cosine function , with a barrier height equal to the dissociation energy at the angle xch * = /2:32finally the pes also accounts for the spherical repulsion between the passive hydrogen atoms hi and h*:33where hh = 2.4 with a2 and a3 being the last two parameters , and h*0 is the corresponding position of h * on the mep ( at the same c h * separation ) . the term ( hh/|(| ) ) allows a cancellation of the steric interaction energy on the mep . the pes is the sum of these three contributions and is defined through the four parameters ai , i = 0 , ... , 3:34 the geometries used to compute the electronic energies are defined by the z - matrix presented in table 3 with x = = 10 ( in order to preserve a nonambiguous definition of the c3 axis ) . the 50 data points collected are divided in two groups : ( i ) one is used to infer the pes model ( noted d * ) , and ( ii ) the second is used to validate it for extrapolation ( noted d ) . the data set d * contains 25 points in the relevant space of the pes for reaction rate calculation and is constituted by:(i)10 points on the mep ( q0 = q1 = 0 ) from s = 2.03.8 .(ii)15 points out of the mep defined by the combination s { 2.0 , 2.4 , 2.8 } and q0 { 20,40,60,80,100 } with q1 = 0.the d data set collects the energies of the corresponding geometries of ( ii ) at q1 = 30 as well as 10 other points at s = 1.6 . the prior intervals , the posteriors means , and 95% cis are presented in table 4 , and the 4 posteriors p(a|d*,m ) are presented in figure figure 9 . we confirm here that the morse potential ( defined for a1 = 1 ) is not the most appropriate to describe the mep , and a value of a1 = 1.25 is the most probable value . the mean value of a3 is surprisingly low for a repulsion term : in a van der waals force field , the exponent of the repulsive part is usually taken between 9 and 12 . 10 points on the mep ( q0 = q1 = 0 ) from s = 2.03.8 . 15 points out of the mep defined by the combination s { 2.0 , 2.4 , 2.8 } and q0 { 20,40,60,80,100 } with q1 = 0 . based on the posterior pdfs , and the posterior mean values ( < . units : a0 , ; a1 : no unit ; and a2 , a3 , and m , kcal / mol . posterior pdf , p(ai|d*,m ) , ( a d ) for respectively i = 0 , 1 , 2 , and 3 . we compare in figure 10 the 95% ci of the posterior mep ( [ v ] ) with the data points of d * ( solid circles ) and d ( open circles ) included in the mep . we also report in this figure the ab initio results at the caspt2/aug - cc - pvqz , full - ci/6 - 31g(d ) , and ccsd(t)/6 - 31g(d ) level of theories presented in the literature . the uncertainty of the pes model is under 2 kcal / mol for separations higher than 2.0 and coincidently encapsulates the results obtained at the full ci and caspt2 level of theory . as already reported , the ccsd(t ) level of theory is not able to properly estimate the energy of the system for intermediate separation . the comparison between the pes model and the direct ab initio data is shown in figure 11 for out - of - mep situations . even for extrapolated values at low separation ( s = 1.6 ) , the model still performs well with the hindering domain being correctly predicted within 10% . comparison between the predicted 95% ci of the mep ( [ v ] ) , the data sets d and d * and the ab initio results presented in literature . lines , [ v ] ; , d * ; and , d. gray symbols , literature results : * , ccsd(t)/6 - 31g(d ) ; + , caspt2/aug - cc - pvqz ; and , full ci/6 - 31g(d ) . comparison between the predicted 95% ci of the pes ( [ v ] ) with the data sets d and d*. lines , [ v ] ; filled symbols , d * ; and empty symbols , d. origin of energy : v(4 ,0,0 ) . the 95% ci ( [ k ] ) and the mean ( < k > ) of the posterior high - pressure reaction are presented in figure 12 with the available experimental measurements and the vtst - vrc theoretical predictions presented by klippenstein et al . and harding et al . the experimental measures have been converted from the k0 values of the original experimental work in the same way presented by klippenstein et al . the cis predicted by our approach are in very good agreement with the experimental values as well as with the theoretical calculations . first , the representation of the dividing surface does not constitute a perfect ds and is associated with an overestimation of the reaction rate . have shown that by using both vtst - vrc and a direct dynamic method , the spherical ds for the ch3 + h ch4 reaction is associated with a 9% overestimation , approximately independent of the temperature . the second limitation is the restriction of the statistical study to the canonical ensemble . in other works , the overestimation of the reaction rate coming from the canonical analysis is probably compensated to some extent by the pes model which predicts a slightly higher hindrance effect at high separation ( the ab initio data are close to the bottom boundary of the ci at the s = 2.8 case in figure 11 ) . to finish this study , we comment on the difference in the pes representation used here and in the works presented by harding et al . and klippenstein et al . in the works of klippenstein et al . , a combined fourier series/3d spline fitting procedure is used to obtain a 4d pes representation ( the umbrella motion is also explicitly considered ) . if the fit is almost exact , it has been achieved using 798 ab initio calculations in the 3d space analyzed here . this model , in addition to having required the manual optimization of almost 20 parameters , does not provide any indication of the associated model error . in the study of harding et al . the number of quantum calculations has not been presented , but assuming that ten points are used to integrate the partition function along q0 and q1 and 10 points to optimize ds and dx , this already results in 10 000 ab initio calculations . we recall that our model , even if associated with a 1.5 uncertainty factor on the reaction rate , is based on 4 parameters and has been set using 25 ab initio calculations for the calibration step ( and 25 to examine the extrapolation capabilities ) . the restriction to the canonical ensemble is another issue which does not need more pes calculations to be overcome . comparison of the high pressure ch3 + h recombination rate predicted by this work ( black lines , [ k ] ; , < k > ) with other theoretical calculations ( gray lines ) and the experimental values ( + , * , ) . gray lines , calculations using different representation of the pes ; full line , on the fly calculation ; dashed line , 4d mathematical fit ; and dotted line , hirst hase pes . we have presented in this work an original approach for the computation of statistical properties of molecular systems involving a large amplitude motion . the objectives were to propose : ( i ) a simple and general procedure to compute the kinetic energy of a lam ; ( ii ) a general procedure to calibrate an analytical pes using ab initio data ; and ( iii ) a rigorous quantification of the uncertainty of the pes model and their propagation to the qoi(s ) . two typical and different test cases have been considered for assessing the methodology : ( i ) the study of 1,3-butadiene , involving nontrivial features , such as coupling of modes or a highly variable kinetic function ; and ( ii ) the study of the ch3 + h recombination , for which a vtst - vrc approach is needed to compute an accurate reaction rate . we proposed to compute the kinetic energy of a lam based on a functional z - matrix formalism , e.g. , a z - matrix , in which the internal dofs are defined with respect to some generalized coordinates . the results obtained are exact within numerical precision and are in total agreement with previous exact methods . indeed , for a typical lam , the internal z - matrix coordinates naturally describe the configurational space of the motion and are much more suited than cartesian coordinates . also , the possibility to include ghost atoms in the z - matrix to represent a virtual pivot point is an additional important advantage when one wishes to study dissociation reactions involving loose complexes . finally , the method applies equally regardless of the number of generalized coordinates or their type ( length , angle , and reaction path - like coordinate ) . furthermore , the calibration of parameters for the analytical pes from ab initio calculations has been achieved using bayesian theory . the two examples treated have allowed us to point out different and interesting features of this approach . the most important one , which is critical to the derivation of analytical models , is that the pes model uncertainty is properly evaluated and can be propagated to the qoi . we have also shown that , providing an adequate pes model is used , narrowing the qoi ci needs significantly fewer data points than other methods , which does not exploit any particular physical contribution of the interaction energy . for instance , in the study of the ch3 + h recombination rate , we were able to use only 25 data points to calibrate the 3d pes in order to compute a reaction rate within an uncertainty factor of 1.5 ( coming from the pes model ) . this corresponds to a typical reduction of one or two orders of magnitude in the amount of data points needed with respect to previous studies . however , it is clear that this approach is relevant when the condition that a satisfactory model of the interaction energy is provided . even for complex force fields , we have shown here that for the two studied applications , accurate models can be based on simple contributions : morse - type potentials for bond stretching , cosine - like functions for bending and torsional motion , and steric repulsion for nonbonded atoms . we believe that these types of potentials should hold for the majority of torsional and simple bond - breaking context studies . finally , for the 1,3- butadiene application , we have discussed the possibility of performing a dual - level inference process . while it is not possible to generalize the results , it was shown that the b3lyp/6 - 31g(d ) level of theory was almost perfectly able to compute the true ( or absolute ) model error , even if the pes is not accurately rendered at this level . as a prior estimation of the model error allows to considerably reduce the required amount of data needed to obtain a given accuracy on the qoi , this property would be of particular interest for the calibration of high - level pess at a minimum computational cost .

we propose a general approach to describe large amplitude motions ( lam ) with multiple degrees of freedom ( dof ) in molecules or reaction intermediates , which is useful for the computation of thermochemical or kinetic data . the kinetic part of the lam lagrangian is derived using a z - matrix internal coordinate representation within a new numerical procedure . this derivation is exact for a classical system , and the uncertainties on the prediction of observable quantities largely arise from uncertainties on the lam potential energy surface ( pes ) itself . in order to rigorously account for these uncertainties , we present an approach based on bayesian theory to infer a parametrized physical model of the pes using ab initio calculations . this framework allows for quantification of uncertainties associated with a pes model as well as the forward propagation of these uncertainties to the quantity of interest . a selection and generalization of some treatments accounting for the coupling of the lam with other internal or external dof are also presented . finally , we discuss and validate the approach with two applications : the calculation of the partition function of 1,3-butadiene and the calculation of the high - pressure reaction rate of the ch3 + h ch4 recombination .

Introduction Theory Applications Conclusions

a reliable prediction of such properties requires tackling two main issues : first , it is necessary to calculate accurate molecular energies at different geometries defining the energetic minima ( stable states ) and saddle points ( transition states , ts ) on the potential energy surface ( pes ) . recent studies show the importance of a rigorous treatment , where quantum effects , multidimensionality , as well as coupling with other internal or external motions can have a non - negligible influence . analytical expressions for the kinetic energy of two rigid counterparts have already been derived in previous work , and it has been shown that this formalism gives a very good approximation of the reaction rate , as long as the used pes is determined with a high level of accuracy . since the kinetic energy operator has an analytic form , the main and most critical issue involved in studies of large amplitude motion is the determination of the pes . the third solution is a global interpolation by a parametrized physical model of the interaction energy and is widely referred to as a force field. explicit introduction of the physical contributions to the interaction energy allows for a considerable decrease in the amount of information required to set up the model . by using a bayesian approach , they have calculated the parameter uncertainties associated with a van der waals type pes and have evaluated their influence on the uncertainties for the thermodynamic data . the purpose of this paper is two - fold : first , we present a general procedure based on a bayesian framework to infer a system - specific force field . this procedure allows the computation of probability density functions ( pdfs ) of the model parameters using a set of ab initio data and also allows their propagatation to the desired qoi ( here , partition function or reaction rate ) . the other concern of this paper is the presentation of a general approach for characterizing the statistical properties of a large amplitude motion ( lam ) . the restriction of previous methods to specific types of motion , largely due to an ad hoc derivation of the kinetic energy of the system , is overcome by introducing a new numerical method based on a functional z - matrix , in which the dof ( dofs ) are written as functions of generalized coordinates . , ( ii ) the geometry relaxation as well as the constraints are easily expressed in the z - matrix formalism , and ( iii ) the numerical implementation is straightforward and robust . to illustrate the advantages and accuracies of the proposed methodology , two calculation tests are presented : ( i ) the partition function of 1,3-butadiene , and ( ii ) the high - pressure limit of the ch3 + h ch4 reaction rate . the particular steps that are emphasized in this work are ( i ) the inference of a pes model ( parametrized by the vector ) from a set of ab initio energies ( pes box ) , ( ii ) the implementation of a z - matrix coordinate system for the computation of the kinetic function ( kinetic box ) , and ( iii ) the lagrangian analysis of the lam and pes uncertainty propagation to the qoi ( stat . in order to compute the partition function , we propose to split the total hamiltonian of a system containing a lam into the following four contributions : ( i ) a large amplitude motion contribution , from which overall rotation and translation are rigorously removed , ( ii ) the small amplitude vibrations , eventually considered loosely coupled to the lam , ( iii ) the overall rotation , loosely coupled to the lam , and ( iv ) the overall translation , rigorously independent of all the other modes . the total partition function of the system is then trivially obtained by simple multiplication of all the contributions:1where t is the temperature and qlm , qhocoupled , qrotcoupled and qtrans are the partition function of the contributions ( i ) to ( iv ) , respectively . in this section , we will address the calculation of qlm , qhocoupled , and qrotcoupled in detail , and the partition function of the overall translation has been discussed extensively elsewhere . acronyms and abbreviations : potential energy surface ( pes ) ; uncertainty quantification ( uq ) ; probability density function ( pdf ) ; thermodynamic ( td ) ; parameters ( param . ) let a lam of a molecular system be described by the positions rat of the atomic nuclei in a system of generalized coordinates q. the lagrangian of the system is written2where v(q ) is the electronic pes and t(q , q ) the kinetic energy , given by the relation:3the matrix a is called the kinetic matrix , its elements are defined by4 the theorem of aston and eidinoff allows a computation of the lam partition function through a configurational integral involving the determinant of the kinetic matrix and the pes:5where kb is the boltzmann constant , t the temperature , n the dimension of q , and the symmetry number of the motion ( see the work of fernndez - ramos et al . the term k(q ) = |a(q)| , called the kinetic function here , completely describes the kinetic part of the lam from a statistical point of view . the partition function defines the statistical properties of the lam in the canonical ensemble ; specifically , the probability density that the system occupies a particular position q in the classical formulation is given by6 in the case of one - dimensional motion , the computation of the partition function is improved by using quantum statistics . an effective temperature - dependent kinetic constant is introduced by the following relation:7the fourier grid hamiltonian algorithm is then employed to compute the eingenvalues ieff(t ) of the effective hamiltonian defined by8 the partition function is finally obtained by a direct count of the eigenvalues ieff(t):9 the calibration of a model can be viewed as the update of model parameters in order to get a better respresentation of observations . when calibrating a physico - mathematical model of the pes with respect to ab initio data , uncertainties are associated with the physical model uncertainties , arising from inadequacies of the physical model ( due to underlying assumptions and simplifications ) . if the data agree within the acceptable tolerance limit , the model ( denoted as mval ) is then directly used for the forward problem : the pdf model parameters are propagated to the quantity of interest . based on a candidate m , all the probabilistic information for the prediction of a vector of quantities of interest q is contained in the posterior predictive pdf for m given by the theorem of total probability:13the above equation obtains the prediction p(q|d , m ) of a vector of quantities of interest q r by summing up the prediction p(q|,d , m ) of each model specified by weighted by its posterior probability p(|d , m)d. the evaluation of the multidimensional integrals of eq 13 can not usually be done analytically . the computation of the kinetic function |a(q)| defined by eq 4 is achieved using the internal z - matrix coordinates . the contribution of the overall rotation and translation is separable through eq 1 , and the kinetic function is calculated so that the total angular ( j ) and linear ( p ) momenta associated to any variation dq are null . the ( relative ) positions of the atoms obtained using the z - matrix definition are denoted by zat(q ) , and their construction rule is straightforward : the first three atoms are defining the orientation of the structure , and the origin of the cartesian coordinates coincides with the center of gravity of the system . at each step i , the angular momentum j is calculated using the current mroti according to20 the matrix mroti is then corrected at the next iteration by21where mrotji , d is the matrix of rotation around the axis j ( current overall angular momentum ) with the counter rotational angle d defined by22where d is an elementary angle ( taken in this paper as 10 radian ) , j is the angular momentum associated with a rotation of d around j of the structure mrotizat(q0 + dq ) . this procedure leads to a progressive annihilation of ji associated with a decrease of the kinetic function . the partition function of the flexible rotor qfr(t ) is based on the rigid rotor ( rr ) expression , calculated at each configuration point of the lam and averaged using the probability density defined in relation ( eq 6):23where qrr(t , q ) is the partition function of a rr of the structure zat(q ) . here , we propose to restrict the integration to the stable configurations involved in the lam . the partition function of the coupled small amplitude vibration under this assumption is given by24where the sum is carried out on all the stable configurations of the lam , qi is the associated configurational point , qho(t , qi ) is the partition function of the small amplitude vibration in the ho approximation of the structure zat(qi ) , and p(qi , t ) is the weighting factor defined by25 the geometry of the molecule at the minimum of the pes is illustrated in figure 2 along with the atom numbering used hereafter . previous studies have shown that it involves a highly asymmetric internal rotor associated with a non - negligible geometry relaxation effect as well as a non - negligible coupling between the torsion and the other dof . because the pitzer values are also based on relaxed geometries , this demonstrates that the pitzer method actually fails to take into account the dynamic influence of the relaxation on the kinetic energy . the model used to describe the pes of the torsional motion accounts for two physical contributions : ( i ) the contribution of the intrinsic torsional energy of the c5c2 sp chemical bond , and ( ii ) the contribution of a repulsive part , representing the steric repulsion between the atoms h9 and h4 during the torsion . using the definition of the repulsive part of a lennard - jones - type potential , and a modified cosine function to account for the sp torsion energy , the pes is defined using the three parameters a0 , a1 , and a2 by26where dvdw is taken equal to 2.4 ( sum of the van der waals radii of the hydrogen atoms ) , and ga0( ) = 1 a0(1 the function ga0 adds dof to model the bonding contribution of the sp torsional energy , and one can verify that ga0( ) = ga0( ) as well as ga0(180 ) = ga0(180 ) the stochastic pes model m contains four parameters to calibrate : the three involved in eq 26 ( a0,a1,a2 ) , and the variance ( m2 ) . in order to study how the ab initio data inform the pes parameters , they are denoted by dnx where x { dft , cc } stands for the level used for the electronic energy calculations , and n is the number of values included ( in uniform repartition in [ 0;180 ] ) . the total partition function of 1,3-butadiene is obtained by multiplication of the lam partition function by the fr and fo partition functions . the 95% ci of the posterior partition function of 1,3-butadiene using the d5cc ( with m2 kept fixed at 1.25 10 kcal / mol ) and d20dft data sets are presented in figure 7 and compared to the results presented by wong and raman ( pert model ) and those presented by vansteenkiste et al . although our predictions slightly overestimate the partition function , the behavior of the curves is very similar , and the results stay very close to each other . the reaction rate of the ch3 + h recombination is computed at the high - pressure limit using variational transition - state theory ( vtst ) with variational reaction coordinate ( vrc ) and spherical dividing surfaces ( ds ) in the canonical ensemble . the optimal ds ( noted ds * ) , defining the ts , is optimized for every temperature in a way that it is associated to a minimal partition function:27where s is the reaction coordinate ( separation ph * ) , and qdsp,s(t ) is the partition function of the ds defined by s and p. the high - pressure reaction rate is calculated using the standard tst assumptions and is given by28where is planck s constant , qch3 and qh the partition functions of the methyl and hydrogen radicals , respectively , and v * is the electronic barrier height associated with the ch3 + h ds*. have shown that its influence is negligible with at most a 2% increase of the reaction rate for temperatures below 2400 k. in this work , the small amplitude motions are then supposed uncoupled to the reactional motion , and the reaction rate expression is simplified to29where qds*2d is the partition function of the h * motion on the ds * , qds*fr the effective overall rotation partition function of the ds , and qch3trans&rot the partition function of the overall translational and rotational motion of ch3 . the kinetic energy of the ds is obtained by defining the functional z - matrix associated with the h * 2d motion . the functional z - matrix which describes the ds the c3 symmetry is imposed to the system , and the relaxation of the ch3 part is taken into account to some extent by using the functions a(|*| ) ( interpolation between the optimized values on the mep ) . illustration of a typical bifaceted dividing surface of the ch3 + h ch4 reaction . the stretching energy vstr is written as a morse - like function of |*| with an origin of energy taken at the products state:31where de = 107.75 kcal / mol is the dissociation energy , req = 1.09 is the equilibrium bond length , and a0 and a1 are the first two parameters of the pes model . the bending energy is modeled by a simple cosine function , with a barrier height equal to the dissociation energy at the angle xch * = /2:32finally the pes also accounts for the spherical repulsion between the passive hydrogen atoms hi and h*:33where hh = 2.4 with a2 and a3 being the last two parameters , and h*0 is the corresponding position of h * on the mep ( at the same c h * separation ) . the pes is the sum of these three contributions and is defined through the four parameters ai , i = 0 , ... , 3:34 the geometries used to compute the electronic energies are defined by the z - matrix presented in table 3 with x = = 10 ( in order to preserve a nonambiguous definition of the c3 axis ) . the comparison between the pes model and the direct ab initio data is shown in figure 11 for out - of - mep situations . the 95% ci ( [ k ] ) and the mean ( < k > ) of the posterior high - pressure reaction are presented in figure 12 with the available experimental measurements and the vtst - vrc theoretical predictions presented by klippenstein et al . have shown that by using both vtst - vrc and a direct dynamic method , the spherical ds for the ch3 + h ch4 reaction is associated with a 9% overestimation , approximately independent of the temperature . in other works , the overestimation of the reaction rate coming from the canonical analysis is probably compensated to some extent by the pes model which predicts a slightly higher hindrance effect at high separation ( the ab initio data are close to the bottom boundary of the ci at the s = 2.8 case in figure 11 ) . we recall that our model , even if associated with a 1.5 uncertainty factor on the reaction rate , is based on 4 parameters and has been set using 25 ab initio calculations for the calibration step ( and 25 to examine the extrapolation capabilities ) . comparison of the high pressure ch3 + h recombination rate predicted by this work ( black lines , [ k ] ; , < k > ) with other theoretical calculations ( gray lines ) and the experimental values ( + , * , ) . we have presented in this work an original approach for the computation of statistical properties of molecular systems involving a large amplitude motion . the objectives were to propose : ( i ) a simple and general procedure to compute the kinetic energy of a lam ; ( ii ) a general procedure to calibrate an analytical pes using ab initio data ; and ( iii ) a rigorous quantification of the uncertainty of the pes model and their propagation to the qoi(s ) . two typical and different test cases have been considered for assessing the methodology : ( i ) the study of 1,3-butadiene , involving nontrivial features , such as coupling of modes or a highly variable kinetic function ; and ( ii ) the study of the ch3 + h recombination , for which a vtst - vrc approach is needed to compute an accurate reaction rate . indeed , for a typical lam , the internal z - matrix coordinates naturally describe the configurational space of the motion and are much more suited than cartesian coordinates . the most important one , which is critical to the derivation of analytical models , is that the pes model uncertainty is properly evaluated and can be propagated to the qoi . for instance , in the study of the ch3 + h recombination rate , we were able to use only 25 data points to calibrate the 3d pes in order to compute a reaction rate within an uncertainty factor of 1.5 ( coming from the pes model ) .

attendance at emergency departments and unplanned hospital readmissions are common for frail older patients after discharge from the hospital.1 patients with chronic illnesses frequently perceived powerlessness in managing their disease after hospital discharge , and they are prompted to seek hospital readmission immediately upon symptom exacerbation.2 unplanned readmission is usually defined as readmission to the hospital within 28 days postdischarge.3,4 in hong kong , it was estimated that the unplanned readmission rate was 16.7% in the general population,3 and more than 20% in the elderly subpopulation.4 the hong kong hospital authority has developed a 14-variable predictive model the hospital administration risk reduction programme for the elderly ( harrpe ) to predict the risk of emergency medical admissions in 28 days after an index hospital visit among elderly patients aged 65 years or above.4 an elderly patient with a harrpe score of 0.2058 was considered as having a high risk of emergency admission . according to the unpublished statistical record from the medical unit of six hospitals under the operation of the hospital authority in 2009 , the average length of stay for patients with a harrpe score of 0.4 is 23 days , which is much higher than the corresponding figure of 5.9 days in the hong kong general population.3 currently , patients are provided with a community nursing service after hospital discharge in hong kong ; it is , therefore , essential to develop effective intervention programs to help prevent unplanned readmission among those elderly patients who are at high risk . the concept of virtual ward is a new model of care that was pioneered in 2004 in the uk.5,6 the aim is to provide clients at high risk of hospital readmission with intensive multidisciplinary and coordinated extensive services so as to facilitate their receiving the necessary care in their own homes , thereby reducing hospital readmissions . since the concept of virtual ward employs the systems , staffing , and daily routine of a hospital ward to deliver care to clients in their home settings,6 the virtual ward does not have a physical ward building . patients admitted to the virtual ward are visited by community nurses for the delivery of routine nursing care normally provided in the hospital , such as measuring their blood sugar levels , administering insulin , and performing wound dressing , at their homes . physicians and other allied health care professionals may also be involved , depending on the patient s health condition.5 evidence supporting the effectiveness of the virtual ward service was reported in the literature . the hospital - at - home care resulted in a reduction in the length of hospitalization compared to inpatient care in a systematic review,7 and it was shown to have a better effect in reducing emergency visits in comparison with usual home care visits in patients with acute or complex conditions requiring care for an anticipated 510 days.8 a 7-year longitudinal study on a large cohort of patients receiving acute hospital services at home reported low rates of unexpected mortality ( 0.15% ) and unplanned returns to hospital ( 4.2%).9 previous studies also supported that patient satisfaction was greater with virtual ward care,6,8 and potential improvements in quality of life ( qol ) , depression , and nutritional status in elderly patients with chronic heart failure have also been reported.10 the greater satisfaction might be attributed to a more personal style of care and a feeling that staying at home was therapeutic.11 the purpose of this study is to evaluate the effectiveness of the virtual ward service as compared to community nursing care on health services utilization and the qol of patients who are at extremely high risk for emergency readmission . the study was conducted from march 2012january 2013 at the kowloon and new territories west clusters under the catchment areas of community nursing services of four regional hospitals of the hong kong hospital authority . subjects were eligible if they had been discharged from hospital , had a harrpe score 0.4 , or had a major functional disability as clinically indicated , and were being supported by home carers who were living with the patients . the study was approved by the institutional review board of kowloon east cluster , kowloon central cluster , kowloon west cluster , and new territories west cluster of hong kong hospital authority . a total of 40 pairs of patients and their carers recruiting from three of the four participating hospitals were assigned to the intervention group , while another 40 matched patient carer dyads recruiting from the remaining hospital were assigned to the control group . carer dyads in the control group ; these included : 1 ) disease type of the patient ; 2 ) frailty level of the patient as measured by the validated clinical frailty index;12,13 3 ) sex of the patient ; 4 ) age of the patient ( 5 years ) ; and 5 ) the carer s relationship with the patient ( maid / spouse / children / relatives ) . this sample size is considered to be adequate for estimating a between - group difference for the pilot study.14 eligible patients were admitted to the three selected hospitals delivering the virtual ward service and their carers were invited to participate in the study . the screening of the subjects was performed by virtual ward nurses at the participating hospitals . the research assistant of the project then conducted the baseline assessment to both the patient and the carer in the intervention group at home before the implementation of the service by the virtual ward team . carer dyad in the intervention group , a matched control dyad was then selected from the fourth hospital by using the above five matching criteria by the nurses in the hospital . the research assistant then administered the baseline assessment to the consented matched control at their homes . participants in the intervention group received the virtual ward service while those in the control group received the usual community nursing service provided to patients discharged from hospitals in hong kong . at 3 months or discharge from the virtual ward ( whichever is earlier ) , a follow - up survey using a standardized questionnaire via face - to - face interviews was administered to both the patients and their carers . three measures that are related to services utilization of the emergency department were reported in the current study : length of the hospitalization via emergency department ; number of unplanned emergency hospital admissions ; and number of emergency attendances in the past 90 days . the patient s qol was measured by the locally validated instrument , namely , the chinese version modified quality - of - life concerns in the end of life questionnaire ( mqolc - e).15,16 the scale consists of 23 items measuring the six dimensions of : 1 ) negative emotions ; 2 ) physical discomfort ; 3 ) value of life ; 4 ) existential distress ; 5 ) care and support ; and 6 ) food - related concerns , on a 4-point likert scale , and the scores ranged from 1 , the least satisfaction to 4 , the most satisfaction toward the condition , with higher scores indicating a better qol . modifications were made to two items on the care and support concern subscale of the instrument regarding perception of the care the patient received . in particular , the two items originally asking the respondents to rate the care they received in hospital have been changed to the care they are currently receiving ( ie , the virtual ward service for subjects in the intervention group and the usual community nursing care in the control group ) . aggregate scores were created for the overall score and each of the six dimensions of the scale by averaging the corresponding items . cronbach s alpha value for the overall score was 0.835 , and those for the six dimensions ranged from 0.791 for value of life to 0.879 for negative emotions in the current sample . the patients in the intervention group received the service provided by the virtual ward health care team that consisted of nurses and physicians in the respective cluster . the virtual ward service focused on the provision of hospital - level care to patients and health care supports to their carers at their homes . patients were then individually assigned to a virtual ward nurse ( primary nurse ) ; and the virtual ward nurse conducted the first home visit as soon as possible within 48 hours after the patient was discharged from the hospital . the virtual ward physician might conduct a physician home visit within the first week after the patient was discharged . based on the health assessment results , the virtual ward nurses planned the schedule of home visits which could range from daily to once every 2 weeks . during the home visits , the virtual ward nurses provided direct patient care and health education , such as monitoring and handling symptom exacerbation , to the patients and their carers . the virtual ward nurses also provided psychosocial supports to both the patients and their carers . the schedule of home visits was individualized , and each patient was visited about four times per week on average during the study . in addition , an extended service beyond office hours and a hotline consultation service were also provided for the patients and their carers by the virtual ward teams . this additional service aims to enable the patients to have fast - track medical consultations by establishing various fast - track services , such as fast - track clinic , direct clinical admission , and enhanced nonemergency ambulance transportation service support . the frequency of calls to the hotline was reported to be at most three times in a week during the study . patients in the control group received community nursing service , referred by the physician in charge , that was based on the patients specific nursing care needs postdischarge ; this included wound dressing , catheterization , and health education for the patients self - care of their chronic diseases . descriptive statistics , including frequencies and percentages for categorical data and means and standard deviations for continuous data , were calculated for the baseline data of the sample . normality of data was checked by using z - tests for skewness and kurtosis.17 paired t - tests for normal data and wilcoxon signed - rank tests for nonnormal data were used to compare the differences in the changes of the outcome variables between the two groups using all available data . we also conducted a sensitivity analysis for hospital service utilization by excluding those patients who had died during the study . all statistical tests were two - sided , and a p - value < 0.05 was considered statistically significant . all the analyses were performed by using spss version 20.0 ( ibm corporation , armonk , ny , usa ) . the study was conducted from march 2012january 2013 at the kowloon and new territories west clusters under the catchment areas of community nursing services of four regional hospitals of the hong kong hospital authority . subjects were eligible if they had been discharged from hospital , had a harrpe score 0.4 , or had a major functional disability as clinically indicated , and were being supported by home carers who were living with the patients . the study was approved by the institutional review board of kowloon east cluster , kowloon central cluster , kowloon west cluster , and new territories west cluster of hong kong hospital authority . a total of 40 pairs of patients and their carers recruiting from three of the four participating hospitals were assigned to the intervention group , while another 40 matched patient carer dyads recruiting from the remaining hospital were assigned to the control group . carer dyads in the control group ; these included : 1 ) disease type of the patient ; 2 ) frailty level of the patient as measured by the validated clinical frailty index;12,13 3 ) sex of the patient ; 4 ) age of the patient ( 5 years ) ; and 5 ) the carer s relationship with the patient ( maid / spouse / children / relatives ) . this sample size is considered to be adequate for estimating a between - group difference for the pilot study.14 eligible patients were admitted to the three selected hospitals delivering the virtual ward service and their carers were invited to participate in the study . the screening of the subjects was performed by virtual ward nurses at the participating hospitals . the research assistant of the project then conducted the baseline assessment to both the patient and the carer in the intervention group at home before the implementation of the service by the virtual ward team . carer dyad in the intervention group , a matched control dyad was then selected from the fourth hospital by using the above five matching criteria by the nurses in the hospital . the research assistant then administered the baseline assessment to the consented matched control at their homes . participants in the intervention group received the virtual ward service while those in the control group received the usual community nursing service provided to patients discharged from hospitals in hong kong . at 3 months or discharge from the virtual ward ( whichever is earlier ) , a follow - up survey using a standardized questionnaire via face - to - face interviews was administered to both the patients and their carers . three measures that are related to services utilization of the emergency department were reported in the current study : length of the hospitalization via emergency department ; number of unplanned emergency hospital admissions ; and number of emergency attendances in the past 90 days . the patient s qol was measured by the locally validated instrument , namely , the chinese version modified quality - of - life concerns in the end of life questionnaire ( mqolc - e).15,16 the scale consists of 23 items measuring the six dimensions of : 1 ) negative emotions ; 2 ) physical discomfort ; 3 ) value of life ; 4 ) existential distress ; 5 ) care and support ; and 6 ) food - related concerns , on a 4-point likert scale , and the scores ranged from 1 , the least satisfaction to 4 , the most satisfaction toward the condition , with higher scores indicating a better qol . modifications were made to two items on the care and support concern subscale of the instrument regarding perception of the care the patient received . in particular , the two items originally asking the respondents to rate the care they received in hospital have been changed to the care they are currently receiving ( ie , the virtual ward service for subjects in the intervention group and the usual community nursing care in the control group ) . aggregate scores were created for the overall score and each of the six dimensions of the scale by averaging the corresponding items . cronbach s alpha value for the overall score was 0.835 , and those for the six dimensions ranged from 0.791 for value of life to 0.879 for negative emotions in the current sample . the patients in the intervention group received the service provided by the virtual ward health care team that consisted of nurses and physicians in the respective cluster . the virtual ward service focused on the provision of hospital - level care to patients and health care supports to their carers at their homes . patients were then individually assigned to a virtual ward nurse ( primary nurse ) ; and the virtual ward nurse conducted the first home visit as soon as possible within 48 hours after the patient was discharged from the hospital . the virtual ward physician might conduct a physician home visit within the first week after the patient was discharged . based on the health assessment results , the virtual ward nurses planned the schedule of home visits which could range from daily to once every 2 weeks . during the home visits , the virtual ward nurses provided direct patient care and health education , such as monitoring and handling symptom exacerbation , to the patients and their carers . the virtual ward nurses also provided psychosocial supports to both the patients and their carers . the schedule of home visits was individualized , and each patient was visited about four times per week on average during the study . in addition , an extended service beyond office hours and a hotline consultation service were also provided for the patients and their carers by the virtual ward teams . this additional service aims to enable the patients to have fast - track medical consultations by establishing various fast - track services , such as fast - track clinic , direct clinical admission , and enhanced nonemergency ambulance transportation service support . the frequency of calls to the hotline was reported to be at most three times in a week during the study . patients in the control group received community nursing service , referred by the physician in charge , that was based on the patients specific nursing care needs postdischarge ; this included wound dressing , catheterization , and health education for the patients self - care of their chronic diseases . descriptive statistics , including frequencies and percentages for categorical data and means and standard deviations for continuous data , were calculated for the baseline data of the sample . normality of data was checked by using z - tests for skewness and kurtosis.17 paired t - tests for normal data and wilcoxon signed - rank tests for nonnormal data were used to compare the differences in the changes of the outcome variables between the two groups using all available data . we also conducted a sensitivity analysis for hospital service utilization by excluding those patients who had died during the study . all statistical tests were two - sided , and a p - value < 0.05 was considered statistically significant . all the analyses were performed by using spss version 20.0 ( ibm corporation , armonk , ny , usa ) . carer dyads in the intervention group and 46 dyads in the control group were invited to participate in the study . for the intervention group , 13 dyads refused , patients in four dyads were readmitted to the hospital , and 32 dyads were excluded because they were unable to be matched with a control dyad . for the control group , six dyads refused to join the study . one dyad in the intervention group withdrew their consents before receiving the virtual ward service , because the patient was expecting to frequently travel to the people s republic of china in the near future and , therefore , could not receive the service . at 3 months , 27 patients in the intervention group and 33 patients in the control group were successfully followed - up . among patients who were lost to follow - up , eleven died and one refused to complete the questionnaire in the intervention group , and six died and one was in a vegetative stage in the control group . thus , a total of 39 matched patient pairs in the two groups were available for the analysis on hospital services utilization . for the qol , due to loss to follow - up and missing data at the item level , the results reported in tables 2 and 3 revealed that the intervention group showed a significantly greater reduction in the number of unplanned emergency hospital admissions ( 1.411.23 versus 0.771.31 ; p=0.049 ) . although there were no significant differences in both the length of hospitalization via emergency admission ( 11.6217.91 versus 4.3826.41 ; p=0.14 ) and the number of emergency attendances ( 1.511.25 versus 1.081.48 ; p=0.29 ) in the past 90 days between the two groups , the decreases in these two clinical measures were greater in the intervention group ( table 2 ) . sensitivity analysis by excluding those patients who had died during the study period also gives similar results on the three clinical outcomes ( table 3 ) . for qol , patients receiving the virtual ward service reported higher mean values in the changes of the overall scores and the scores in all the six dimensions of mqolc - e from the baseline to the follow - up than patients receiving the community nursing service ( table 4 ) . significant differences were observed in the overall qol scores ( 0.600.56 versus 0.070.56 ; p=0.02 ) and the three dimensions of food - related concerns ( 0.820.87 versus 0.140.95 ; p=0.003 ) , negative emotions ( 0.730.74 versus 0.021.03 ; p=0.01 ) , and existential distress ( 0.721.06 versus 0.150.81 ; p=0.04 ) . our pilot study shows that for frail patients with chronic diseases , the virtual ward service , with physicians and nurses undertaking visits to the patient s home , results in a significantly greater reduction in the number of admissions via emergency admissions as compared to matched controls receiving the usual community nursing service . the result on frequency of admission via emergency admission remained significant in the sensitivity analysis by excluding those patients who died during the study period . although non - significant , the reductions in length of stay via emergency admission and emergency attendance were also greater in the intervention group . consistent with a previous study , our study also showed that health care provided at home by a multidisciplinary team involving physicians , nurses , and carers is effective in reducing emergency visits than is the usual care provided by community nurses and carers.8 compared to a previous local randomized controlled trial ( rct ) which examined the effectiveness of nursing home visits driven by a protocol which used the omaha scheme and the standard community nursing service,18 our subjects were older and frailer , and they had to be taken care of , which means that they are expected to be at a higher risk of emergency hospitalization . their study showed that home visits driven by a protocol that used the omaha scheme had a similar effect as the basic community nursing service on the frequency of hospitalization . the current results highlight the potentially important role of the main feature of targeting those patients who are at high risk of emergency hospitalization in the virtual ward services , which distinguishes it from other programs designed to prevent hospitalization . our study had provided new evidence for the efficacy of the virtual ward service , as compared to home visits by community nurses , in reducing the number of readmissions . this suggests that providing a more intensive care at home by a team of multidisciplinary health care professionals might be more effective than a low intervention dose , ie , home visits delivered by community nurses only for frail older patients.18,19 this observation also echoed the views that patients with chronic , functional , and relatively intractable health problems require a higher level of intervention.20 however , further studies using an rct with adequate power on testing the effectiveness of the virtual ward service on the target population are warranted . the mortality rate in the intervention group was higher than that in the control group , although the difference was not significant ( mcnemar test , p=0.27 ) . it is possible that some of the patients were at their end stage of life and that the patients as well as their carers might prefer to stay at home and forgo life - sustaining treatment.21 although the accessibility of hospitals and the low cost for a hospital pay in hong kong might be important factors for unplanned readmission,16 both patients and their carers may not opt for this option unless necessary . hospitalization inevitably induces stress both to patients and their family carers.22,23 repeated emergency hospital admissions are also a significant and disruptive event for families , because it may herald the possibility of an uncertain future and anticipated loss to both the families and the patients.24 thus , it is possible that the frail patients or their carers may feel that it is safer for the patients to stay in the hospital if their conditions are out of control even though both of them may not be well - prepared for such readmission.25 furthermore , many forms of care and support , particularly comfort measures like feeding and bathing , that family members normally provide to older adults with multiple comorbidities or impaired functional ability are to be continued but in a hospital setting.24 this situation is especially true in chinese society , because the chinese have a strong sense of moral obligation to take care of a sick family member.26 hence , it is very important to manage the patients condition by providing timely professional advice , such as a change of medication prescription , so that some emergency admissions could be avoided . the close monitoring of the patients health condition and the prompt adjustment of medication orders are risk factors of readmission27 that can be remedied by better health care coordination , such as that provided by the virtual ward service . patients receiving the virtual ward service reported greater improvements in the overall score and all the six dimension scores in qol as compared to patients receiving the community nursing service ; this is in line with the findings from a previous study on elderly patients with chronic heart failure.10 in particular , the changes in the overall qol score and the three dimensions of food - related concerns , negative emotions , and existential distress were statistically significant between the two groups . the results might suggest that the virtual ward service may exert positive effects on the qol of the patients by providing individualized care , health education , and emotional support to the patients and their carers so that the conditions of the patients can be better monitored . however , the results from our nonrandomized matched control study should be interpreted with caution because of potential bias due to patients loss to follow - ups . first , this is a pilot study , and it may have produced imprecise estimates of the differences in the outcome variables between the groups . nevertheless , our study has produced an estimate of the effect size of the virtual ward service to the usual community nursing service for future studies . second , although we have successfully recruited matched controls by using the five criteria for comparison , it is still possible that we have not controlled other possible confounders , such as the characteristics of the carers and the study site , and this might have induced bias into the study results . however , we were not able to recruit control subjects in the experimental hospitals due to the time constraint of this pilot study , and the small sample size precludes the inclusion of the carers characteristics in the analysis . further studies should consider using rct with a larger sample size , to examine the effectiveness of the virtual ward service in the target population . third , we have high attrition rates in patients in both groups , and this might have introduced bias in the results of qol . fourth , we have a mixture of disease - specific patient groups including chronic obstructive pulmonary disease ( copd ) , chronic heart failure , cancer , and other diseases in our study . each patient group might require different types of interventions at home , and some of the required interventions might not be covered by the virtual ward service . thus , it may be possible that the impact of the service might differ in accordance with the disease types of the patients . given the encouraging results obtained in this pilot study , it is worthwhile to further investigate the effect of the virtual ward service on patients with some specific diseases , such as copd and chronic heart failure , who are at high risk of emergency hospitalization individually . finally , we did not collect cost information of the virtual ward and the usual community nursing service for this pilot study due to time and financial constraints . because the virtual ward service is still available at the three experimental hospitals , one of the main focuses of further studies should be on the cost - effectiveness of the service as compared to the community nursing care . supported by the results of this pilot study , the virtual ward service is effective in reducing unplanned hospital readmissions and improving the patients qol . it is also potentially effective in reducing the patients length of stay via emergency admissions and emergency attendances . these results support the potential benefits of virtual ward service for frail elderly patients who live with their carers .

introductionattendance at emergency departments and unplanned hospital readmissions are common for frail older patients after discharge from hospitals . a virtual ward service was piloted to deliver hospital - at - home services by community nurses and geriatricians to frail older patients immediately after their discharge from hospital to reduce emergency services utilization.objectivesthis study examined the impacts of the virtual ward service on changes in the patients emergency attendance and medical readmissions , and their quality of life ( qol).methodsa matched - control quasi - experimental study was conducted at four hospitals , with three providing the virtual ward service ( intervention ) and one providing the usual community nursing care ( control ) . subjects in the intervention group were those who are at high risk of readmission and who are supported by home carers recruited from the three hospitals providing the virtual ward service . matched control patients were those recruited from the hospital providing usual care . outcome measures include emergency attendance and medical readmission in the past 90 days as identified from medical records , and patient - reported qol as measured by the modified quality - of - life concerns in the end of life questionnaire ( chinese version ) . wilcoxon signed - rank tests compared the changes in the outcome variables between groups.resultsa total of 39 patients in each of the two groups were recruited . the virtual ward group showed a greater significant reduction in the number of unplanned emergency hospital readmissions ( 1.411.23 versus 0.771.31 ; p=0.049 ) and a significant improvement in their overall qol ( n=18 ; 0.600.56 versus 0.070.56 ; p=0.02 ) , but there was no significant difference in the number of emergency attendances ( 1.511.25 versus 1.081.48 ; p=0.29).conclusionthe study results support the effectiveness of the virtual ward service in reducing unplanned emergency medical readmissions and in improving the qol in frail older patients after discharge .

Introduction Materials and methods Subjects Measures Virtual ward program Statistical analysis Results Discussion Conclusion

attendance at emergency departments and unplanned hospital readmissions are common for frail older patients after discharge from the hospital.1 patients with chronic illnesses frequently perceived powerlessness in managing their disease after hospital discharge , and they are prompted to seek hospital readmission immediately upon symptom exacerbation.2 unplanned readmission is usually defined as readmission to the hospital within 28 days postdischarge.3,4 in hong kong , it was estimated that the unplanned readmission rate was 16.7% in the general population,3 and more than 20% in the elderly subpopulation.4 the hong kong hospital authority has developed a 14-variable predictive model the hospital administration risk reduction programme for the elderly ( harrpe ) to predict the risk of emergency medical admissions in 28 days after an index hospital visit among elderly patients aged 65 years or above.4 an elderly patient with a harrpe score of 0.2058 was considered as having a high risk of emergency admission . according to the unpublished statistical record from the medical unit of six hospitals under the operation of the hospital authority in 2009 , the average length of stay for patients with a harrpe score of 0.4 is 23 days , which is much higher than the corresponding figure of 5.9 days in the hong kong general population.3 currently , patients are provided with a community nursing service after hospital discharge in hong kong ; it is , therefore , essential to develop effective intervention programs to help prevent unplanned readmission among those elderly patients who are at high risk . the concept of virtual ward is a new model of care that was pioneered in 2004 in the uk.5,6 the aim is to provide clients at high risk of hospital readmission with intensive multidisciplinary and coordinated extensive services so as to facilitate their receiving the necessary care in their own homes , thereby reducing hospital readmissions . since the concept of virtual ward employs the systems , staffing , and daily routine of a hospital ward to deliver care to clients in their home settings,6 the virtual ward does not have a physical ward building . patients admitted to the virtual ward are visited by community nurses for the delivery of routine nursing care normally provided in the hospital , such as measuring their blood sugar levels , administering insulin , and performing wound dressing , at their homes . physicians and other allied health care professionals may also be involved , depending on the patient s health condition.5 evidence supporting the effectiveness of the virtual ward service was reported in the literature . the hospital - at - home care resulted in a reduction in the length of hospitalization compared to inpatient care in a systematic review,7 and it was shown to have a better effect in reducing emergency visits in comparison with usual home care visits in patients with acute or complex conditions requiring care for an anticipated 510 days.8 a 7-year longitudinal study on a large cohort of patients receiving acute hospital services at home reported low rates of unexpected mortality ( 0.15% ) and unplanned returns to hospital ( 4.2%).9 previous studies also supported that patient satisfaction was greater with virtual ward care,6,8 and potential improvements in quality of life ( qol ) , depression , and nutritional status in elderly patients with chronic heart failure have also been reported.10 the greater satisfaction might be attributed to a more personal style of care and a feeling that staying at home was therapeutic.11 the purpose of this study is to evaluate the effectiveness of the virtual ward service as compared to community nursing care on health services utilization and the qol of patients who are at extremely high risk for emergency readmission . subjects were eligible if they had been discharged from hospital , had a harrpe score 0.4 , or had a major functional disability as clinically indicated , and were being supported by home carers who were living with the patients . a total of 40 pairs of patients and their carers recruiting from three of the four participating hospitals were assigned to the intervention group , while another 40 matched patient carer dyads recruiting from the remaining hospital were assigned to the control group . carer dyads in the control group ; these included : 1 ) disease type of the patient ; 2 ) frailty level of the patient as measured by the validated clinical frailty index;12,13 3 ) sex of the patient ; 4 ) age of the patient ( 5 years ) ; and 5 ) the carer s relationship with the patient ( maid / spouse / children / relatives ) . this sample size is considered to be adequate for estimating a between - group difference for the pilot study.14 eligible patients were admitted to the three selected hospitals delivering the virtual ward service and their carers were invited to participate in the study . the research assistant of the project then conducted the baseline assessment to both the patient and the carer in the intervention group at home before the implementation of the service by the virtual ward team . carer dyad in the intervention group , a matched control dyad was then selected from the fourth hospital by using the above five matching criteria by the nurses in the hospital . participants in the intervention group received the virtual ward service while those in the control group received the usual community nursing service provided to patients discharged from hospitals in hong kong . at 3 months or discharge from the virtual ward ( whichever is earlier ) , a follow - up survey using a standardized questionnaire via face - to - face interviews was administered to both the patients and their carers . three measures that are related to services utilization of the emergency department were reported in the current study : length of the hospitalization via emergency department ; number of unplanned emergency hospital admissions ; and number of emergency attendances in the past 90 days . the patient s qol was measured by the locally validated instrument , namely , the chinese version modified quality - of - life concerns in the end of life questionnaire ( mqolc - e).15,16 the scale consists of 23 items measuring the six dimensions of : 1 ) negative emotions ; 2 ) physical discomfort ; 3 ) value of life ; 4 ) existential distress ; 5 ) care and support ; and 6 ) food - related concerns , on a 4-point likert scale , and the scores ranged from 1 , the least satisfaction to 4 , the most satisfaction toward the condition , with higher scores indicating a better qol . in particular , the two items originally asking the respondents to rate the care they received in hospital have been changed to the care they are currently receiving ( ie , the virtual ward service for subjects in the intervention group and the usual community nursing care in the control group ) . the patients in the intervention group received the service provided by the virtual ward health care team that consisted of nurses and physicians in the respective cluster . patients were then individually assigned to a virtual ward nurse ( primary nurse ) ; and the virtual ward nurse conducted the first home visit as soon as possible within 48 hours after the patient was discharged from the hospital . in addition , an extended service beyond office hours and a hotline consultation service were also provided for the patients and their carers by the virtual ward teams . patients in the control group received community nursing service , referred by the physician in charge , that was based on the patients specific nursing care needs postdischarge ; this included wound dressing , catheterization , and health education for the patients self - care of their chronic diseases . normality of data was checked by using z - tests for skewness and kurtosis.17 paired t - tests for normal data and wilcoxon signed - rank tests for nonnormal data were used to compare the differences in the changes of the outcome variables between the two groups using all available data . subjects were eligible if they had been discharged from hospital , had a harrpe score 0.4 , or had a major functional disability as clinically indicated , and were being supported by home carers who were living with the patients . a total of 40 pairs of patients and their carers recruiting from three of the four participating hospitals were assigned to the intervention group , while another 40 matched patient carer dyads recruiting from the remaining hospital were assigned to the control group . carer dyads in the control group ; these included : 1 ) disease type of the patient ; 2 ) frailty level of the patient as measured by the validated clinical frailty index;12,13 3 ) sex of the patient ; 4 ) age of the patient ( 5 years ) ; and 5 ) the carer s relationship with the patient ( maid / spouse / children / relatives ) . this sample size is considered to be adequate for estimating a between - group difference for the pilot study.14 eligible patients were admitted to the three selected hospitals delivering the virtual ward service and their carers were invited to participate in the study . the research assistant of the project then conducted the baseline assessment to both the patient and the carer in the intervention group at home before the implementation of the service by the virtual ward team . carer dyad in the intervention group , a matched control dyad was then selected from the fourth hospital by using the above five matching criteria by the nurses in the hospital . participants in the intervention group received the virtual ward service while those in the control group received the usual community nursing service provided to patients discharged from hospitals in hong kong . at 3 months or discharge from the virtual ward ( whichever is earlier ) , a follow - up survey using a standardized questionnaire via face - to - face interviews was administered to both the patients and their carers . three measures that are related to services utilization of the emergency department were reported in the current study : length of the hospitalization via emergency department ; number of unplanned emergency hospital admissions ; and number of emergency attendances in the past 90 days . the patient s qol was measured by the locally validated instrument , namely , the chinese version modified quality - of - life concerns in the end of life questionnaire ( mqolc - e).15,16 the scale consists of 23 items measuring the six dimensions of : 1 ) negative emotions ; 2 ) physical discomfort ; 3 ) value of life ; 4 ) existential distress ; 5 ) care and support ; and 6 ) food - related concerns , on a 4-point likert scale , and the scores ranged from 1 , the least satisfaction to 4 , the most satisfaction toward the condition , with higher scores indicating a better qol . in particular , the two items originally asking the respondents to rate the care they received in hospital have been changed to the care they are currently receiving ( ie , the virtual ward service for subjects in the intervention group and the usual community nursing care in the control group ) . the patients in the intervention group received the service provided by the virtual ward health care team that consisted of nurses and physicians in the respective cluster . patients were then individually assigned to a virtual ward nurse ( primary nurse ) ; and the virtual ward nurse conducted the first home visit as soon as possible within 48 hours after the patient was discharged from the hospital . during the home visits , the virtual ward nurses provided direct patient care and health education , such as monitoring and handling symptom exacerbation , to the patients and their carers . the virtual ward nurses also provided psychosocial supports to both the patients and their carers . in addition , an extended service beyond office hours and a hotline consultation service were also provided for the patients and their carers by the virtual ward teams . patients in the control group received community nursing service , referred by the physician in charge , that was based on the patients specific nursing care needs postdischarge ; this included wound dressing , catheterization , and health education for the patients self - care of their chronic diseases . normality of data was checked by using z - tests for skewness and kurtosis.17 paired t - tests for normal data and wilcoxon signed - rank tests for nonnormal data were used to compare the differences in the changes of the outcome variables between the two groups using all available data . carer dyads in the intervention group and 46 dyads in the control group were invited to participate in the study . for the intervention group , 13 dyads refused , patients in four dyads were readmitted to the hospital , and 32 dyads were excluded because they were unable to be matched with a control dyad . one dyad in the intervention group withdrew their consents before receiving the virtual ward service , because the patient was expecting to frequently travel to the people s republic of china in the near future and , therefore , could not receive the service . at 3 months , 27 patients in the intervention group and 33 patients in the control group were successfully followed - up . among patients who were lost to follow - up , eleven died and one refused to complete the questionnaire in the intervention group , and six died and one was in a vegetative stage in the control group . thus , a total of 39 matched patient pairs in the two groups were available for the analysis on hospital services utilization . for the qol , due to loss to follow - up and missing data at the item level , the results reported in tables 2 and 3 revealed that the intervention group showed a significantly greater reduction in the number of unplanned emergency hospital admissions ( 1.411.23 versus 0.771.31 ; p=0.049 ) . although there were no significant differences in both the length of hospitalization via emergency admission ( 11.6217.91 versus 4.3826.41 ; p=0.14 ) and the number of emergency attendances ( 1.511.25 versus 1.081.48 ; p=0.29 ) in the past 90 days between the two groups , the decreases in these two clinical measures were greater in the intervention group ( table 2 ) . for qol , patients receiving the virtual ward service reported higher mean values in the changes of the overall scores and the scores in all the six dimensions of mqolc - e from the baseline to the follow - up than patients receiving the community nursing service ( table 4 ) . significant differences were observed in the overall qol scores ( 0.600.56 versus 0.070.56 ; p=0.02 ) and the three dimensions of food - related concerns ( 0.820.87 versus 0.140.95 ; p=0.003 ) , negative emotions ( 0.730.74 versus 0.021.03 ; p=0.01 ) , and existential distress ( 0.721.06 versus 0.150.81 ; p=0.04 ) . our pilot study shows that for frail patients with chronic diseases , the virtual ward service , with physicians and nurses undertaking visits to the patient s home , results in a significantly greater reduction in the number of admissions via emergency admissions as compared to matched controls receiving the usual community nursing service . consistent with a previous study , our study also showed that health care provided at home by a multidisciplinary team involving physicians , nurses , and carers is effective in reducing emergency visits than is the usual care provided by community nurses and carers.8 compared to a previous local randomized controlled trial ( rct ) which examined the effectiveness of nursing home visits driven by a protocol which used the omaha scheme and the standard community nursing service,18 our subjects were older and frailer , and they had to be taken care of , which means that they are expected to be at a higher risk of emergency hospitalization . the current results highlight the potentially important role of the main feature of targeting those patients who are at high risk of emergency hospitalization in the virtual ward services , which distinguishes it from other programs designed to prevent hospitalization . our study had provided new evidence for the efficacy of the virtual ward service , as compared to home visits by community nurses , in reducing the number of readmissions . this suggests that providing a more intensive care at home by a team of multidisciplinary health care professionals might be more effective than a low intervention dose , ie , home visits delivered by community nurses only for frail older patients.18,19 this observation also echoed the views that patients with chronic , functional , and relatively intractable health problems require a higher level of intervention.20 however , further studies using an rct with adequate power on testing the effectiveness of the virtual ward service on the target population are warranted . it is possible that some of the patients were at their end stage of life and that the patients as well as their carers might prefer to stay at home and forgo life - sustaining treatment.21 although the accessibility of hospitals and the low cost for a hospital pay in hong kong might be important factors for unplanned readmission,16 both patients and their carers may not opt for this option unless necessary . hospitalization inevitably induces stress both to patients and their family carers.22,23 repeated emergency hospital admissions are also a significant and disruptive event for families , because it may herald the possibility of an uncertain future and anticipated loss to both the families and the patients.24 thus , it is possible that the frail patients or their carers may feel that it is safer for the patients to stay in the hospital if their conditions are out of control even though both of them may not be well - prepared for such readmission.25 furthermore , many forms of care and support , particularly comfort measures like feeding and bathing , that family members normally provide to older adults with multiple comorbidities or impaired functional ability are to be continued but in a hospital setting.24 this situation is especially true in chinese society , because the chinese have a strong sense of moral obligation to take care of a sick family member.26 hence , it is very important to manage the patients condition by providing timely professional advice , such as a change of medication prescription , so that some emergency admissions could be avoided . the close monitoring of the patients health condition and the prompt adjustment of medication orders are risk factors of readmission27 that can be remedied by better health care coordination , such as that provided by the virtual ward service . patients receiving the virtual ward service reported greater improvements in the overall score and all the six dimension scores in qol as compared to patients receiving the community nursing service ; this is in line with the findings from a previous study on elderly patients with chronic heart failure.10 in particular , the changes in the overall qol score and the three dimensions of food - related concerns , negative emotions , and existential distress were statistically significant between the two groups . the results might suggest that the virtual ward service may exert positive effects on the qol of the patients by providing individualized care , health education , and emotional support to the patients and their carers so that the conditions of the patients can be better monitored . first , this is a pilot study , and it may have produced imprecise estimates of the differences in the outcome variables between the groups . nevertheless , our study has produced an estimate of the effect size of the virtual ward service to the usual community nursing service for future studies . however , we were not able to recruit control subjects in the experimental hospitals due to the time constraint of this pilot study , and the small sample size precludes the inclusion of the carers characteristics in the analysis . further studies should consider using rct with a larger sample size , to examine the effectiveness of the virtual ward service in the target population . each patient group might require different types of interventions at home , and some of the required interventions might not be covered by the virtual ward service . given the encouraging results obtained in this pilot study , it is worthwhile to further investigate the effect of the virtual ward service on patients with some specific diseases , such as copd and chronic heart failure , who are at high risk of emergency hospitalization individually . finally , we did not collect cost information of the virtual ward and the usual community nursing service for this pilot study due to time and financial constraints . because the virtual ward service is still available at the three experimental hospitals , one of the main focuses of further studies should be on the cost - effectiveness of the service as compared to the community nursing care . supported by the results of this pilot study , the virtual ward service is effective in reducing unplanned hospital readmissions and improving the patients qol . these results support the potential benefits of virtual ward service for frail elderly patients who live with their carers .

telomeres , unique structures at the physical ends of linear eukaryotic chromosomes , were first described almost 70 yearsago by hermann muller in his classic studies of the fruit fly drosophilia melanogaster ( 1 ) . he noted that chromosomal inversions resulting from ionizing radiation ( ir)-induced double - strand breaks ( dsbs ) never involved the very end of a chromosome rejoining with some other part of a chromosome . telomere , which comes from greek telos meaning end and meros meaning part based on this chromosome end protection phenomenon . shortly thereafter , barbara mcclintock observed that while broken ends of maize chromosome fused , forming dicentric chromosomes , unbroken chromosomes rarely fused ; i.e. natural chromosomal termini are not the absence of interstitial telomere sequence within ir - induced dicentrics was later verified in human cells ( 3 ) . these studies demonstrate that normally cells accurately distinguish telomeric ends from random dsb ends and protect the former from illegitimate end - joining reactions . how cells make this critical distinction continues to be an active area of research today , especially as the dividing lines between the two types of ends have become less , rather than more clear . recent discoveries that certain dsb repair proteins act to preserve rather than to join the natural ends of mammalian chromosomes ( 47 ) , have provided impetus for the union of two seemingly disparate scientific fields , dna repair and telomere biology . here , we focus on the creation of dysfunctional mammalian telomeres in various repair deficient backgrounds that result from either the loss of end - capping structure or the loss of terminal sequence ( shortening ) , and the consequences of this loss of function . telomeres serve multiple functions in preserving chromosome stability , including protecting the ends of chromosomes from degradation and preventing chromosomal end fusion . the dna component of telomeres consists of tandem arrays of short , repetitive g - rich sequence [ ttaggg in vertebrates , ( 8,9 ) ] , oriented 5-to-3 towards the end of the chromosome ( 10 ) , ending in an essential 3 single - stranded overhang that ranges in length from 50 to 400 nt ( 1113 ) . electron microscopy studies suggest this overhang can loop back and integrate into the duplex repeat tract , forming a t - loop ( 14 ) , an attractive , although not necessarily exclusive , architectural solution to the end - capping dilemma . telomeres gradually shorten in replicating cells due to end - processing and the end replication problem ( 1518 ) , so in order for continuous cell division to occur , they must be replenished . the addition of telomeric repeats de novo is accomplished by the reverse transcriptase telomerase ( 19,20 ) . thus , telomere length maintenance is a state of equilibrium between telomere loss and re - addition . precisely how telomere length regulation occurs is unknown , but it does appear that the shortest telomeres are preferentially targeted for elongation by telomerase ( 21 ) . telomere length is maintained in germ line cells , however , most human somatic cells do not express sufficient telomerase activity to prevent telomere loss as they divide . as a result , telomeres eventually shorten to the point where they initiate a cell cycle arrest in g1 , a state termed replicative senescence ( 22 ) . telomere shortening can , therefore , limit the number of times somatic cells divide , contributing not only to aging phenotypes , but also providing an effective tumor suppressor mechanism . cells that lose the ability to senesce because of mutations in p53 protein continue to divide , eventually entering , cells that constitutively express telomerase can continue to divide almost indefinitely ( 23,24 ) . consistent with the requirement for telomere maintenance as a step in carcinogenesis , most tumor cells express telomerase ( 25 ) . however , an alternative mechanism for telomere maintenance has also been described ( 26,27 ) , which is observed in some tumors ( 28 ) and involves recombination ( 26,29 ) . although telomerase is responsible for addition of telomeric sequence with every cell cycle , a plethora of other proteins also play important roles in the regulation of telomere length maintenance and in the formation of a protective end - cap that prevents chromosome fusion ( 30,31 ) . proteins that directly bind the double - stranded telomeric repeats include the ttaggg repeat factors trf1 ( 32,33 ) and trf2 ( 34,35 ) . pot1 ( protection of telomeres 1 ) specifically recognizes telomeric single - stranded dna , belongs to a family of oligosaccharide / oligonucleotide - binding ( ob)-fold - containing proteins and is highly conserved among eukaryotes ( 36,37 ) . telomere - associated proteins that do not bind dna directly include tin2 ( trf1-interacting nuclear protein 2 ) , which associates with trf1 ( 38 ) and trf2 ( 39,40 ) . the trf1 complex contains both tin2 and pot1 and acts to regulate telomere - length homeostasis . tpp1 , the recently proposed name ( 31 ) for a pot1-interacting protein identified independently in three laboratories [ tint1 ( 41 ) , ptop ( 42 ) and pip1 ( 43 ) ] , recruits pot1 to telomeres and so is also involved in telomere length regulation . rap1 ( human repressor activator protein ) ( 44 ) is recruited to telomeres by trf2 and has been shown to negatively regulate telomere length in vivo ( 45 ) . six proteins have been proposed to form an essential , dynamic complex at human telomeres : trf1 , trf2 , pot1 , tin2 , tpp1 and rap1 . this mammalian telomeric core complex serves to form and protect the telomere , and has been termed both the telosome ( 42 ) and alternately , shelterin ( 31 ) . other proteins , many of which are more commonly associated with dna repair , are also found at telomeric ends [ reviewed in ( 46 ) ] . examples include dna - pk ( ku70/ku86/dna - pkcs ) , the mrn complex ( mre11/rad50/nbs1 ) , parp1/2 , tankyrase 1/2 , atm , ercc1/xpf , rad51d , wrn and blm . trf2 has been shown to bind to atm , blocking a damage response at telomeres ( 47 ) . interestingly , it has recently been proposed that normally , functional human telomeres must be recognized as dna damage in the g2 phase of the cell cycle , in order to recruit the processing machinery necessary for formation of a functional telomere ( 48 ) . the interplay between telomeres and dsbs may be better understood not by viewing the striking differences between them , but instead by viewing the obvious similarity both are dna ends , the very substrate telomere and damage response / repair proteins specifically recognize and bind . functional telomeres are essential for continuous cellular proliferation , and therefore loss of chromosomal end - capping has consequences in both aging and carcinogenesis ( 49,50 ) . we have shown that effective end - capping of mammalian telomeres requires the non - homologous end - joining ( nhej ) protein dna - dependent protein kinase ( dna - pk ) ( 4 ) . mutation of any of the genes comprising dna - pk , i.e. ku70 , ku86 , or the catalytic subunit , dna - pkcs ( 51 ) , leads to spontaneous chromosomal end - to - end fusions that maintain large blocks of telomeric sequence at the points of fusion ( figure 1 ) . dna - pk has since been shown to associate with human telomeric dna in vivo ( 52,53 ) . events at the extreme terminus of the chromosome that normally serve to create a functionally protected telomere fail in the absence of dna - pk , resulting in inappropriate end - to - end fusion events of uncapped telomeres fusing not only to each other , but also to ir - induced dsbs ( 54 ) . we have also shown that the kinase activity of dna - pkcs is required for effective telomere protection , just as it is for nhej ( 55,56 ) . however , as with nhej , the critical in vivo substrates are not known . the in vivo consequences of inappropriate interstitial blocks of telomere sequence are also unknown , but they most likely affect chromatin stability , and in vitro studies have demonstrated they increase chromosomal instability ( 57 ) . we find that telomere fusions contribute significantly to the background level of chromosomal aberrations , and that they occur despite the presence of ample telomere sequence . thus , they are obviously not a consequence of telomere shortening , nor are they telomere associations [ defined as distinct telomere signals separated by less than approximately one - third the width of a chromatid , ( 58 ) ] . banding studies demonstrated that any chromosome could be involved in the telomere fusions ( end - capping failure is not chromosome specific ) , and further , that some of the telomere fusions were clonal , suggestive of covalent linkages ( s. bouffler , unpublished data ) . utilizing the strand - specific co - fish technique ( 59,60 ) , we found that the telomere fusions in dna - pkcs - deficient cells exclusively involved telomeres synthesized via leading - strand dna synthesis , suggesting a crucial difference in the post - replicative protection of telomeres that is linked to their mode of replication ( 61 ) . additional support for a model of end - specific differences in telomeric end - protection was provided by the demonstration of preferential loss of lagging - strand telomeres with wrn deficiency in human cell lines ( 62 ) . it protects the 3 single - stranded g - rich overhang and is involved in t - loop formation , perhaps facilitating invasion of the 3 single - stranded overhang ( 14 ) . inhibition of trf2 induces a dramatic telomere fusion phenotype ( 65 ) resulting from failure of end - protection preferentially at leading - strand telomeres ( 61 ) . end - capping failure occurs after replication , as evidenced by the presence of numerous chromatid - type telomere fusions ( 61 ) ( figure 2 ) . it has also been shown that dna damage foci form at telomeres uncapped by trf2 inhibition ( telomere dysfunction - induced foci ; tifs ) , and consistent with the cytogenetic results , uncapping of telomeres occurs in late s / g2 , i.e. after replication ( 66 ) . in addition to loss of end - capping function , chromosomal fusion can also result from the absence of sufficient telomeric repeat sequences to form a functional telomere . the most obvious mechanism for the loss of telomeric repeat sequences is attrition due to the failure to compensate for the gradual loss of telomeric repeat sequences during cell division . this failure to maintain telomere homeostasis can result from either insufficient telomerase activity or alterations in other telomere - associated proteins required for the recruitment of telomerase to the telomere . the gradual loss of telomeric repeat sequence due to insufficient telomerase activity is exemplified by the telomere shortening that occurs in telomerase - deficient somatic cells with each cell division , although due to cell senescence or apoptosis , this does not normally result in chromosome instability . in addition to gradual loss , telomeric repeat sequences can also be lost through stochastic processes , in which large blocks of telomeric repeat sequences are lost in single events . stochastic events leading to telomere loss can occur through a variety of different mechanisms , the most obvious being large deletions involving recombination , problems encountered during dna synthesis or inefficient dna repair . replication forks stall near telomeres in yeast , and require the rrm3 helicase , which promotes replication through regions of non - histone chromatin ( 67,68 ) . mammalian telomeres may also pose problems for dna replication , since the mammalian telomere - binding proteins trf1 and trf2 can inhibit replication fork movement ( 69 ) . studies in yeast have also demonstrated that telomeric regions are deficient in repair of dsbs . the introduction of dsbs at different sites along a chromosome with the i - scei endonuclease demonstrated that dsbs near telomeres are not repaired efficiently by nhej , but instead result in complex chromosome rearrangements ( 70 ) . mammalian telomeres have been shown to be deficient in repair of single - strand breaks ( 71 ) and get damaged from ultraviolet light ( 72 ) . studies with mouse es cells ( 73,74 ) and human tumor cell lines ( j. p. murnane , unpublished data ) also show that a single dsb generated by i - scei often results in complex chromosome rearrangements not commonly observed at i - scei - induced dsbs at other locations within chromosomes ( 7577 ) . cells containing mutations in various proteins known to be involved in telomere maintenance have provided valuable insights into the mechanisms of telomere loss . one such protein is wrn , which is responsible for the human genetic disease werner syndrome ( ws ) , an autosomal recessive genetic disease presenting a wide range of phenotypic abnormalities , including characteristics of premature aging ( 78 ) . wrn is a member of the recq dna helicase family and has both a 3 to 5 helicase activity and a 3 to 5 exonuclease activity ( 7981 ) . cells isolated from individuals with ws exhibit shortened life span in culture ( 82 ) , as well as an increased rate of dna rearrangements , including translocations , deletions and dicentrics ( 8388 ) . wrn binds to a number of proteins involved in recombination , including replication protein a ( rpa ) ( 89,90 ) , pcna and topoisomerase i ( 91 ) , dna polymerase delta ( 92 ) , and co - localizes with rad51 ( 93 ) and the mre11/rad50/nbs1 ( mrn ) complex in response to dsbs ( 94 ) . consistent with these protein interactions , ws cells have been shown to have a defect in homologous recombination ( hr ) ( 95,96 ) . in addition , the observations that ws cells have a prolonged s - phase ( 97 ) and wrn co - localizes with rpa in cells arrested in s - phase with hydroxyurea ( 98 ) , suggest that ws cells have a defect in resolving stalled replication forks ( 80,98,99 ) . wrn has also been demonstrated to bind the dna - pk complex ( 94,100103 ) and fen-1 ( 104 ) , proteins involved in nhej . moreover , the activity of wrn is influenced by its binding to the dna - pk complex ( 101,102 ) . thus , in addition to a role in hr , wrn also appears to have a role in the nhej pathway for dsb repair . the role of telomeres in cellular senescence initially led to the proposal that the shortened life - span of ws cells in culture might be due to accelerated telomere shortening ( 105 ) . ws cells were found to have accelerated telomere shortening , although the premature senescence in ws cells was found to occur when telomeres were longer than in senescent normal cells ( 106 ) . however , a subsequent study found that ws cells at senescence have telomeres that are similar in length to normal senescent cells ( 107 ) . combined with the fact that ws cells can be immortalized by expression of telomerase ( 107109 ) , these results suggest that ws cells have a defect in telomere maintenance that leads to premature senescence . although due to their long telomeres , mice deficient in wrn alone do not demonstrate the premature aging phenotype observed in humans with ws , later generations of telomerase - deficient mice with shortened telomeres demonstrate classic ws - like premature aging , accelerated replicative senescence , and genomic instability ( 110 ) . the mechanism responsible for telomere shortening in wrn - deficient cells has yet to be determined . one study found that cells expressing dominant - negative wrn showed an increase in chromosome ends without detectable telomeres even though no difference in average telomere length was observed ( 111 ) , indicating that telomere loss in ws is due to a stochastic process . the association of wrn with the dna - pk complex involved in nhej would suggest that telomere loss in ws cells is due to a deficiency in repair of dsbs near telomeres . however , the dna - pk complex also has a role in telomere capping ( 46,53,112,113 ) . in addition , wrn has been found to bind to trf2 ( 114 ) , which is essential for maintaining the cap on the end of the chromosome ( 65 ) , and wrn is required for d - loop resolution regulated by trf1 and trf2 ( 114 ) . based on these observations , another possible mechanism for telomere loss in wrn - deficient cells would involve t - loop deletions similar to those observed in cells deficient in trf2 ( 115 ) . however , loss of end - capping structure would not lead directly to loss of telomeric repeat sequences . a critical clue to the mechanism of telomere loss in ws cells comes from a recent study demonstrating the preferential loss of the lagging - strand of telomeres ( 62 ) , suggesting that telomere loss in ws cells is a result of the requirement for wrn in replication of the g - rich dna found in the lagging strand . this model is consistent with the involvement of wrn in dna replication and/or resolution of stalled replication forks ( 80,98,99 ) , since failure to resolve stalled replication forks can result in dna dsbs ( 116,117 ) . the importance of wrn in replication and/or recombination of telomeres is also apparent from its requirement in suppression of sister chromatid exchange ( sce ) specifically within telomeric dna ( t - sce ) ( 118 ) and activation of the alt pathway that involves recombination ( 119 ) , similar to that proposed for the yeast homolog for wrn , sgs1 ( 120122 ) . mutations in nbs1 are responsible for the autosomal recessive disease nijmegen breakage syndrome ( nbs ) , which displays a wide range of phenotypic abnormalities , including premature aging , increased cancer incidence , chromosomal instability and sensitivity to ir ( 123,124 ) . nbs1 is part of the mrn complex that also contains the mre11 and rad50 proteins ( 125,126 ) . the mrn complex is a key player in the cellular response to dsbs in that association of the mrn complex with dsbs is required for the localization and activation of atm ( 127 ) , which in turn phosphorylates nbs1 ( 128130 ) . as a result , similar to cells deficient in atm , mammalian cells deficient in nbs1 lack the s - phase cell cycle checkpoint ( 131 ) and are sensitive to ir ( 132 ) . in addition to its roles in dna recombination and repair , the mrn complex also functions in telomere maintenance . inhibition of nbs1 by rnai resulted in an increased frequency of telomere association ( 58 ) . primary fibroblasts from individuals with nbs have shortened telomeres , which are proposed to play a role in the pathology of this disease ( 133 ) . in this respect nbs is similar to at , where accelerated telomere shortening is observed in primary fibroblasts ( 134 ) , although no difference in telomere length is evident in immortal cells actively maintaining their telomeres ( 135 ) . in fact , mice with combined knockouts in both atm and the rna component of telomerase show accelerated telomere loss and premature aging , leading to the hypothesis that telomere loss is the reason for some of the phenotypic abnormalities observed in at ( 136 ) . like wrn , similar to wrn , the mrn complex interacts with trf2 ( 137 ) , and therefore is likely to function in proper end - cap formation . however , although mre11 and rad50 are found at the telomere throughout the cell cycle , nbs1 is associated with the telomere only during s - phase , suggesting that it is involved in telomere replication . thus , like wrn , a defect in nbs1 may promote telomere loss through problems in dna replication or the resolution of stalled replication forks in telomeric regions . in fact , wrn associates with mrn ( 94 ) , and both dominant - negative nbs1 ( 138 ) and wrn ( 111 ) have been found to cause similar increases in the rate of telomere loss with no change in average telomere length . selectable marker genes adjacent to telomeres have been used to study the consequences of telomere loss in mammalian cells ( 139 ) . this approach has the advantage of following the changes in individual chromosomes from the initial event , rather than attempting to reconstruct the sequence of events involved in the generation of complex rearrangements . these marked telomeres contain a herpes simplex virus thymidine kinase ( hsv - tk ) selectable - marker gene to select for loss of the telomere , as well as an 18 bp recognition site for the i - scei endonuclease to introduce dsbs , which has been widely used to study dna repair and recombination in mammalian cells ( 7577 ) . using this system , the types of chromosome rearrangements resulting from telomere loss have been followed in both mouse es cells ( 73 ) and the ej-30 human tumor cell line ( 140142 ) . in both the mouse es cells and ej-30 , telomere loss resulted in either a telomere added directly on to the end of the broken chromosome or inverted repeats resulting from sister chromatid fusion . while the addition of a new telomere resulted in stabilization of the marker chromosome , sister chromatid fusion was followed by breakage / fusion / bridge ( b / f / b ) cycles and amplification of subtelomeric dna ( figure 3 ) . these b / f / b cycles occur when the chromosome that has lost a telomere is replicated , and the sister chromatids fuse together at their ends . the fused sister chromatids then form a bridge that breaks during anaphase when the two centromeres are pulled in opposite directions . following dna replication in the next cell cycle , the sister chromatids fuse once again , and therefore these b / f / b cycles continue until the marker chromosome acquires a new telomere . because neither direct telomere addition nor sister chromatid fusion have been observed at dsbs generated by i - scei at interstitial sites ( 7577 ) , these results suggest that there is something different about the processing of dsbs occurring near telomeres . consistent with this conclusion , dsbs generated by i - scei are poorly repaired by nhej near telomeres in yeast , and result in complex chromosome rearrangements ( 70 ) . moreover , direct telomere addition on to the ends of broken chromosomes in yeast preferentially occurs near existing telomeric repeat sequences ( 143 ) . one important difference between the mouse es cells and the ej-30 human tumor cell line is that while telomeres in mouse es cells are highly stable ( loss of the hsv - tk gene < 10 events / cell / generation ) , the telomeres in the human ej-30 tumor cell line are lost at a relatively high rate ( 10 events / cell / generation ) . similar results were observed with other human tumor cell lines ( j. p. murnane , unpublished data ) . this observation is consistent with other studies demonstrating that cancer cells commonly have telomere instability ( 144,145 ) . therefore , although chromosomal rearrangements due to telomere loss in cancer cells are commonly thought to result from the extensive chromosome fusion that occurs during crisis ( 146,147 ) , a high rate of telomere loss is often observed even in human tumors and tumor cell lines that express telomerase ( 73,140,144,148,149 ) , suggesting that many tumor cells have a fundamental defect that promotes telomere loss . another important difference between the mouse es cells and human tumor cell lines is that while the direct addition of a telomere at the site of the break is a common event it mouse es cells ( 74 ) , telomere loss in human tumor cell lines often results in sister chromatid fusion followed by b / f / b cycles ( 140 ) . in addition , while b / f / b cycles last only a few generations in mouse es cells ( 73,74 ) , in ej-30 they can last for many cell generations ( 73,140,141 ) . this inability to terminate b / f / b cycles is likely to contribute to the chromosome instability resulting from telomere loss in human tumor cells . the prolonged b / f / b cycles in the ej-30 human tumor cell line results in extensive dna amplification and terminal deletions of dna on the end of the marker chromosome that lost its telomere ( 141 ) . the fused sister chromatids most often break within 1 mb of the site of fusion , which was confirmed by a subsequent study that found that anaphase bridges formed by sister chromatid fusions most often break near their center , regardless of length ( 150 ) . as a result , the region amplified by b / f / b cycles without selection is most often relatively small ( i.e. < 1 mb ) . however , some breaks also occur far from the site of fusion , resulting in large duplications and deletions ( 73,140,141 ) . this type of break is involved in the amplification of selectable marker genes located far from the end of the chromosome , and has been shown to occur at the location of fragile sites ( 151,152 ) . thus , the loss of a telomere can result in the amplification of genes anywhere on the arm of a chromosome . b / f / b cycles end when the chromosome acquires a telomere and again becomes stable . the most common mechanism for telomere acquisition in both the mouse es cells and the ej-30 tumor cell line was through translocation of the ends of other chromosomes ( 139,142 ) . in ej-30 these translocations were either non - reciprocal ( nrt ) or involved duplications , as determined by the status of the donor chromosome , i.e. with nrts , one of the homologs of the donor chromosome is missing part or all of an arm and its telomere , whereas with duplications , both homologs are intact . both types of events have important consequences for the genome as a whole . the translocations involving duplications commonly involve large portions of the arms of other chromosomes , and therefore generate allelic imbalances involving a large number of genes . on the other hand , nrts result in the loss of a telomere on the donor chromosome , resulting in its instability and the eventual acquisition of a telomere through translocations from other chromosomes . in fact , in one cell this transfer of instability was found to involve six different chromosomes , demonstrating that the loss of a single telomere can result in instability involving multiple chromosomes . therefore , in addition to amplification of dna at the end of a chromosome that has lost a telomere , once initiated , b / f / b cycles can result in other rearrangements , not only involving the chromosome that initially lost its telomere , but other chromosomes as well . in view of the fact that b / f / b cycles can continue for many cell generations , the loss of even a single telomere can generate a wide variety of chromosomal changes in the cell population . taken together , the above results demonstrate that loss of telomeric function whether due to loss of sequence or loss of structure and the ensuing instability and chromosomal rearrangements , can be expected to have a dramatic impact on the stability of the genome . therefore , in view of increasing evidence implicating genomic instability in carcinogenesis ( 153,154 ) , telomere loss is likely to be a significant contributing factor . a role for loss of functional telomeres in the chromosome instability commonly associated with cancer is supported by the large increase in human - like carcinomas in mice deficient in both telomerase and p53 , and the presence of chromosome rearrangements typical of b / f / b cycles in these tumors ( 146,147,155,156 ) . understanding the mechanisms of telomere maintenance and the various factors that promote telomere instability should therefore provide valuable insights into both human genetic disease and cancer . strand - specific co - fish detection of leading- ( red ) and lagging- ( green ) strand telomeres demonstrating chromosomal telomere - telomere fusion in a dna - pkcs deficient background , indicative of end - capping failure owing to loss of structure , not loss of sequence . co - fish detection of chromatid - type telomere fusions in trf2 deficient cells demonstrating preferential failure of end - capping at leading - strand ( red ) telomeres . b / f / b cycles as a mechanism for chromosome instability resulting from telomere loss . b / f / b cycles are initiated when sister chromatids fuse following the loss of a telomere . owing to the presence of two centromeres , the fused sister chromatids break when the cell attempts to divide up its sister chromatids at anaphase . because the break does not occur exactly at the site of the fusion , one daughter cell will receive a copy of the chromosome with an inverted repeat at its end , while the other daughter cell will have a copy of the chromosome with a terminal deletion . owing to the lack of a telomere , these chromosomes will again undergo sister chromatid fusion after dna replication , resulting in additional amplification and terminal deletions . the location of telomeres ( squares ) , centromeres ( circles ) and orientation of the subtelomeric sequences ( horizontal arrows ) are shown .

telomeres are composed of repetitive g - rich sequence and an abundance of associated proteins that together form a dynamic cap that protects chromosome ends and allows them to be distinguished from deleterious dsbs . telomere - associated proteins also function to regulate telomerase , the ribonucleoprtotein responsible for addition of the species - specific terminal repeat sequence . loss of telomere function is an important mechanism for the chromosome instability commonly found in cancer . dysfunctional telomeres can result either from alterations in the telomere - associated proteins required for end - capping function , or from alterations that promote the gradual or sudden loss of sufficient repeat sequence necessary to maintain proper telomere structure . regardless of the mechanism , loss of telomere function can result in sister chromatid fusion and prolonged breakage / fusion / bridge ( b / f / b ) cycles , leading to extensive dna amplification and large terminal deletions . b / f / b cycles terminate primarily when the unstable chromosome acquires a new telomere , most often by translocation of the ends of other chromosomes , thereby providing a mechanism for transfer of instability from one chromosome to another . thus , the loss of a single telomere can result in on - going instability , affect multiple chromosomes , and generate many of the types of rearrangements commonly associated with human cancer .

TELOMERES AND THEIR FUNCTIONS FAILURE OF TELOMERIC FUNCTION DUE TO LOSS OF END-CAPPING STRUCTURE FAILURE OF TELOMERE FUNCTION DUE TO LOSS OF TELOMERIC REPEAT SEQUENCES MECHANISMS OF GENOMIC INSTABILITY RESULTING FROM TELOMERE LOSS Figures and Tables

telomeres , unique structures at the physical ends of linear eukaryotic chromosomes , were first described almost 70 yearsago by hermann muller in his classic studies of the fruit fly drosophilia melanogaster ( 1 ) . shortly thereafter , barbara mcclintock observed that while broken ends of maize chromosome fused , forming dicentric chromosomes , unbroken chromosomes rarely fused ; i.e. these studies demonstrate that normally cells accurately distinguish telomeric ends from random dsb ends and protect the former from illegitimate end - joining reactions . how cells make this critical distinction continues to be an active area of research today , especially as the dividing lines between the two types of ends have become less , rather than more clear . recent discoveries that certain dsb repair proteins act to preserve rather than to join the natural ends of mammalian chromosomes ( 47 ) , have provided impetus for the union of two seemingly disparate scientific fields , dna repair and telomere biology . here , we focus on the creation of dysfunctional mammalian telomeres in various repair deficient backgrounds that result from either the loss of end - capping structure or the loss of terminal sequence ( shortening ) , and the consequences of this loss of function . telomeres serve multiple functions in preserving chromosome stability , including protecting the ends of chromosomes from degradation and preventing chromosomal end fusion . the dna component of telomeres consists of tandem arrays of short , repetitive g - rich sequence [ ttaggg in vertebrates , ( 8,9 ) ] , oriented 5-to-3 towards the end of the chromosome ( 10 ) , ending in an essential 3 single - stranded overhang that ranges in length from 50 to 400 nt ( 1113 ) . electron microscopy studies suggest this overhang can loop back and integrate into the duplex repeat tract , forming a t - loop ( 14 ) , an attractive , although not necessarily exclusive , architectural solution to the end - capping dilemma . although telomerase is responsible for addition of telomeric sequence with every cell cycle , a plethora of other proteins also play important roles in the regulation of telomere length maintenance and in the formation of a protective end - cap that prevents chromosome fusion ( 30,31 ) . proteins that directly bind the double - stranded telomeric repeats include the ttaggg repeat factors trf1 ( 32,33 ) and trf2 ( 34,35 ) . telomere - associated proteins that do not bind dna directly include tin2 ( trf1-interacting nuclear protein 2 ) , which associates with trf1 ( 38 ) and trf2 ( 39,40 ) . the trf1 complex contains both tin2 and pot1 and acts to regulate telomere - length homeostasis . this mammalian telomeric core complex serves to form and protect the telomere , and has been termed both the telosome ( 42 ) and alternately , shelterin ( 31 ) . other proteins , many of which are more commonly associated with dna repair , are also found at telomeric ends [ reviewed in ( 46 ) ] . interestingly , it has recently been proposed that normally , functional human telomeres must be recognized as dna damage in the g2 phase of the cell cycle , in order to recruit the processing machinery necessary for formation of a functional telomere ( 48 ) . functional telomeres are essential for continuous cellular proliferation , and therefore loss of chromosomal end - capping has consequences in both aging and carcinogenesis ( 49,50 ) . we have shown that effective end - capping of mammalian telomeres requires the non - homologous end - joining ( nhej ) protein dna - dependent protein kinase ( dna - pk ) ( 4 ) . ku70 , ku86 , or the catalytic subunit , dna - pkcs ( 51 ) , leads to spontaneous chromosomal end - to - end fusions that maintain large blocks of telomeric sequence at the points of fusion ( figure 1 ) . events at the extreme terminus of the chromosome that normally serve to create a functionally protected telomere fail in the absence of dna - pk , resulting in inappropriate end - to - end fusion events of uncapped telomeres fusing not only to each other , but also to ir - induced dsbs ( 54 ) . the in vivo consequences of inappropriate interstitial blocks of telomere sequence are also unknown , but they most likely affect chromatin stability , and in vitro studies have demonstrated they increase chromosomal instability ( 57 ) . thus , they are obviously not a consequence of telomere shortening , nor are they telomere associations [ defined as distinct telomere signals separated by less than approximately one - third the width of a chromatid , ( 58 ) ] . banding studies demonstrated that any chromosome could be involved in the telomere fusions ( end - capping failure is not chromosome specific ) , and further , that some of the telomere fusions were clonal , suggestive of covalent linkages ( s. bouffler , unpublished data ) . utilizing the strand - specific co - fish technique ( 59,60 ) , we found that the telomere fusions in dna - pkcs - deficient cells exclusively involved telomeres synthesized via leading - strand dna synthesis , suggesting a crucial difference in the post - replicative protection of telomeres that is linked to their mode of replication ( 61 ) . additional support for a model of end - specific differences in telomeric end - protection was provided by the demonstration of preferential loss of lagging - strand telomeres with wrn deficiency in human cell lines ( 62 ) . it protects the 3 single - stranded g - rich overhang and is involved in t - loop formation , perhaps facilitating invasion of the 3 single - stranded overhang ( 14 ) . end - capping failure occurs after replication , as evidenced by the presence of numerous chromatid - type telomere fusions ( 61 ) ( figure 2 ) . in addition to loss of end - capping function , chromosomal fusion can also result from the absence of sufficient telomeric repeat sequences to form a functional telomere . the most obvious mechanism for the loss of telomeric repeat sequences is attrition due to the failure to compensate for the gradual loss of telomeric repeat sequences during cell division . this failure to maintain telomere homeostasis can result from either insufficient telomerase activity or alterations in other telomere - associated proteins required for the recruitment of telomerase to the telomere . the gradual loss of telomeric repeat sequence due to insufficient telomerase activity is exemplified by the telomere shortening that occurs in telomerase - deficient somatic cells with each cell division , although due to cell senescence or apoptosis , this does not normally result in chromosome instability . stochastic events leading to telomere loss can occur through a variety of different mechanisms , the most obvious being large deletions involving recombination , problems encountered during dna synthesis or inefficient dna repair . the introduction of dsbs at different sites along a chromosome with the i - scei endonuclease demonstrated that dsbs near telomeres are not repaired efficiently by nhej , but instead result in complex chromosome rearrangements ( 70 ) . studies with mouse es cells ( 73,74 ) and human tumor cell lines ( j. p. murnane , unpublished data ) also show that a single dsb generated by i - scei often results in complex chromosome rearrangements not commonly observed at i - scei - induced dsbs at other locations within chromosomes ( 7577 ) . cells containing mutations in various proteins known to be involved in telomere maintenance have provided valuable insights into the mechanisms of telomere loss . one such protein is wrn , which is responsible for the human genetic disease werner syndrome ( ws ) , an autosomal recessive genetic disease presenting a wide range of phenotypic abnormalities , including characteristics of premature aging ( 78 ) . thus , in addition to a role in hr , wrn also appears to have a role in the nhej pathway for dsb repair . the mechanism responsible for telomere shortening in wrn - deficient cells has yet to be determined . in addition , wrn has been found to bind to trf2 ( 114 ) , which is essential for maintaining the cap on the end of the chromosome ( 65 ) , and wrn is required for d - loop resolution regulated by trf1 and trf2 ( 114 ) . however , loss of end - capping structure would not lead directly to loss of telomeric repeat sequences . a critical clue to the mechanism of telomere loss in ws cells comes from a recent study demonstrating the preferential loss of the lagging - strand of telomeres ( 62 ) , suggesting that telomere loss in ws cells is a result of the requirement for wrn in replication of the g - rich dna found in the lagging strand . this model is consistent with the involvement of wrn in dna replication and/or resolution of stalled replication forks ( 80,98,99 ) , since failure to resolve stalled replication forks can result in dna dsbs ( 116,117 ) . the importance of wrn in replication and/or recombination of telomeres is also apparent from its requirement in suppression of sister chromatid exchange ( sce ) specifically within telomeric dna ( t - sce ) ( 118 ) and activation of the alt pathway that involves recombination ( 119 ) , similar to that proposed for the yeast homolog for wrn , sgs1 ( 120122 ) . mutations in nbs1 are responsible for the autosomal recessive disease nijmegen breakage syndrome ( nbs ) , which displays a wide range of phenotypic abnormalities , including premature aging , increased cancer incidence , chromosomal instability and sensitivity to ir ( 123,124 ) . the mrn complex is a key player in the cellular response to dsbs in that association of the mrn complex with dsbs is required for the localization and activation of atm ( 127 ) , which in turn phosphorylates nbs1 ( 128130 ) . primary fibroblasts from individuals with nbs have shortened telomeres , which are proposed to play a role in the pathology of this disease ( 133 ) . in fact , mice with combined knockouts in both atm and the rna component of telomerase show accelerated telomere loss and premature aging , leading to the hypothesis that telomere loss is the reason for some of the phenotypic abnormalities observed in at ( 136 ) . like wrn , similar to wrn , the mrn complex interacts with trf2 ( 137 ) , and therefore is likely to function in proper end - cap formation . however , although mre11 and rad50 are found at the telomere throughout the cell cycle , nbs1 is associated with the telomere only during s - phase , suggesting that it is involved in telomere replication . in fact , wrn associates with mrn ( 94 ) , and both dominant - negative nbs1 ( 138 ) and wrn ( 111 ) have been found to cause similar increases in the rate of telomere loss with no change in average telomere length . selectable marker genes adjacent to telomeres have been used to study the consequences of telomere loss in mammalian cells ( 139 ) . these marked telomeres contain a herpes simplex virus thymidine kinase ( hsv - tk ) selectable - marker gene to select for loss of the telomere , as well as an 18 bp recognition site for the i - scei endonuclease to introduce dsbs , which has been widely used to study dna repair and recombination in mammalian cells ( 7577 ) . using this system , the types of chromosome rearrangements resulting from telomere loss have been followed in both mouse es cells ( 73 ) and the ej-30 human tumor cell line ( 140142 ) . in both the mouse es cells and ej-30 , telomere loss resulted in either a telomere added directly on to the end of the broken chromosome or inverted repeats resulting from sister chromatid fusion . while the addition of a new telomere resulted in stabilization of the marker chromosome , sister chromatid fusion was followed by breakage / fusion / bridge ( b / f / b ) cycles and amplification of subtelomeric dna ( figure 3 ) . these b / f / b cycles occur when the chromosome that has lost a telomere is replicated , and the sister chromatids fuse together at their ends . the fused sister chromatids then form a bridge that breaks during anaphase when the two centromeres are pulled in opposite directions . following dna replication in the next cell cycle , the sister chromatids fuse once again , and therefore these b / f / b cycles continue until the marker chromosome acquires a new telomere . because neither direct telomere addition nor sister chromatid fusion have been observed at dsbs generated by i - scei at interstitial sites ( 7577 ) , these results suggest that there is something different about the processing of dsbs occurring near telomeres . consistent with this conclusion , dsbs generated by i - scei are poorly repaired by nhej near telomeres in yeast , and result in complex chromosome rearrangements ( 70 ) . moreover , direct telomere addition on to the ends of broken chromosomes in yeast preferentially occurs near existing telomeric repeat sequences ( 143 ) . one important difference between the mouse es cells and the ej-30 human tumor cell line is that while telomeres in mouse es cells are highly stable ( loss of the hsv - tk gene < 10 events / cell / generation ) , the telomeres in the human ej-30 tumor cell line are lost at a relatively high rate ( 10 events / cell / generation ) . therefore , although chromosomal rearrangements due to telomere loss in cancer cells are commonly thought to result from the extensive chromosome fusion that occurs during crisis ( 146,147 ) , a high rate of telomere loss is often observed even in human tumors and tumor cell lines that express telomerase ( 73,140,144,148,149 ) , suggesting that many tumor cells have a fundamental defect that promotes telomere loss . another important difference between the mouse es cells and human tumor cell lines is that while the direct addition of a telomere at the site of the break is a common event it mouse es cells ( 74 ) , telomere loss in human tumor cell lines often results in sister chromatid fusion followed by b / f / b cycles ( 140 ) . in addition , while b / f / b cycles last only a few generations in mouse es cells ( 73,74 ) , in ej-30 they can last for many cell generations ( 73,140,141 ) . this inability to terminate b / f / b cycles is likely to contribute to the chromosome instability resulting from telomere loss in human tumor cells . the prolonged b / f / b cycles in the ej-30 human tumor cell line results in extensive dna amplification and terminal deletions of dna on the end of the marker chromosome that lost its telomere ( 141 ) . the fused sister chromatids most often break within 1 mb of the site of fusion , which was confirmed by a subsequent study that found that anaphase bridges formed by sister chromatid fusions most often break near their center , regardless of length ( 150 ) . as a result , the region amplified by b / f / b cycles without selection is most often relatively small ( i.e. this type of break is involved in the amplification of selectable marker genes located far from the end of the chromosome , and has been shown to occur at the location of fragile sites ( 151,152 ) . thus , the loss of a telomere can result in the amplification of genes anywhere on the arm of a chromosome . b / f / b cycles end when the chromosome acquires a telomere and again becomes stable . the most common mechanism for telomere acquisition in both the mouse es cells and the ej-30 tumor cell line was through translocation of the ends of other chromosomes ( 139,142 ) . with nrts , one of the homologs of the donor chromosome is missing part or all of an arm and its telomere , whereas with duplications , both homologs are intact . both types of events have important consequences for the genome as a whole . the translocations involving duplications commonly involve large portions of the arms of other chromosomes , and therefore generate allelic imbalances involving a large number of genes . on the other hand , nrts result in the loss of a telomere on the donor chromosome , resulting in its instability and the eventual acquisition of a telomere through translocations from other chromosomes . in fact , in one cell this transfer of instability was found to involve six different chromosomes , demonstrating that the loss of a single telomere can result in instability involving multiple chromosomes . therefore , in addition to amplification of dna at the end of a chromosome that has lost a telomere , once initiated , b / f / b cycles can result in other rearrangements , not only involving the chromosome that initially lost its telomere , but other chromosomes as well . in view of the fact that b / f / b cycles can continue for many cell generations , the loss of even a single telomere can generate a wide variety of chromosomal changes in the cell population . taken together , the above results demonstrate that loss of telomeric function whether due to loss of sequence or loss of structure and the ensuing instability and chromosomal rearrangements , can be expected to have a dramatic impact on the stability of the genome . a role for loss of functional telomeres in the chromosome instability commonly associated with cancer is supported by the large increase in human - like carcinomas in mice deficient in both telomerase and p53 , and the presence of chromosome rearrangements typical of b / f / b cycles in these tumors ( 146,147,155,156 ) . understanding the mechanisms of telomere maintenance and the various factors that promote telomere instability should therefore provide valuable insights into both human genetic disease and cancer . strand - specific co - fish detection of leading- ( red ) and lagging- ( green ) strand telomeres demonstrating chromosomal telomere - telomere fusion in a dna - pkcs deficient background , indicative of end - capping failure owing to loss of structure , not loss of sequence . co - fish detection of chromatid - type telomere fusions in trf2 deficient cells demonstrating preferential failure of end - capping at leading - strand ( red ) telomeres . b / f / b cycles as a mechanism for chromosome instability resulting from telomere loss . b / f / b cycles are initiated when sister chromatids fuse following the loss of a telomere . owing to the presence of two centromeres , the fused sister chromatids break when the cell attempts to divide up its sister chromatids at anaphase . because the break does not occur exactly at the site of the fusion , one daughter cell will receive a copy of the chromosome with an inverted repeat at its end , while the other daughter cell will have a copy of the chromosome with a terminal deletion . owing to the lack of a telomere , these chromosomes will again undergo sister chromatid fusion after dna replication , resulting in additional amplification and terminal deletions .

down syndrome ( ds ) results from the triplication of genes on human chromosome 21 ( hsa21 ) and is associated with a range of phenotypes including craniofacial changes [ 1 , 2 ] , cardiac defects , susceptibility to leukemia but with reduced occurrence of solid cancers [ 4 , 5 ] , and intellectual disability [ 6 , 7 ] . while the presence and severity of these individual phenotypes vary among ds individuals , every individual with ds has some degree of cognitive impairment . these impairments limit the independence of ds subjects and adversely impact their quality of life . consequently , understanding the genetic causes of cognitive dysfunction in ds has been the focus of much research in this field . the phenomenon of synaptic plasticity has been strongly linked to cognitive processes , such as learning and memory [ 8 , 9 ] . synaptic plasticity refers to the dynamic nature of synapses , sites of communication between neurons , in which the structure , composition , or function of the synapse changes in response to network activity . depending on the timing and strength of pre- and postsynaptic activity , synapses can either be strengthened or weakened providing a potential mechanism for memory formation and storage . structurally , synaptic connections on excitatory neurons are typically formed on the heads of dendritic spines . the morphology of the spines enables compartmentalization of signaling cascades and facilitates manipulation of the structure and composition of the cell membrane by second messenger systems [ 12 , 13 ] . thus , not only is the number of spines important , as individual locations for excitatory synaptic transmission , but the shape of the individual spines also has a critical functional role . the link between synaptic plasticity and cognitive processes such as learning and memory is frequently studied within the hippocampus , a structure involved in diverse cognitive processes such as those related to acquisition , coding , storing , and recalling information in physical or perceived spatial environments [ 1416 ] . multiple lines of evidence indicate that long - lasting up- or downregulation of functional synaptic strengths , referred to as long - term potentiation ( ltp ) and long - term depression ( ltd ) , respectively , are fundamental synaptic mechanisms underlying hippocampal contributions to these processes . thus , dendritic and synaptic abnormalities in the hippocampus , either morphological or functional , would be expected to significantly impact spatial cognition . indeed , neuropsychological investigations requiring the use of spatial information in problem solving indicate that deficits in hippocampal - mediated learning and memory processes are hallmarks of ds [ 17 , 18 ] . in this paper , we will provide an overview of the morphological and behavioral evidence for altered synaptic plasticity in ds with a focus on the hippocampus and discuss the insights provided by mouse models of this neurodevelopmental disorder into the potential molecular mechanisms contributing to these deficits . the basis for altered synaptic plasticity in ds can be found in changes in the physical structure of the dendrites . alterations in the shape and densities of dendrites would be expected to adversely affect the information storage capacity of neural networks by reducing the number of potential sites for plasticity to occur . consistent with this idea and the observed deficits in cognition associated with ds , examination of postmortem brain tissue from ds individuals reveals profound alterations in dendritic and neuronal densities and morphology across many regions of the brain beginning in utero and persisting throughout life . the neocortical development of ds fetuses appears normal up to at least gestational week 22 [ 1921 ] . by 40 weeks gestation , less discrete lamination is observed in the neocortex of ds fetuses with lower and higher cell densities observed in the visual cortex and superior temporal neocortex , respectively [ 19 , 20 ] . in the hippocampus , deficits begin to appear slightly earlier as ds fetuses ( 17 to 21 weeks of gestation ) show altered morphology , reduced neuron numbers , enhanced apoptosis , and reduced cell proliferation [ 2224 ] . these changes may result , in part , from reductions in serotonin , dopamine , and gaba levels in the fetal ds cortex since , during development , neurotransmitters such as these can act as neurotrophic factors assisting with neuronal migration , axon guidance , and neurite development . during the early postnatal period , significant deficits in brain weight and gross morphology as well as myelination and neuronal densities and morphology appear . initially , dendritic expansion is enhanced in ds infants , but , by the first to second year of life , this trend reverses to become a deficit [ 19 , 28 ] which persists into adulthood [ 19 , 29 ] . dendritic spine numbers are reduced , and morphology altered in ds [ 30 , 31 ] . consistent with adverse changes in dendrite morphology , synaptogenesis is also aberrant in ds fetuses [ 19 , 32 , 33 ] and remains deficient in adulthood . mri studies reveal that ds children and young adults have smaller overall brain volumes [ 35 , 36 ] with particular deficits noted in the hippocampus [ 36 , 37 ] . hippocampal volume , that continues to decrease with age in ds individuals , was found to be inversely correlated with the degree of cognitive impairment . cognitive tests such as the cambridge neuropsychological testing automated battery ( cantab ) and the arizona cognitive test battery ( actb ) , the latter specifically tailored to address ds deficits , indicate that hippocampal function is particularly impacted by the ds genetic condition [ 17 , 39 ] . these morphological and cognitive deficits are consistent with aberrant synaptic plasticity , and , indeed , while difficult to measure directly in human subjects , evidence suggests that plasticity is reduced at least in the motor cortex of ds individuals . additionally , functional mri ( fmri ) during cognitive processing tasks reveals abnormal neural activation patterns in ds children and young adults [ 41 , 42 ] . examination of resting glucose metabolism in the cerebral cortex of ds individuals found enhanced uptake in brain regions associated with cognition suggesting cellular hyperactivity in those areas . to better understand the functional consequences resulting from altered network morphologies as well as investigate potential alterations in intracellular signaling cascades contributing to aberrant plasticity over the past few decades , several mouse models of down syndrome have been developed to further our understanding of the link between enhanced gene dosage and ds phenotypes such as altered plasticity and cognition . the tc1 mouse model carries an almost complete , freely segregating copy of hsa21 , but the chromosome is present in only approximately 50% of cells making this a mosaic model of ds . it is important to note that , in spite of the mosaicism and gene deletions , many ds phenotypes have been replicated in this model [ 46 , 48 , 49 ] . other mouse models have taken advantage of the homology between regions of hsa21 and mouse chromosomes 10 , 16 , and 17 ( mmu10 , 16 , 17 ) making models in which these genes are triplicated highly useful in understanding the genetic basis of ds phenotypes [ 50 , 51 ] . a mouse model trisomic for all hsa21 homologous segments was recently developed and holds great promise for furthering our understanding of ds . as this is a relatively new model , however , most research has been conducted using the ts65dn segmental trisomic mouse [ 5355 ] which is trisomic for more than 50% genes of hsa21 homologs [ 56 , 57 ] and has well - documented ds - like deficits in behavioral tasks such as those relying upon declarative memory ( novel object recognition and spontaneous alternation tasks ) and the proper encoding and recollection of spatial information ( radial arm and morris water mazes ) [ 5863 ] . while the ts65dn mouse is the only mouse model of ds to have a freely segregating supernumerary chromosome , they are also trisomic for 60 genes that do not have hsa21 homologs , and the impact of overexpression of these genes on ts65dn phenotypes remains to be determined . similar to the ts65dn mouse but with smaller triplicated mmu16 segments are the ts1cje and ts1rhr mouse models . these mice display phenotypes similar to ts65dn mice including hippocampal dysfunction ; however , the severity of the deficits is reduced [ 6569 ] . the reduced severity of ds - like deficits in mice with fewer trisomic genes highlights one of the powerful aspects of mouse models : the ability to control expression of certain hsa21 homologs to assess their contribution to specific ds phenotypes . those deficits associated with the hippocampus , whose function is notably altered in ds individuals [ 17 , 39 ] , will be the focus of the remainder of this paper . mouse models of ds , including the ts65dn strain , show similar detrimental changes in neuronal and dendritic morphologies observed in humans . the neocortex of ts65dn mice contains fewer excitatory neurons but an increased number of a subset of inhibitory neurons relative to euploid controls , a phenotype that was reversed by normalizing the expression levels of olig1/2 . additionally , regions both in the neocortex and hippocampus have decreased spine densities with larger spine volumes . in the dentate granule cells of the hippocampus , there is a shift of inhibitory synaptic connections away from the dendritic shafts and onto the necks . such a change would be expected to increase the efficacy of inhibitory synaptic transmission given the significantly reduced volume of the spine neck compared to the shaft . at a finer resolution , symmetric ( presumed inhibitory synapses ) have greater opposition lengths in ts65dn while asymmetric synapses were unaltered , again supporting a shift towards excess inhibition in these mice . similar but less severe changes are observed in ts1cje mice . beyond suppressing excitatory synaptic activity , the altered spine morphology and shift towards excess inhibition in trisomy would be expected to suppress plasticity - related signaling cascades that frequently rely on depolarization - mediated calcium influx into the postsynaptic structural domains . synaptic plasticity in the hippocampus is often investigated in the context of long - term potentiation ( ltp ) in which high - frequency activation of specific inputs in the hippocampus results in a long - lasting potentiation of synaptic responses along the excited afferent pathway . first described in the anesthetized rabbit , this phenomenon is believed to be a fundamental mechanism underlying memory formation [ 8 , 74 ] and is suppressed in ts65dn ( depicted in figure 1 for the ca1 region of the hippocampus ) [ 44 , 75 , 76 ] and ts1cje [ 66 , 67 ] but not in ts1rhr mice ( however , see ) as well as mice trisomic for hsa21 syntenic regions of mmu16 and mmu17 or those carrying an almost complete copy of hsa21 [ 46 , 48 ] . as outlined above , structural changes suggest that inhibition is exaggerated in the trisomic hippocampus . consistent with this idea is the observation that ltp in the dentate gyrus and ca1 regions of ts65dn hippocampal slices , induced by high - frequency stimulation and theta burst protocols , respectively , can be rescued by the gabaa antagonists picrotoxin [ 75 , 76 ] . blockade of gabaa receptors in hippocampal slices from ts1cje and ts1rhr mice rescues ltp deficits in the dentate gyrus in these ds mouse models as well [ 67 , 68 ] . in addition to suppressed ltp , hippocampi from ts65dn mice show enhanced long - term depression ( ltd ) in response to sustained activation of excitatory synapses [ 45 , 78 ] . this latter effect can be reversed with the uncompetitive nmda receptor antagonist memantine and also improves the cognitive performance of ts65dn mice [ 7981 ] . these results draw a clear link between altered synaptic plasticity in the hippocampus and cognitive performance in the ts65dn mouse model of down syndrome . changes in intracellular calcium concentrations are important triggers for many intracellular signaling cascades including those underlying ltp and ltd . for example , the presence of a calcium chelator that buffers intracellular calcium levels in postsynaptic neurons prevents the induction of ltp consistent with the hypothesis that a postsynaptic rise in intracellular calcium levels is necessary for ltp . when strongly depolarized , the magnesium block of nmda channels is lifted providing the main ( but not exclusive ) mechanism for calcium entry into the postsynaptic cell . elevated intracellular calcium levels trigger a cascade of intracellular messengers that ultimately lead to the induction and maintenance of synaptic plasticity ( both ltp and ltd depending on the kinetics ) . an excellent overview of this process can be found in several reviews [ 82 , 84 ] , and only key components known to be affected by trisomy ( figure 2 ) will be discussed here . activation of postsynaptic nmda receptors ( nmdars ) concomitant with the depolarization of the postsynaptic membrane is sufficient to relieve the magnesium block of nmda channels leading to an influx of calcium into the intracellular postsynaptic space . in the case of ltp , the rise of intracellular calcium leads to the activation of calcium calmodulin - dependent protein kinase ii ( camkii ) , a necessary step for initiating nmdar - dependent ltp . blocking camkii prevents induction of ltp , [ 82 , 85 , 86 ] , while constitutively active forms can induce ltp . during all phases of ltp ( induction , early , and late ) , camkii phosphorylated at threonine 286 ( thr286 ) can become constitutively active providing a potential switch for initiating and then maintaining potentiation . alternatively , phosphorylation of thr305/306 can inhibit the expression of ltp by interfering with the binding of calcium / calmodulin [ 90 , 91 ] . indeed , cognitive deficits associated with angelman syndrome were reversed in a mouse model of the disorder by reducing the levels of camkii phosphorylated at thr305/306 . thus , depending on the site of phosphorylation , camkii can facilitate or suppress initiation and maintenance of ltp . in ts65dn mice , we found that baseline levels of camkii phosphorylated at thr286 are elevated in the hippocampus . excessive basal phosphorylation of the camkii site leading to constitutive activation could leave the ds modeling trisomic network in a saturated state unable to shift to more potentiated levels . one of the substrates targeted by camkii during the initial expression of ltp is the serine 831 residue on glur1 subunits of ampa receptors [ 94 , 95 ] . this phosphorylation leads to an increase in conductance of the ampa channel providing a rapid mechanism for enhancing glutamatergic synaptic strength . in ts65dn mice , we find that baseline levels of phosphorylated serine 831 in synaptically located glur1 receptors are elevated . this apparent increase in ampa channel conductance appears not to have any significant effect on baseline excitatory synaptic transmission which is normal in the ts65dn hippocampus [ 45 , 75 , 76 , 93 ] . however , it could also partially occlude the initiation of ltp in these mice by leaving ts65dn hippocampal excitatory synapses with fewer ampa channels available for potentiation . this finding would be consistent with our observation of increased camkii in ts65dn hippocampus noted above and the suggestion that some components of the ltp network are in an apparent saturated state in these mice . in particular , evidence suggests that it is involved in initiating the protein synthesis required for the late phase of ltp [ 97 , 98 ] . blocking pka activity suppresses the late phase of ltp ( lasting beyond 3 hours ) while leaving the early phase of ltp ( less than 3 hours ) unaffected [ 99 , 100 ] . transgenic mice in which pka activity is reduced have significantly decreased late - phase ltp in ca1 but normal early ltp and perform poorly on tasks requiring long- but not short - term memory formation . pka plays a role in ltd where its substrates , such as glur1 , show increased dephosphorylation following induction [ 102 , 103 ] . dephosphorylation of glur1 subunits should reduce the conductance levels of affected ampa receptors resulting in a reduction of synaptic strength . pka also enhances the activity of rcan1 , an inhibitor of calcineurin which contributes to ampa receptor internalization and reductions in nmda receptor mean open time . in ts65dn mice , we found that pka activity is reduced in the hippocampus , which should adversely affect ltp by reducing protein expression required for the late phase . with respect to ltd , reduced pka activity would result in more ampa receptors remaining in a high conductance state and less facilitation of rcan1 activity . this latter effect is offset , however , by the overexpression of the gene encoding rcan1 in ds and ts65dn mice . how these factors contribute to the enhancement of ltd in ts65dn hippocampi [ 45 , 78 ] remains to be determined . in addition to glur1 subunits , both camkii and pka converge on another common effector , the mitogen - activated protein kinase ( mapk / erk ) , that is associated with a host of synaptic - plasticity - related cellular processes . in the case of hippocampal ltp , there is a rapid increase in the amount of phosphorylated erk following induction and blocking erk activation prevents expression of ltp . cultured hippocampal neurons undergo phosphorylated erk - dependent spine generation following ltp conditioning stimuli implicating this pathway in spine formation . additionally , it is believed that lateral diffusion of extrasynaptic ampa receptors containing glur1 subunits into the postsynaptic density ( psd ) is a major contributor to ltp expression . this process is assisted by ras / erk phosphorylation of stargazin on extrasynaptic ampa receptors enabling them to be structurally secured at the synapse to psd95 . in the ts65dn hippocampus , erk phosphorylation this would be expected to adversely affect the insertion of new ampa receptors into the psd as well as morphological restructuring of synaptic spines observed after ltp in normal mice [ 113 , 114 ] . brain - derived neurotrophic factor ( bdnf ) contributes to ltp by stimulating protein synthesis . in activating postsynaptic trkb receptors , bdnf stimulates the pi3k pathway which can initiate translation through mammalian target of rapamycin ( mtor ) thereby enhancing synthesis of proteins such as camkii , glur1 , and nmda receptor subunit 1 . in ts65dn mice , we found that pi3k phosphorylation failed to increase following an ltp induction protocol suggesting this pathway is perturbed by trisomy . consistent with this notion , in ds individuals , bdnf blood plasma levels are approximately 5 times higher than in age - matched controls . as bdnf readily crosses the blood - brain barrier , these levels likely reflect those present in the cns as well . examination of bdnf levels in ds mouse models presents a complex picture . in ts65dn mice , in the hippocampus , both no difference and a reduction compared to control have been reported . in the latter case , the reduction in bdnf levels was associated with decreased neurogenesis and was reversible through treatment with fluoxetine . in the ts1cje mouse model of ds , bdnf is overexpressed in the hippocampus , particularly in the dentate gyrus and ca1 regions and in the dendrites of dissociated hippocampal neurons grown in culture . increased bdnf levels in ts1cje mice hippocampi were associated with greater levels of phosphorylated akt - mtor and expression of glur1 protein which could not be further enhanced with exogenous supplemental bdnf suggesting this pathway related to synaptic plasticity is saturated in these mice preventing further contributions to ltp . the discrepancies between observations in ts65dn and ts1cje bdnf levels may reflect how bdnf expression is distributed in these structures , elevated in some subregions or subcellular compartments while diminished in others , resulting in an increased functional effect despite reduced global levels . conversely , the differences in observed bdnf levels could be related to the different numbers of genes overexpressed in these two mouse lines [ 53 , 65 , 122 ] or , as mice of differing age groups were used in the studies , may reflect differences in expression levels as a function of age . however , the observation that rapamycin has a restorative effect on phosphorylated akt - mtor levels in ts1cje suggests a potential therapeutic mechanism for improving cognition in ds individuals possibly by normalizing a pathway involved in synaptic plasticity . as mentioned above this initiating step relies heavily upon depolarization of the postsynaptic membrane to relieve the voltage - dependent magnesium block of nmda channels . any phenomenon that reduces the ability of the postsynaptic membrane to depolarize would thus be expected to adversely affect plasticity . through its coupling to gaba b receptors , the type 2 g - protein - activated inward rectifying potassium ( girk2 ) channel may act to dampen the expression of ltp in ts65dn hippocampus through a shunting mechanism . girk2 is encoded by the gene kcnj6 which is located on the chromosomal segment triplicated in ds and ts65dn mice , and , consequently , elevated expression levels have been found in the ts65dn hippocampus [ 50 , 123 ] . at a cellular level , overexpression of girk2 leads to a more hyperpolarized resting potential in cultured hippocampal neurons and ca1 pyramidal neurons in vitro . selectively reducing the expression level of girk2 by crossing euploid and ts65dn mice with mice heterozygous for girk2 ( girk2 ) resulted in a gene dosage - dependent change in the resting membrane potential and facilitation of ltp in girk2 knockout mice . selective overexpression of girk2 alone in mice results in cognitive deficits , reduced depotentiation ( a functional reversal of potentiation at a synapse ) , and enhanced ltd . these effects on ltp and ltd could be mediated through gabab receptors which , in pyramidal neurons , are in closest proximity to girk2-contaning potassium channels near glutamatergic synapses on dendritic spines . gabab receptors are functionally linked to girk channels , and , indeed , whole - cell gabab - mediated potassium currents are exaggerated in ts65dn hippocampal neurons [ 50 , 124 , 128 ] . in ca1 , these exaggerated currents have a greater functional impact on the distal dendrites of pyramidal neurons as opposed to those located more proximally . a similar enhancement of gabab - mediated currents is also found in the dentate gyrus where the presynaptic release probability of gaba is increased . thus , girk channels , activated by gabab and other g - protein coupled receptors , appear to act as a break on synaptic plasticity in the ts65dn hippocampus . the hippocampus receives major inputs from the entorhinal cortex ( ec ) which converge on ca1 pyramidal through two main pathways : the perforant pathway ( pp ) and the temporoammonic ( ta ) pathway . the pp pathway passes through the dentate gyrus to pyramidal neurons in ca3 before impinging upon the relatively proximal dendrites of ca1 pyramidal neurons in stratum radiatum ( sr ) . conversely , inputs to ca1 from ta target the distal dendrites located in stratum lacunosum molecular ( slm ) . in the normal hippocampus , frequency - based synaptic plasticity at the ca3-ca1 synapse , coupled with a feed - forward inhibition loop from stratum oriens alveus interneurons that suppress inputs to distal ca1 dendrites , enables segregation of information flow through these two pathways . during high - frequency synaptic activity , the ca3-ca1 synapse would be expected to undergo ltp , increasing the excitatory drive to ca1 pyramidal neurons and , consequently , enhanced suppression of inputs to distal ca1 dendrites by the feed - forward inhibition loop ( figure 3(a ) ) . thus , ta inputs that target distal ca1 dendrites would be suppressed , and information flows through the ca3-ca1 pathway enhanced during during high - frequency events . conversely , during low - frequency synaptic activity , the ca3-ca1 synapse would be expected to undergo ltd and become less effective . inhibition of distal ca1 synapses would then be decreased and information flow through the ta pathway would likely be enhanced ( figure 3(b ) ) . diminished ltp resulting from trisomy would then interfere with this frequency - based segregation of information flow through the hippocampus . without ltp , feed - forward inhibition would cause suppression of information flow through the ta pathway causing inputs from the two pathways to become superimposed upon and interfere with each other ( figure 3(c ) ) . in contrast , the flow of information during low frequency signaling would likely remain intact , or potentially facilitated , since enhanced ltd at ca3-ca1 would prevent interference from this pathway ( figure 3(d ) ) . electroencephalogram ( eeg ) recordings from ds individuals suggest that such a preferential suppression of high - frequency information flow may result from overexpression of hsa21 genes . compared to controls , ds individuals have increased power at low eeg frequencies and a corresponding reduction in power at higher frequencies . while it is not clear that hippocampal activity is accurately reflected in eeg recordings , abnormal eegs findings are consistent with aberrant processing of high - frequency information in ds individuals and ts65dn mice . a number of studies using the ts65dn mouse model of ds have examined the possibility of pharmacologically reversing cognitive deficits ( reviewed in ) . of particular note with respect to hippocampal plasticity are those targeting the excess gabaergic inhibitory tone or nmda receptors whose activation , as outlined above , is a critical step in initiating ltp and ltd . application of the gabaa receptor antagonist , picrotoxin to hippocampal slices from ts65dn mice rescues ltp in the dentate gyrus and ca1 region . chronic administration of low doses of picrotoxin or other gabaa receptor antagonists ( pentylenetetrazole or bilobalide ) improves cognition in ts65dn mice suggesting that the efficacy of this class of pharmacological agents could be tested for reversing impaired cognition in ds . however , as overinhibition of gabaa receptors can induce seizures , translating these findings to humans requires great caution . careful screening of similar drugs or design of pharmacological compounds with similar blocking capabilities but reduced propensities for inducing seizures may prove to be effective treatments . currently , a small molecule targeting gabaa receptors developed by f. hoffmann - la roche ltd ( pharmaceutical pipeline molecule rg1662 http://www.roche.com/roche_pharma_pipeline.htm ) is in clinical trials with the goal of safely improving cognition in ds individuals . [ 134 , 135 ] are currently investigating a similar promising inhibitor that targets the alpha-5 subunit of gabaa receptors . another pharmacological avenue targets aberrant nmda receptor - mediated signaling apparently present in ts65dn mice . the uncompetitive nmda receptor antagonist memantine improves the cognitive performance of ts65dn mice and normalizes hippocampal ltd . memantine is an fda approved and fairly well - tolerated drug already in use for treating dementia in alzheimer disease . clinical trials assessing the safety , tolerability , and efficacy in alleviating ds cognitive phenotypes are currently underway . in addition to pharmacological approaches , behavioral therapies have been shown to improve cognition in ts65dn mice . when housed in enriched environments ( larger cage with novel objects such as toys and running wheels ) , trisomic mice performed as well as euploid littermates in the morris water maze and had normalized hippocampal ltp [ 136 , 137 ] . interestingly , environmental enrichment was effective for trisomic females but not trisomic males potentially due to social and physical factors associated with the new environments . the benefits of environmental enrichment appear to be linked in part to regulation of excess inhibition in the neocortex and hippocampus . release of gaba from synaptosomes isolated from the hippocampus and neocortex is elevated in ts65dn mice , an effect that is reversed by environmental enrichment . in adult rats with amblyopia ( via monocular deprivation during critical period ) , environmental enrichment reversed visual deficits reduced gaba levels in the visual cortex contralateral to the deprived eye while increasing plasticity . it thus appears possible to regulate aberrant levels of inhibition in trisomic mice behaviorally without pharmacological intervention and achieve similar behavioral outcomes without the concerns associated with nonspecific actions or adverse side - effects of drugs . deficits in neurogenesis in the dentate gyrus and forebrain subventricular zone in ts65dn mice are also reversed following environmental enrichment and may add an additional therapeutic layer to the beneficial effect of a decrease in inhibitory tone . a structural benefit of environmental enrichment appears lacking ; however , as , unlike euploid mice , this treatment has failed to significantly increase dendritic branching and spine density in ts65dn mice . early behavioral intervention techniques designed to improve development in ds children show great promise [ 141 , 142 ] suggesting that this comparatively easily translatable therapeutic approach , either used alone or in combination with pharmacological agents , could potentially increase cognitive capacities in ds individuals . this belief is bolstered by experiments where drugs that normalize aberrant plasticity in hippocampal slices isolated from mouse models of ds also confer improvements in cognition in in intact adult mice . the initiation and maintenance of plastic changes involve structural and compositional modifications of synapses that depend on intracellular signaling cascades . the reduced excitatory neuronal densities and deficits in dendritic morphologies present in individuals with ds diminish the capacity of their neural networks in general to undergo neuroplastic adaptations . combined with the deficits in signaling pathways reported in ts65dn mice , evidence strongly suggests that synaptic plasticity is severely impaired in ds neural networks . by understanding how plasticity is perturbed , we can design therapies to reverse these phenotypes and ultimately improve cognition in ds individuals .

down syndrome ( ds ) is caused by the overexpression of genes on triplicated regions of human chromosome 21 ( hsa21 ) . while the resulting physiological and behavioral phenotypes vary in their penetrance and severity , all individuals with ds have variable but significant levels of cognitive disability . at the core of cognitive processes is the phenomenon of synaptic plasticity , a functional change in the strength at points of communication between neurons . a wide variety of evidence from studies on ds individuals and mouse models of ds indicates that synaptic plasticity is adversely affected in human trisomy 21 and mouse segmental trisomy 16 , respectively , an outcome that almost certainly extensively contributes to the cognitive impairments associated with ds . in this review , we will highlight some of the neurophysiological changes that we believe reduce the ability of trisomic neurons to undergo neuroplasticity - related adaptations . we will focus primarily on hippocampal networks which appear to be particularly impacted in ds and where consequently the majority of cellular and neuronal network research has been performed using ds animal models , in particular the ts65dn mouse . finally , we will postulate on how altered plasticity may contribute to the ds cognitive disability .

1. Introduction 2. Evidence for Altered Synaptic Plasticity in DS: A Neurodevelopmental Impact 3. Modeling DS Cognitive Impairment 4. Potential Impact of Altered Plasticity on Hippocampal Processing 5. Cognitive Therapies Targeting Plasticity 6. Conclusion

down syndrome ( ds ) results from the triplication of genes on human chromosome 21 ( hsa21 ) and is associated with a range of phenotypes including craniofacial changes [ 1 , 2 ] , cardiac defects , susceptibility to leukemia but with reduced occurrence of solid cancers [ 4 , 5 ] , and intellectual disability [ 6 , 7 ] . while the presence and severity of these individual phenotypes vary among ds individuals , every individual with ds has some degree of cognitive impairment . consequently , understanding the genetic causes of cognitive dysfunction in ds has been the focus of much research in this field . the phenomenon of synaptic plasticity has been strongly linked to cognitive processes , such as learning and memory [ 8 , 9 ] . synaptic plasticity refers to the dynamic nature of synapses , sites of communication between neurons , in which the structure , composition , or function of the synapse changes in response to network activity . the morphology of the spines enables compartmentalization of signaling cascades and facilitates manipulation of the structure and composition of the cell membrane by second messenger systems [ 12 , 13 ] . thus , not only is the number of spines important , as individual locations for excitatory synaptic transmission , but the shape of the individual spines also has a critical functional role . the link between synaptic plasticity and cognitive processes such as learning and memory is frequently studied within the hippocampus , a structure involved in diverse cognitive processes such as those related to acquisition , coding , storing , and recalling information in physical or perceived spatial environments [ 1416 ] . multiple lines of evidence indicate that long - lasting up- or downregulation of functional synaptic strengths , referred to as long - term potentiation ( ltp ) and long - term depression ( ltd ) , respectively , are fundamental synaptic mechanisms underlying hippocampal contributions to these processes . thus , dendritic and synaptic abnormalities in the hippocampus , either morphological or functional , would be expected to significantly impact spatial cognition . indeed , neuropsychological investigations requiring the use of spatial information in problem solving indicate that deficits in hippocampal - mediated learning and memory processes are hallmarks of ds [ 17 , 18 ] . in this paper , we will provide an overview of the morphological and behavioral evidence for altered synaptic plasticity in ds with a focus on the hippocampus and discuss the insights provided by mouse models of this neurodevelopmental disorder into the potential molecular mechanisms contributing to these deficits . the basis for altered synaptic plasticity in ds can be found in changes in the physical structure of the dendrites . consistent with this idea and the observed deficits in cognition associated with ds , examination of postmortem brain tissue from ds individuals reveals profound alterations in dendritic and neuronal densities and morphology across many regions of the brain beginning in utero and persisting throughout life . by 40 weeks gestation , less discrete lamination is observed in the neocortex of ds fetuses with lower and higher cell densities observed in the visual cortex and superior temporal neocortex , respectively [ 19 , 20 ] . these changes may result , in part , from reductions in serotonin , dopamine , and gaba levels in the fetal ds cortex since , during development , neurotransmitters such as these can act as neurotrophic factors assisting with neuronal migration , axon guidance , and neurite development . initially , dendritic expansion is enhanced in ds infants , but , by the first to second year of life , this trend reverses to become a deficit [ 19 , 28 ] which persists into adulthood [ 19 , 29 ] . consistent with adverse changes in dendrite morphology , synaptogenesis is also aberrant in ds fetuses [ 19 , 32 , 33 ] and remains deficient in adulthood . hippocampal volume , that continues to decrease with age in ds individuals , was found to be inversely correlated with the degree of cognitive impairment . cognitive tests such as the cambridge neuropsychological testing automated battery ( cantab ) and the arizona cognitive test battery ( actb ) , the latter specifically tailored to address ds deficits , indicate that hippocampal function is particularly impacted by the ds genetic condition [ 17 , 39 ] . these morphological and cognitive deficits are consistent with aberrant synaptic plasticity , and , indeed , while difficult to measure directly in human subjects , evidence suggests that plasticity is reduced at least in the motor cortex of ds individuals . examination of resting glucose metabolism in the cerebral cortex of ds individuals found enhanced uptake in brain regions associated with cognition suggesting cellular hyperactivity in those areas . to better understand the functional consequences resulting from altered network morphologies as well as investigate potential alterations in intracellular signaling cascades contributing to aberrant plasticity over the past few decades , several mouse models of down syndrome have been developed to further our understanding of the link between enhanced gene dosage and ds phenotypes such as altered plasticity and cognition . it is important to note that , in spite of the mosaicism and gene deletions , many ds phenotypes have been replicated in this model [ 46 , 48 , 49 ] . other mouse models have taken advantage of the homology between regions of hsa21 and mouse chromosomes 10 , 16 , and 17 ( mmu10 , 16 , 17 ) making models in which these genes are triplicated highly useful in understanding the genetic basis of ds phenotypes [ 50 , 51 ] . as this is a relatively new model , however , most research has been conducted using the ts65dn segmental trisomic mouse [ 5355 ] which is trisomic for more than 50% genes of hsa21 homologs [ 56 , 57 ] and has well - documented ds - like deficits in behavioral tasks such as those relying upon declarative memory ( novel object recognition and spontaneous alternation tasks ) and the proper encoding and recollection of spatial information ( radial arm and morris water mazes ) [ 5863 ] . while the ts65dn mouse is the only mouse model of ds to have a freely segregating supernumerary chromosome , they are also trisomic for 60 genes that do not have hsa21 homologs , and the impact of overexpression of these genes on ts65dn phenotypes remains to be determined . similar to the ts65dn mouse but with smaller triplicated mmu16 segments are the ts1cje and ts1rhr mouse models . the reduced severity of ds - like deficits in mice with fewer trisomic genes highlights one of the powerful aspects of mouse models : the ability to control expression of certain hsa21 homologs to assess their contribution to specific ds phenotypes . those deficits associated with the hippocampus , whose function is notably altered in ds individuals [ 17 , 39 ] , will be the focus of the remainder of this paper . mouse models of ds , including the ts65dn strain , show similar detrimental changes in neuronal and dendritic morphologies observed in humans . the neocortex of ts65dn mice contains fewer excitatory neurons but an increased number of a subset of inhibitory neurons relative to euploid controls , a phenotype that was reversed by normalizing the expression levels of olig1/2 . in the dentate granule cells of the hippocampus , there is a shift of inhibitory synaptic connections away from the dendritic shafts and onto the necks . such a change would be expected to increase the efficacy of inhibitory synaptic transmission given the significantly reduced volume of the spine neck compared to the shaft . synaptic plasticity in the hippocampus is often investigated in the context of long - term potentiation ( ltp ) in which high - frequency activation of specific inputs in the hippocampus results in a long - lasting potentiation of synaptic responses along the excited afferent pathway . first described in the anesthetized rabbit , this phenomenon is believed to be a fundamental mechanism underlying memory formation [ 8 , 74 ] and is suppressed in ts65dn ( depicted in figure 1 for the ca1 region of the hippocampus ) [ 44 , 75 , 76 ] and ts1cje [ 66 , 67 ] but not in ts1rhr mice ( however , see ) as well as mice trisomic for hsa21 syntenic regions of mmu16 and mmu17 or those carrying an almost complete copy of hsa21 [ 46 , 48 ] . as outlined above , structural changes suggest that inhibition is exaggerated in the trisomic hippocampus . consistent with this idea is the observation that ltp in the dentate gyrus and ca1 regions of ts65dn hippocampal slices , induced by high - frequency stimulation and theta burst protocols , respectively , can be rescued by the gabaa antagonists picrotoxin [ 75 , 76 ] . blockade of gabaa receptors in hippocampal slices from ts1cje and ts1rhr mice rescues ltp deficits in the dentate gyrus in these ds mouse models as well [ 67 , 68 ] . this latter effect can be reversed with the uncompetitive nmda receptor antagonist memantine and also improves the cognitive performance of ts65dn mice [ 7981 ] . these results draw a clear link between altered synaptic plasticity in the hippocampus and cognitive performance in the ts65dn mouse model of down syndrome . elevated intracellular calcium levels trigger a cascade of intracellular messengers that ultimately lead to the induction and maintenance of synaptic plasticity ( both ltp and ltd depending on the kinetics ) . activation of postsynaptic nmda receptors ( nmdars ) concomitant with the depolarization of the postsynaptic membrane is sufficient to relieve the magnesium block of nmda channels leading to an influx of calcium into the intracellular postsynaptic space . in the case of ltp , the rise of intracellular calcium leads to the activation of calcium calmodulin - dependent protein kinase ii ( camkii ) , a necessary step for initiating nmdar - dependent ltp . indeed , cognitive deficits associated with angelman syndrome were reversed in a mouse model of the disorder by reducing the levels of camkii phosphorylated at thr305/306 . in ts65dn mice , we found that baseline levels of camkii phosphorylated at thr286 are elevated in the hippocampus . excessive basal phosphorylation of the camkii site leading to constitutive activation could leave the ds modeling trisomic network in a saturated state unable to shift to more potentiated levels . one of the substrates targeted by camkii during the initial expression of ltp is the serine 831 residue on glur1 subunits of ampa receptors [ 94 , 95 ] . in ts65dn mice , we find that baseline levels of phosphorylated serine 831 in synaptically located glur1 receptors are elevated . this apparent increase in ampa channel conductance appears not to have any significant effect on baseline excitatory synaptic transmission which is normal in the ts65dn hippocampus [ 45 , 75 , 76 , 93 ] . in particular , evidence suggests that it is involved in initiating the protein synthesis required for the late phase of ltp [ 97 , 98 ] . dephosphorylation of glur1 subunits should reduce the conductance levels of affected ampa receptors resulting in a reduction of synaptic strength . pka also enhances the activity of rcan1 , an inhibitor of calcineurin which contributes to ampa receptor internalization and reductions in nmda receptor mean open time . in ts65dn mice , we found that pka activity is reduced in the hippocampus , which should adversely affect ltp by reducing protein expression required for the late phase . this latter effect is offset , however , by the overexpression of the gene encoding rcan1 in ds and ts65dn mice . how these factors contribute to the enhancement of ltd in ts65dn hippocampi [ 45 , 78 ] remains to be determined . in addition to glur1 subunits , both camkii and pka converge on another common effector , the mitogen - activated protein kinase ( mapk / erk ) , that is associated with a host of synaptic - plasticity - related cellular processes . additionally , it is believed that lateral diffusion of extrasynaptic ampa receptors containing glur1 subunits into the postsynaptic density ( psd ) is a major contributor to ltp expression . this process is assisted by ras / erk phosphorylation of stargazin on extrasynaptic ampa receptors enabling them to be structurally secured at the synapse to psd95 . in the ts65dn hippocampus , erk phosphorylation this would be expected to adversely affect the insertion of new ampa receptors into the psd as well as morphological restructuring of synaptic spines observed after ltp in normal mice [ 113 , 114 ] . consistent with this notion , in ds individuals , bdnf blood plasma levels are approximately 5 times higher than in age - matched controls . examination of bdnf levels in ds mouse models presents a complex picture . in ts65dn mice , in the hippocampus , both no difference and a reduction compared to control have been reported . in the latter case , the reduction in bdnf levels was associated with decreased neurogenesis and was reversible through treatment with fluoxetine . in the ts1cje mouse model of ds , bdnf is overexpressed in the hippocampus , particularly in the dentate gyrus and ca1 regions and in the dendrites of dissociated hippocampal neurons grown in culture . increased bdnf levels in ts1cje mice hippocampi were associated with greater levels of phosphorylated akt - mtor and expression of glur1 protein which could not be further enhanced with exogenous supplemental bdnf suggesting this pathway related to synaptic plasticity is saturated in these mice preventing further contributions to ltp . conversely , the differences in observed bdnf levels could be related to the different numbers of genes overexpressed in these two mouse lines [ 53 , 65 , 122 ] or , as mice of differing age groups were used in the studies , may reflect differences in expression levels as a function of age . however , the observation that rapamycin has a restorative effect on phosphorylated akt - mtor levels in ts1cje suggests a potential therapeutic mechanism for improving cognition in ds individuals possibly by normalizing a pathway involved in synaptic plasticity . as mentioned above this initiating step relies heavily upon depolarization of the postsynaptic membrane to relieve the voltage - dependent magnesium block of nmda channels . any phenomenon that reduces the ability of the postsynaptic membrane to depolarize would thus be expected to adversely affect plasticity . girk2 is encoded by the gene kcnj6 which is located on the chromosomal segment triplicated in ds and ts65dn mice , and , consequently , elevated expression levels have been found in the ts65dn hippocampus [ 50 , 123 ] . selectively reducing the expression level of girk2 by crossing euploid and ts65dn mice with mice heterozygous for girk2 ( girk2 ) resulted in a gene dosage - dependent change in the resting membrane potential and facilitation of ltp in girk2 knockout mice . selective overexpression of girk2 alone in mice results in cognitive deficits , reduced depotentiation ( a functional reversal of potentiation at a synapse ) , and enhanced ltd . thus , girk channels , activated by gabab and other g - protein coupled receptors , appear to act as a break on synaptic plasticity in the ts65dn hippocampus . in the normal hippocampus , frequency - based synaptic plasticity at the ca3-ca1 synapse , coupled with a feed - forward inhibition loop from stratum oriens alveus interneurons that suppress inputs to distal ca1 dendrites , enables segregation of information flow through these two pathways . during high - frequency synaptic activity , the ca3-ca1 synapse would be expected to undergo ltp , increasing the excitatory drive to ca1 pyramidal neurons and , consequently , enhanced suppression of inputs to distal ca1 dendrites by the feed - forward inhibition loop ( figure 3(a ) ) . electroencephalogram ( eeg ) recordings from ds individuals suggest that such a preferential suppression of high - frequency information flow may result from overexpression of hsa21 genes . compared to controls , ds individuals have increased power at low eeg frequencies and a corresponding reduction in power at higher frequencies . while it is not clear that hippocampal activity is accurately reflected in eeg recordings , abnormal eegs findings are consistent with aberrant processing of high - frequency information in ds individuals and ts65dn mice . a number of studies using the ts65dn mouse model of ds have examined the possibility of pharmacologically reversing cognitive deficits ( reviewed in ) . application of the gabaa receptor antagonist , picrotoxin to hippocampal slices from ts65dn mice rescues ltp in the dentate gyrus and ca1 region . chronic administration of low doses of picrotoxin or other gabaa receptor antagonists ( pentylenetetrazole or bilobalide ) improves cognition in ts65dn mice suggesting that the efficacy of this class of pharmacological agents could be tested for reversing impaired cognition in ds . careful screening of similar drugs or design of pharmacological compounds with similar blocking capabilities but reduced propensities for inducing seizures may prove to be effective treatments . currently , a small molecule targeting gabaa receptors developed by f. hoffmann - la roche ltd ( pharmaceutical pipeline molecule rg1662 http://www.roche.com/roche_pharma_pipeline.htm ) is in clinical trials with the goal of safely improving cognition in ds individuals . when housed in enriched environments ( larger cage with novel objects such as toys and running wheels ) , trisomic mice performed as well as euploid littermates in the morris water maze and had normalized hippocampal ltp [ 136 , 137 ] . the benefits of environmental enrichment appear to be linked in part to regulation of excess inhibition in the neocortex and hippocampus . release of gaba from synaptosomes isolated from the hippocampus and neocortex is elevated in ts65dn mice , an effect that is reversed by environmental enrichment . in adult rats with amblyopia ( via monocular deprivation during critical period ) , environmental enrichment reversed visual deficits reduced gaba levels in the visual cortex contralateral to the deprived eye while increasing plasticity . it thus appears possible to regulate aberrant levels of inhibition in trisomic mice behaviorally without pharmacological intervention and achieve similar behavioral outcomes without the concerns associated with nonspecific actions or adverse side - effects of drugs . deficits in neurogenesis in the dentate gyrus and forebrain subventricular zone in ts65dn mice are also reversed following environmental enrichment and may add an additional therapeutic layer to the beneficial effect of a decrease in inhibitory tone . early behavioral intervention techniques designed to improve development in ds children show great promise [ 141 , 142 ] suggesting that this comparatively easily translatable therapeutic approach , either used alone or in combination with pharmacological agents , could potentially increase cognitive capacities in ds individuals . this belief is bolstered by experiments where drugs that normalize aberrant plasticity in hippocampal slices isolated from mouse models of ds also confer improvements in cognition in in intact adult mice . the reduced excitatory neuronal densities and deficits in dendritic morphologies present in individuals with ds diminish the capacity of their neural networks in general to undergo neuroplastic adaptations . combined with the deficits in signaling pathways reported in ts65dn mice , evidence strongly suggests that synaptic plasticity is severely impaired in ds neural networks . by understanding how plasticity is perturbed , we can design therapies to reverse these phenotypes and ultimately improve cognition in ds individuals .

hypertension ( ht ) is one of the most common risk factors for cardiovascular disease . a longstanding increase in blood pressure ( bp ) it has been demonstrated that changes in atrial size and fibrosis cause dispersion of refractoriness and alterations in atrial conduction . as a result , arrhythmia , pulsed wave tissue doppler imaging ( tdi ) is one of the noninvasive , easy applicable and accessible methods to evaluate atrial electromechanical conduction properties . sequential analysis of atrial electromechanical coupling by tdi allows analysis of atrial electromechanical coupling between different regions . total atrial activation time and interatrial electromechanical delay ( defined by the dispersion of right and left atrial electromechanical coupling ) are mostly used tdi - derived parameters . previous studies have shown that prolongation of these parameters promote af . the aims of this study were ( 1 ) to identify early changes in atrial electromechanical conduction among hypertensive patients using tdi , ( 2 ) to assess the parameters that affect atrial electromechanical conduction . a total of 76 patients ( 41 male , mean age 52.6 9.0 years ) , with grade i or ii ht according to the european society of cardiology / european society of ht classification and a disease duration of maximum 6 months and 41 age - matched healthy volunteers ( 22 male , mean age 49.8 7.9 years ) were included in the study . patients with atrioventricular or intraventricular conduction defects on electrocardiogram ( ecg ) , left ventricular ( lv ) ejection fraction ( ef ) < 50% , significant valvular abnormalities , history of coronary artery disease , suspicion of secondary ht , history of diabetes mellitus , thyroid disorders , pulmonary diseases , and renal failure , electrolyte imbalance , technically insufficient echocardiographic and ecg data were excluded . all subjects were in sinus rhythm and none of them were taking medications with antiarrhythmic properties ( including beta - blockers and nondihydropyridine calcium channel blockers ) , tricyclic antidepressants , and antipsychotic drugs causing prolongation of pr duration . the maximal treadmill exercise test according to bruce protocol was performed to the majority of study subjects to rule out the coronary artery disease . all study groups understood the purpose , process of the study and provided written informed consent before participation to the study . standard 12-lead ecg recordings were obtained using a recorder ( philips , pagewriter trim ii ecg machine , davis medical electronics , california , usa ) set at a rate of 50 mm / s paper speed and 2 mv / cm standardization in the supine position . p - wave duration was measured manually with calipers and magnifying glass by a qualified investigator who was blinded to the study . the onset of the p - wave was defined as the junction between isoelectric line and the start of the p - wave first upward deflection . the offset of the p - wave was defined as the junction between the end of the p - wave downward deflection and the isoelectric line . maximum p - wave duration ( pmax ) in any of the 12-lead surface ecgs was measured . transthoracic echocardiographic examinations were performed using commercially available cardiac ultrasound scanner ( acuson sequoia 512 system with 2.54.0 mhz transducer , siemens mountain view , california , usa ) in the left lateral position according to the criteria of the american society of echocardiography . loops from five cardiac cycles were acquired at end - expiration during quiet breathing . during echocardiography , the lv mass ( lvm ) was calculated with the devereux formula as lvm ( g ) = 1.04 ( [ lvid + pwt + ivst]lvid ) 14 ( lvid : lv internal dimension ; pwt : posterior wall thickness ; ivst : ivs thickness ) and indexed to the body surface area ( dividing lvm by body surface area ) . left atrial volumes were determined at ventricular end - systole according to area - length method using apical 4- and 2-chamber views . the peak early diastolic velocity ( e ) was measured from the transmitral pulsed wave doppler spectra . the pulsed wave tdi were performed with a sample volume of 5 mm and the monitor sweep speed was set at 100 mm / s to optimize the spectral display of myocardial velocities . the average pulsed wave tdi - derived early diastolic myocardial velocity ( e ) was obtained from the septal and lateral sides of the mitral annulus . then , e / e ratio was calculated to provide an estimation of lv filling pressures . the myocardial performance index ( mpi ) , which is a noninvasive doppler measurement of global ventricular function , was calculated as the ratio of isovolumic contraction and relaxation times to ejection time . all doppler spectral velocities were recorded at sweep speed of 100 mm / s and values were averaged over 3 consecutive beats . standard two - dimensional apical 4-chamber views were obtained to visualize the right atrium ( ra ) and left atrium ( la ) free walls . pulsed wave tdi recordings were applied , and the sample volume was placed as parallel as possible to ensure the optimal angle of imaging at the ra free wall , the interatrial septum ( ias ) , and the la free wall just below the level of the atrioventricular annulus . the time intervals from the onset of the p - wave on the surface ecg to the beginning of the late diastolic tdi signal ( a ) , which is called atrial electromechanical coupling time , were measured from the la ( pla ) , ias ( pis ) , and ra ( pra ) . the following parameters were calculated : left intra - atrial delay ( la delay ) was defined as the differences between pla and pis ; right intra - atrial delay ( ra delay ) was defined as the differences between pis and pra ; interatrial delay was defined as the difference between pla and pra . the intraobserver variability was assessed in twenty subjects at random from the patient study group by repeating measurements under the same basal conditions . to test the interobserver variability , correlation coefficients ( pearson 's r ) for intraobserver variability were 0.89 , 0.87 , 0.90 for pla , pis and pra ; and interobserver variability were 0.83 , 0.81 , and 0.85 for pla , pis , and pra , respectively . the statistical analyses were performed with the spss package , version 11.0 ( spss inc . , all continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were defined as percentages . continuous variables with normal distributions were compared using the unpaired student 's t - test . pearson 's correlation coefficients were used to assess the strength of the relationship between continuous variables . a stepwise , multiple regression analysis was used to identify significant determinants of electromechanical delay . thus , all predetermined independent variables that correlated with a p < 0.1 in the pearson 's correlations were inserted into stepwise , linear regression analysis . a total of 76 patients ( 41 male , mean age 52.6 9.0 years ) , with grade i or ii ht according to the european society of cardiology / european society of ht classification and a disease duration of maximum 6 months and 41 age - matched healthy volunteers ( 22 male , mean age 49.8 7.9 years ) were included in the study . patients with atrioventricular or intraventricular conduction defects on electrocardiogram ( ecg ) , left ventricular ( lv ) ejection fraction ( ef ) < 50% , significant valvular abnormalities , history of coronary artery disease , suspicion of secondary ht , history of diabetes mellitus , thyroid disorders , pulmonary diseases , and renal failure , electrolyte imbalance , technically insufficient echocardiographic and ecg data were excluded . all subjects were in sinus rhythm and none of them were taking medications with antiarrhythmic properties ( including beta - blockers and nondihydropyridine calcium channel blockers ) , tricyclic antidepressants , and antipsychotic drugs causing prolongation of pr duration . the maximal treadmill exercise test according to bruce protocol was performed to the majority of study subjects to rule out the coronary artery disease . all study groups understood the purpose , process of the study and provided written informed consent before participation to the study . standard 12-lead ecg recordings were obtained using a recorder ( philips , pagewriter trim ii ecg machine , davis medical electronics , california , usa ) set at a rate of 50 mm / s paper speed and 2 mv / cm standardization in the supine position . p - wave duration was measured manually with calipers and magnifying glass by a qualified investigator who was blinded to the study . the onset of the p - wave was defined as the junction between isoelectric line and the start of the p - wave first upward deflection . the offset of the p - wave was defined as the junction between the end of the p - wave downward deflection and the isoelectric line . maximum p - wave duration ( pmax ) in any of the 12-lead surface ecgs was measured . transthoracic echocardiographic examinations were performed using commercially available cardiac ultrasound scanner ( acuson sequoia 512 system with 2.54.0 mhz transducer , siemens mountain view , california , usa ) in the left lateral position according to the criteria of the american society of echocardiography . loops from five cardiac cycles were acquired at end - expiration during quiet breathing . during echocardiography , the lv mass ( lvm ) was calculated with the devereux formula as lvm ( g ) = 1.04 ( [ lvid + pwt + ivst]lvid ) 14 ( lvid : lv internal dimension ; pwt : posterior wall thickness ; ivst : ivs thickness ) and indexed to the body surface area ( dividing lvm by body surface area ) . left atrial volumes were determined at ventricular end - systole according to area - length method using apical 4- and 2-chamber views . the peak early diastolic velocity ( e ) was measured from the transmitral pulsed wave doppler spectra . the pulsed wave tdi were performed with a sample volume of 5 mm and the monitor sweep speed was set at 100 mm / s to optimize the spectral display of myocardial velocities . the average pulsed wave tdi - derived early diastolic myocardial velocity ( e ) was obtained from the septal and lateral sides of the mitral annulus . then , e / e ratio was calculated to provide an estimation of lv filling pressures . the myocardial performance index ( mpi ) , which is a noninvasive doppler measurement of global ventricular function , was calculated as the ratio of isovolumic contraction and relaxation times to ejection time . all doppler spectral velocities were recorded at sweep speed of 100 mm / s and values were averaged over 3 consecutive beats . standard two - dimensional apical 4-chamber views were obtained to visualize the right atrium ( ra ) and left atrium ( la ) free walls . pulsed wave tdi recordings were applied , and the sample volume was placed as parallel as possible to ensure the optimal angle of imaging at the ra free wall , the interatrial septum ( ias ) , and the la free wall just below the level of the atrioventricular annulus . the time intervals from the onset of the p - wave on the surface ecg to the beginning of the late diastolic tdi signal ( a ) , which is called atrial electromechanical coupling time , were measured from the la ( pla ) , ias ( pis ) , and ra ( pra ) . the following parameters were calculated : left intra - atrial delay ( la delay ) was defined as the differences between pla and pis ; right intra - atrial delay ( ra delay ) was defined as the differences between pis and pra ; interatrial delay was defined as the difference between pla and pra . the intraobserver variability was assessed in twenty subjects at random from the patient study group by repeating measurements under the same basal conditions . to test the interobserver variability , correlation coefficients ( pearson 's r ) for intraobserver variability were 0.89 , 0.87 , 0.90 for pla , pis and pra ; and interobserver variability were 0.83 , 0.81 , and 0.85 for pla , pis , and pra , respectively . the statistical analyses were performed with the spss package , version 11.0 ( spss inc . , all continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were defined as percentages . continuous variables with normal distributions were compared using the unpaired student 's t - test . pearson 's correlation coefficients were used to assess the strength of the relationship between continuous variables . a stepwise , multiple regression analysis was used to identify significant determinants of electromechanical delay . thus , all predetermined independent variables that correlated with a p < 0.1 in the pearson 's correlations were inserted into stepwise , linear regression analysis . the two groups were similar with regards to age , sex , body mass index , and smoking status . by definition , hypertensive subjects had higher bp . most of the patients ( 56.6% ) were given medication within the 90 days after index diagnosis with at least one of the antihypertensive drugs ( i.e. , angiotensin-2 receptor blockers , angiotensin - converting enzyme inhibitors , dihydropyridine calcium channel blockers and diuretics ) . maximum p - wave duration was not different between the groups ( 98.6 11.7 ms vs. 98.1 9.9 ms , p = 0.824 , respectively ) . only nine patients with ht ( 12% ) and three subjects in the control group ( 7% ) had p - wave durations 110 ms . patients characteristics of the study population data are presented as n ( % ) or mean standard deviation . bmi : body mass index ; ace - i : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; ccb : calcium channel blocker ; ns : not significant ; bp : blood pressure ; 1mmhg=0.133kpa . lvm index , la volume index , e / e and mpi ratios were higher in the hypertensive group ( p < 0.001 , p = 0.014 , p = 0.011 and p < 0.001 , respectively ) . lv : left ventricle ; ra : right atrium ; la : left atrium ; ef : ejection fraction ; e / e : early diastolic velocity / early diastolic myocardial velocity ; mpi : myocardial performance index ; ns : not significant . the results of atrial electromechanical coupling parameters measured by tdi are summarized in table 3 . pra and pis did not differ significantly between the groups ( p > 0.05 ) . pla was significantly prolonged in the hypertensive group . to determine the influential factors of pla , we examined potential variables which thought to be echocardiographically relevant : lvm index , la dimension , la volume index , e / e ratio , mpi , and pmax . we found a significant correlation between pla and pmax ( r = 0.289 , p = 0.002 ) , la volume index ( r = 0.274 , p = 0.003 ) and lvm index ( r = 0.347 , p = 0.001 ) . the stepwise linear regression analysis showed that the lvm index and pmax were independent predictors of pla ( r = 0.436 , p < 0.001 , and p = 0.002 , respectively ) . atrial electromechanical coupling findings measured by tissue doppler imaging data are presented as mean standard deviation . pla : left atrial electromechanical coupling time ; pis : interatrial septum electromechanical coupling time ; pra : right atrial electromechanical coupling time ; la : left intraatrial ; ra : right intraatrial ; ns : not significant . although there was a positive correlation between the interatrial delay and lvm index ( r = 0.316 , p = 0.001 ) , there was no significant correlation between the interatrial delay and la volume index , la diameter , ra diameter , lv mpi and e / e ratio . in stepwise linear regression analysis , lvm index was demonstrated to be independently associated with interatrial delay ( r = 0.348 , p = 0.001 ) . the la delay and however , there was no relationship between la delay and pmax , la volume index , e / e ratio , lv mpi ratio , or lvm index . the two groups were similar with regards to age , sex , body mass index , and smoking status . by definition , hypertensive subjects had higher bp . most of the patients ( 56.6% ) were given medication within the 90 days after index diagnosis with at least one of the antihypertensive drugs ( i.e. , angiotensin-2 receptor blockers , angiotensin - converting enzyme inhibitors , dihydropyridine calcium channel blockers and diuretics ) . maximum p - wave duration was not different between the groups ( 98.6 11.7 ms vs. 98.1 9.9 ms , p = 0.824 , respectively ) . only nine patients with ht ( 12% ) and three subjects in the control group ( 7% ) had p - wave durations 110 ms . patients characteristics of the study population data are presented as n ( % ) or mean standard deviation . bmi : body mass index ; ace - i : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; ccb : calcium channel blocker ; ns : not significant ; bp : blood pressure ; 1mmhg=0.133kpa . lvm index , la volume index , e / e and mpi ratios were higher in the hypertensive group ( p < 0.001 , p = 0.014 , p = 0.011 and p < 0.001 , respectively ) . lv : left ventricle ; ra : right atrium ; la : left atrium ; ef : ejection fraction ; e / e : early diastolic velocity / early diastolic myocardial velocity ; mpi : myocardial performance index ; ns : not significant . the results of atrial electromechanical coupling parameters measured by tdi are summarized in table 3 . pra and pis did not differ significantly between the groups ( p > 0.05 ) . pla was significantly prolonged in the hypertensive group . to determine the influential factors of pla , we examined potential variables which thought to be echocardiographically relevant : lvm index , la dimension , la volume index , e / e ratio , mpi , and pmax . we found a significant correlation between pla and pmax ( r = 0.289 , p = 0.002 ) , la volume index ( r = 0.274 , p = 0.003 ) and lvm index ( r = 0.347 , p = 0.001 ) . the stepwise linear regression analysis showed that the lvm index and pmax were independent predictors of pla ( r = 0.436 , p < 0.001 , and p = 0.002 , respectively ) . atrial electromechanical coupling findings measured by tissue doppler imaging data are presented as mean standard deviation . pla : left atrial electromechanical coupling time ; pis : interatrial septum electromechanical coupling time ; pra : right atrial electromechanical coupling time ; la : left intraatrial ; ra : right intraatrial ; ns : not significant . although there was a positive correlation between the interatrial delay and lvm index ( r = 0.316 , p = 0.001 ) , there was no significant correlation between the interatrial delay and la volume index , la diameter , ra diameter , lv mpi and e / e ratio . in stepwise linear regression analysis , lvm index was demonstrated to be independently associated with interatrial delay ( r = 0.348 , p = 0.001 ) . the la delay and however , there was no relationship between la delay and pmax , la volume index , e / e ratio , lv mpi ratio , or lvm index . atrial conduction disorders and resultant abnormalities are associated with a higher risk of paroxysmal atrial tachyarrhythmia . in most studies , however , atrial electromechanical coupling and intra- and inter - atrial conduction delays can be performed noninvasively by tdi , which has already been proved useful for evaluating atrial impairment in patients with paroxysmal af and various diseases such as mitral stenosis , type 2 diabetes mellitus , scleroderma . in this study , we used tdi to assess atrial electromechanical conduction properties in patients with ht and without history of af . contrary to la size and la volume index , prolonged atrial conduction times indicate both electrical and structural remodelling of the atria . we hypothesized that pulsed wave tdi would identify early abnormalities in atrial conduction . in our study , it was shown that pla , intra- and inter - atrial conduction delays were prolonged . the prolongation of atrial conduction times in this study confirmed the previous findings of emiroglu et al . however , in that study , the study population included hypertensive patients for at least 4 years and whose bp remained elevated while our study population included patients with grade i and ii ht with disease duration of maximum 6 months . similarly to our results , ermis et al . , showed that intra- and inter - atrial conduction times were prolonged in prehypertensive patients . on the basis of these findings , we speculate that intra- and inter - atrial electromechanical delay and pla might occur at early times of ht . our study also showed that lvm index and pmax were independent predictors of pla which is in line with the study of emiroglu et al . prolonged total atrial conduction times are often present in patients with increased p - wave duration on 12-lead ecg . prolonged p - wave duration is also known as an electrophysiological marker to evaluate the inhomogeneous conduction of sinus impulses . in the present study , although pmax did not differ significantly between the groups , there was significant difference in the tdi - derived pla . the absence of prolonged p - wave on the 12-lead ecg might be because of the study population , which many of them recently diagnosed as hypertensive , and having normal atrial dimension and volume . thus , tdi could demonstrate earlier period of atrial electromechanical impairment before appearance of obvious prolongation of p - wave on the 12-lead ecg . moreover , the main difference of our study from the previous ones is that the previous studies mostly included patients with documented paroxysmal af , which means atrial electrical abnormalities have already developed . we demonstrated that lvm index is an independent predictor of both prolonged interatrial delay and pla . the structural and electrophysiological changes in the myocardium caused by ht might play role in this relationship . the baseline severity of lv hypertrophy has been reported to be a significant predictor of the development of the new - onset af in many studies . previous studies have shown that reducing bp with blockade of the renin - angiotensin system , reduce af incidence in patients with ht . potential mechanisms of af prevention supported by these studies include regression of lv muscle mass . it is possible to consider that la structural remodeling ( i.e. , altered atrial tissue architecture or interstitial fibrosis ) concomitant to lv hypertrophy might lead to nonhomogeneity of atrial conduction , dispersion of atrial refractoriness and conduction delay which is associated with a higher risk of atrial tachyarrhythmia . taken together these findings with our results , we suggest that la structural remodelling detected by tdi might be used to predict atrial tachyarrhythmia . further studies of the relationship of changes in atrial conduction to changing values of lvm index during antihypertensive therapy will hopefully provide greater insights into this potential mechanistic link between lvm regression and the development of atrial tachyarrhythmia . as this was a cross - sectional study , follow - up of the patients for arrhythmic episodes and the relationship between bp control and abnormal atrial electrical activity could not be done . therefore , we can not comment if prolongation of intra- and inter - atrial delay precipitated af in patients with ht with a disease duration of 6 months . also , the population size was relatively small . moreover , we included the patients with known ht with a disease duration of maximum 6 months according to the patients reports . for this reason , long - term follow - up and large - scale prospective studies are needed to determine the clinical predictive value of prolonged intra- and interatrial electromechanical delays in this population . the results of our study showed prolonged pla and intra- , inter - atrial delays might be an important marker of early atrial structural and electromechanical changes in patients with grade i and ii ht and without history of af and with disease duration of maximum 6 months . tdi is a noninvasive method to assess the atrial electromechanical remodeling even in patients with normal pmax on surface 12-lead ecg .

background : hypertension ( ht ) is associated with atrial electrophysiological abnormalities . echocardiographic pulsed wave tissue doppler imaging ( tdi ) is one of the noninvasive methods for evaluation of atrial electromechanical properties . the aims of our study were to investigate the early changes in atrial electromechanical conduction in patients with ht and to assess the parameters that affect atrial electromechanical conduction.methods:seventy-six patients with ht ( 41 males , mean age 52.6 9.0 years ) and 41 controls ( 22 males , mean age 49.8 7.9 years ) were included in the study . atrial electromechanical coupling at the right ( pra ) , left ( pla ) , interatrial septum ( pis ) were measured with tdi . intra- ( right : pis - pra , left : pla - pis ) and inter - atrial ( pla - pra ) electromechanical delays were calculated . maximum p - wave duration ( pmax ) was calculated from 12-lead electrocardiogram.results:atrial electromechanical coupling at pla ( 76.6 14.1 ms vs. 82.9 15.8 ms , p = 0.036 ) , left intra - atrial ( 10.9 5.0 ms vs. 14.0 9.7 ms , p = 0.023 ) , right intra - atrial ( 10.6 7.8 ms vs. 14.5 10.1 ms , p = 0.035 ) , and interatrial electromechanical ( 21.4 9.8 ms vs. 28.3 12.7 ms , p = 0.003 ) delays were significantly longer in patients with ht . the linear regression analysis showed that left ventricular ( lv ) mass index and pmax were significantly associated with pla ( p = 0.001 and p = 0.002 , respectively ) , and the lv mass index was the only related factor for interatrial delay ( p = 0.001).conclusions : intra- and interatrial electromechanical delay , pla were significantly prolonged in hypertensive patients . lv mass index and pmax were significantly associated with pla , and the lv mass index was the only related factor for interatrial delay . the atrial tdi can be a valuable method to assess the early changes of atrial electromechanical conduction properties in those patients .

I M Study population Electrocardiography Transthoracic echocardiography Atrial tissue Doppler imaging Reproducibility of measurements Statistical analysis R Patient characteristics Transthoracic echocardiographic parameters and atrial tissue Doppler imaging D Financial support and sponsorship Conflicts of interest

hypertension ( ht ) is one of the most common risk factors for cardiovascular disease . as a result , arrhythmia , pulsed wave tissue doppler imaging ( tdi ) is one of the noninvasive , easy applicable and accessible methods to evaluate atrial electromechanical conduction properties . sequential analysis of atrial electromechanical coupling by tdi allows analysis of atrial electromechanical coupling between different regions . total atrial activation time and interatrial electromechanical delay ( defined by the dispersion of right and left atrial electromechanical coupling ) are mostly used tdi - derived parameters . the aims of this study were ( 1 ) to identify early changes in atrial electromechanical conduction among hypertensive patients using tdi , ( 2 ) to assess the parameters that affect atrial electromechanical conduction . a total of 76 patients ( 41 male , mean age 52.6 9.0 years ) , with grade i or ii ht according to the european society of cardiology / european society of ht classification and a disease duration of maximum 6 months and 41 age - matched healthy volunteers ( 22 male , mean age 49.8 7.9 years ) were included in the study . patients with atrioventricular or intraventricular conduction defects on electrocardiogram ( ecg ) , left ventricular ( lv ) ejection fraction ( ef ) < 50% , significant valvular abnormalities , history of coronary artery disease , suspicion of secondary ht , history of diabetes mellitus , thyroid disorders , pulmonary diseases , and renal failure , electrolyte imbalance , technically insufficient echocardiographic and ecg data were excluded . all subjects were in sinus rhythm and none of them were taking medications with antiarrhythmic properties ( including beta - blockers and nondihydropyridine calcium channel blockers ) , tricyclic antidepressants , and antipsychotic drugs causing prolongation of pr duration . all study groups understood the purpose , process of the study and provided written informed consent before participation to the study . p - wave duration was measured manually with calipers and magnifying glass by a qualified investigator who was blinded to the study . the onset of the p - wave was defined as the junction between isoelectric line and the start of the p - wave first upward deflection . the offset of the p - wave was defined as the junction between the end of the p - wave downward deflection and the isoelectric line . maximum p - wave duration ( pmax ) in any of the 12-lead surface ecgs was measured . transthoracic echocardiographic examinations were performed using commercially available cardiac ultrasound scanner ( acuson sequoia 512 system with 2.54.0 mhz transducer , siemens mountain view , california , usa ) in the left lateral position according to the criteria of the american society of echocardiography . during echocardiography , the lv mass ( lvm ) was calculated with the devereux formula as lvm ( g ) = 1.04 ( [ lvid + pwt + ivst]lvid ) 14 ( lvid : lv internal dimension ; pwt : posterior wall thickness ; ivst : ivs thickness ) and indexed to the body surface area ( dividing lvm by body surface area ) . the peak early diastolic velocity ( e ) was measured from the transmitral pulsed wave doppler spectra . the pulsed wave tdi were performed with a sample volume of 5 mm and the monitor sweep speed was set at 100 mm / s to optimize the spectral display of myocardial velocities . the average pulsed wave tdi - derived early diastolic myocardial velocity ( e ) was obtained from the septal and lateral sides of the mitral annulus . the myocardial performance index ( mpi ) , which is a noninvasive doppler measurement of global ventricular function , was calculated as the ratio of isovolumic contraction and relaxation times to ejection time . standard two - dimensional apical 4-chamber views were obtained to visualize the right atrium ( ra ) and left atrium ( la ) free walls . pulsed wave tdi recordings were applied , and the sample volume was placed as parallel as possible to ensure the optimal angle of imaging at the ra free wall , the interatrial septum ( ias ) , and the la free wall just below the level of the atrioventricular annulus . the time intervals from the onset of the p - wave on the surface ecg to the beginning of the late diastolic tdi signal ( a ) , which is called atrial electromechanical coupling time , were measured from the la ( pla ) , ias ( pis ) , and ra ( pra ) . the following parameters were calculated : left intra - atrial delay ( la delay ) was defined as the differences between pla and pis ; right intra - atrial delay ( ra delay ) was defined as the differences between pis and pra ; interatrial delay was defined as the difference between pla and pra . to test the interobserver variability , correlation coefficients ( pearson 's r ) for intraobserver variability were 0.89 , 0.87 , 0.90 for pla , pis and pra ; and interobserver variability were 0.83 , 0.81 , and 0.85 for pla , pis , and pra , respectively . , all continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were defined as percentages . pearson 's correlation coefficients were used to assess the strength of the relationship between continuous variables . a stepwise , multiple regression analysis was used to identify significant determinants of electromechanical delay . thus , all predetermined independent variables that correlated with a p < 0.1 in the pearson 's correlations were inserted into stepwise , linear regression analysis . a total of 76 patients ( 41 male , mean age 52.6 9.0 years ) , with grade i or ii ht according to the european society of cardiology / european society of ht classification and a disease duration of maximum 6 months and 41 age - matched healthy volunteers ( 22 male , mean age 49.8 7.9 years ) were included in the study . patients with atrioventricular or intraventricular conduction defects on electrocardiogram ( ecg ) , left ventricular ( lv ) ejection fraction ( ef ) < 50% , significant valvular abnormalities , history of coronary artery disease , suspicion of secondary ht , history of diabetes mellitus , thyroid disorders , pulmonary diseases , and renal failure , electrolyte imbalance , technically insufficient echocardiographic and ecg data were excluded . all subjects were in sinus rhythm and none of them were taking medications with antiarrhythmic properties ( including beta - blockers and nondihydropyridine calcium channel blockers ) , tricyclic antidepressants , and antipsychotic drugs causing prolongation of pr duration . p - wave duration was measured manually with calipers and magnifying glass by a qualified investigator who was blinded to the study . the onset of the p - wave was defined as the junction between isoelectric line and the start of the p - wave first upward deflection . the offset of the p - wave was defined as the junction between the end of the p - wave downward deflection and the isoelectric line . maximum p - wave duration ( pmax ) in any of the 12-lead surface ecgs was measured . transthoracic echocardiographic examinations were performed using commercially available cardiac ultrasound scanner ( acuson sequoia 512 system with 2.54.0 mhz transducer , siemens mountain view , california , usa ) in the left lateral position according to the criteria of the american society of echocardiography . during echocardiography , the lv mass ( lvm ) was calculated with the devereux formula as lvm ( g ) = 1.04 ( [ lvid + pwt + ivst]lvid ) 14 ( lvid : lv internal dimension ; pwt : posterior wall thickness ; ivst : ivs thickness ) and indexed to the body surface area ( dividing lvm by body surface area ) . the peak early diastolic velocity ( e ) was measured from the transmitral pulsed wave doppler spectra . the average pulsed wave tdi - derived early diastolic myocardial velocity ( e ) was obtained from the septal and lateral sides of the mitral annulus . the myocardial performance index ( mpi ) , which is a noninvasive doppler measurement of global ventricular function , was calculated as the ratio of isovolumic contraction and relaxation times to ejection time . standard two - dimensional apical 4-chamber views were obtained to visualize the right atrium ( ra ) and left atrium ( la ) free walls . pulsed wave tdi recordings were applied , and the sample volume was placed as parallel as possible to ensure the optimal angle of imaging at the ra free wall , the interatrial septum ( ias ) , and the la free wall just below the level of the atrioventricular annulus . the time intervals from the onset of the p - wave on the surface ecg to the beginning of the late diastolic tdi signal ( a ) , which is called atrial electromechanical coupling time , were measured from the la ( pla ) , ias ( pis ) , and ra ( pra ) . the following parameters were calculated : left intra - atrial delay ( la delay ) was defined as the differences between pla and pis ; right intra - atrial delay ( ra delay ) was defined as the differences between pis and pra ; interatrial delay was defined as the difference between pla and pra . to test the interobserver variability , correlation coefficients ( pearson 's r ) for intraobserver variability were 0.89 , 0.87 , 0.90 for pla , pis and pra ; and interobserver variability were 0.83 , 0.81 , and 0.85 for pla , pis , and pra , respectively . pearson 's correlation coefficients were used to assess the strength of the relationship between continuous variables . thus , all predetermined independent variables that correlated with a p < 0.1 in the pearson 's correlations were inserted into stepwise , linear regression analysis . most of the patients ( 56.6% ) were given medication within the 90 days after index diagnosis with at least one of the antihypertensive drugs ( i.e. maximum p - wave duration was not different between the groups ( 98.6 11.7 ms vs. 98.1 9.9 ms , p = 0.824 , respectively ) . only nine patients with ht ( 12% ) and three subjects in the control group ( 7% ) had p - wave durations 110 ms . lvm index , la volume index , e / e and mpi ratios were higher in the hypertensive group ( p < 0.001 , p = 0.014 , p = 0.011 and p < 0.001 , respectively ) . the results of atrial electromechanical coupling parameters measured by tdi are summarized in table 3 . pla was significantly prolonged in the hypertensive group . to determine the influential factors of pla , we examined potential variables which thought to be echocardiographically relevant : lvm index , la dimension , la volume index , e / e ratio , mpi , and pmax . we found a significant correlation between pla and pmax ( r = 0.289 , p = 0.002 ) , la volume index ( r = 0.274 , p = 0.003 ) and lvm index ( r = 0.347 , p = 0.001 ) . the stepwise linear regression analysis showed that the lvm index and pmax were independent predictors of pla ( r = 0.436 , p < 0.001 , and p = 0.002 , respectively ) . atrial electromechanical coupling findings measured by tissue doppler imaging data are presented as mean standard deviation . pla : left atrial electromechanical coupling time ; pis : interatrial septum electromechanical coupling time ; pra : right atrial electromechanical coupling time ; la : left intraatrial ; ra : right intraatrial ; ns : not significant . although there was a positive correlation between the interatrial delay and lvm index ( r = 0.316 , p = 0.001 ) , there was no significant correlation between the interatrial delay and la volume index , la diameter , ra diameter , lv mpi and e / e ratio . in stepwise linear regression analysis , lvm index was demonstrated to be independently associated with interatrial delay ( r = 0.348 , p = 0.001 ) . most of the patients ( 56.6% ) were given medication within the 90 days after index diagnosis with at least one of the antihypertensive drugs ( i.e. maximum p - wave duration was not different between the groups ( 98.6 11.7 ms vs. 98.1 9.9 ms , p = 0.824 , respectively ) . only nine patients with ht ( 12% ) and three subjects in the control group ( 7% ) had p - wave durations 110 ms . patients characteristics of the study population data are presented as n ( % ) or mean standard deviation . lvm index , la volume index , e / e and mpi ratios were higher in the hypertensive group ( p < 0.001 , p = 0.014 , p = 0.011 and p < 0.001 , respectively ) . the results of atrial electromechanical coupling parameters measured by tdi are summarized in table 3 . pla was significantly prolonged in the hypertensive group . to determine the influential factors of pla , we examined potential variables which thought to be echocardiographically relevant : lvm index , la dimension , la volume index , e / e ratio , mpi , and pmax . we found a significant correlation between pla and pmax ( r = 0.289 , p = 0.002 ) , la volume index ( r = 0.274 , p = 0.003 ) and lvm index ( r = 0.347 , p = 0.001 ) . the stepwise linear regression analysis showed that the lvm index and pmax were independent predictors of pla ( r = 0.436 , p < 0.001 , and p = 0.002 , respectively ) . atrial electromechanical coupling findings measured by tissue doppler imaging data are presented as mean standard deviation . pla : left atrial electromechanical coupling time ; pis : interatrial septum electromechanical coupling time ; pra : right atrial electromechanical coupling time ; la : left intraatrial ; ra : right intraatrial ; ns : not significant . although there was a positive correlation between the interatrial delay and lvm index ( r = 0.316 , p = 0.001 ) , there was no significant correlation between the interatrial delay and la volume index , la diameter , ra diameter , lv mpi and e / e ratio . in stepwise linear regression analysis , lvm index was demonstrated to be independently associated with interatrial delay ( r = 0.348 , p = 0.001 ) . in most studies , however , atrial electromechanical coupling and intra- and inter - atrial conduction delays can be performed noninvasively by tdi , which has already been proved useful for evaluating atrial impairment in patients with paroxysmal af and various diseases such as mitral stenosis , type 2 diabetes mellitus , scleroderma . in this study , we used tdi to assess atrial electromechanical conduction properties in patients with ht and without history of af . in our study , it was shown that pla , intra- and inter - atrial conduction delays were prolonged . however , in that study , the study population included hypertensive patients for at least 4 years and whose bp remained elevated while our study population included patients with grade i and ii ht with disease duration of maximum 6 months . , showed that intra- and inter - atrial conduction times were prolonged in prehypertensive patients . on the basis of these findings , we speculate that intra- and inter - atrial electromechanical delay and pla might occur at early times of ht . our study also showed that lvm index and pmax were independent predictors of pla which is in line with the study of emiroglu et al . prolonged total atrial conduction times are often present in patients with increased p - wave duration on 12-lead ecg . prolonged p - wave duration is also known as an electrophysiological marker to evaluate the inhomogeneous conduction of sinus impulses . the absence of prolonged p - wave on the 12-lead ecg might be because of the study population , which many of them recently diagnosed as hypertensive , and having normal atrial dimension and volume . thus , tdi could demonstrate earlier period of atrial electromechanical impairment before appearance of obvious prolongation of p - wave on the 12-lead ecg . moreover , the main difference of our study from the previous ones is that the previous studies mostly included patients with documented paroxysmal af , which means atrial electrical abnormalities have already developed . the baseline severity of lv hypertrophy has been reported to be a significant predictor of the development of the new - onset af in many studies . previous studies have shown that reducing bp with blockade of the renin - angiotensin system , reduce af incidence in patients with ht . , altered atrial tissue architecture or interstitial fibrosis ) concomitant to lv hypertrophy might lead to nonhomogeneity of atrial conduction , dispersion of atrial refractoriness and conduction delay which is associated with a higher risk of atrial tachyarrhythmia . further studies of the relationship of changes in atrial conduction to changing values of lvm index during antihypertensive therapy will hopefully provide greater insights into this potential mechanistic link between lvm regression and the development of atrial tachyarrhythmia . as this was a cross - sectional study , follow - up of the patients for arrhythmic episodes and the relationship between bp control and abnormal atrial electrical activity could not be done . therefore , we can not comment if prolongation of intra- and inter - atrial delay precipitated af in patients with ht with a disease duration of 6 months . for this reason , long - term follow - up and large - scale prospective studies are needed to determine the clinical predictive value of prolonged intra- and interatrial electromechanical delays in this population . the results of our study showed prolonged pla and intra- , inter - atrial delays might be an important marker of early atrial structural and electromechanical changes in patients with grade i and ii ht and without history of af and with disease duration of maximum 6 months . tdi is a noninvasive method to assess the atrial electromechanical remodeling even in patients with normal pmax on surface 12-lead ecg .

for over a decade , improving hospital performance on patient safety and patient outcomes have been national priorities in the united states.1 , 2 , 3 , 4 , 5 , 6 , 7 hospitals with high inhospital adverse event , mortality , or unplanned readmission rates are considered to provide poorer quality of care.7 , 8 , 9 studies estimate that the excess annual cost attributed to measurable medical errors is around $ 17 billion10 and that unplanned readmissions result in an additional $ 15 billion in annual medicare expenditures.4 although extensive nationwide efforts have focused on improving patient safety and outcomes for acute myocardial infarction ( ami ) in particular,11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 with some recent data showing that patient safety and outcomes for acute cardiovascular disease have improved,19 , 20 , 21 inhospital adverse events , shortterm mortality , and unplanned readmission rates for ami patients remain high , with considerable variation across hospitals.22 efforts to improve patient safety , reduce hospital mortality , and reduce unplanned readmission rates are largely pursued independently . their impact on one another is unclear or thought to be small.23 , 24 , 25 , 26 previous studies have shown that patients with 1 or more adverse events are more likely to have higher mortality or to be readmitted , but these studies were limited to a few measures and local data sources.27 , 28 , 29 , 30 , 31 , 32 they also focused on patientlevel analyses , rather than connecting hospitallevel performance on patient safety with other important hospitallevel outcomes . an association between adverse events and outcomes of individual patients indicates that adverse events may lead to worse outcomes , but it may not reflect hospital performance . a patient experiencing more adverse events may be sicker , and these events may not reflect the performance of the hospital that treated that patient . depending on case mix , a hospital with a high raw adverse event rate may also have a high raw mortality or readmission rate , but a high raw adverse event rate does not indicate that the hospital has worse performance in patient safety . how hospital performance on patient safety associates with hospital performance on outcomes is unclear from a patientlevel analysis . without evidence of a link between patient safety and mortality and readmission at the hospital level , hospitals may not view their investment in improving safety as benefiting readmission rates or even mortality and hence may not recognize harm reduction as a potential strategy for lessening these key patient outcomes . accordingly , we sought to investigate the association at the hospitallevel between a wide range of inhospital adverse event rates and both mortality and readmission rates for medicare feeforservice patients with ami , an acute condition that may be more sensitive to inhospital adverse events . to accomplish this analysis on a national scale , we used data from the agency for healthcare research and quality ( ahrq ) 's medicare patient safety monitoring system ( mpsms ) , the nation 's largest randomly selected hospital medical recordabstracted patient safety database,33 and data from the centers for medicare & medicaid services ( cms ) , which includes hospital performance on mortality and readmissions for over 4000 medicarecertified hospitals , to assess the relationship between hospital performance on patient safety and hospital performance on 30day allcause mortality and unplanned readmissions for medicare feeforservice patients discharged with ami . the mpsms data , at the individual patient level , includes 21 inhospital adverse event measures19 ( table 1 ) jointly developed by federal agencies and private health care organizations.33 starting from 2009 , mpsms medical records were drawn from the cms validation sample for processofcare measures required for the hospital inpatient quality reporting program , which includes a multistage random sample of allpayer patients hospitalized for ami , heart failure , pneumonia , and major surgical conditions . all hospitals in the mpsms data were randomly selected . hospital inpatient quality reporting program validation sample also includes 200 hospitals targeted based on past data quality or performance ( selected after the random sample was drawn ) ; these were not included in mpsms data . approximately 17 500 records were randomly selected from 4000 hospitals for 2009 and 34 000 records from 1400 hospitals for 2010 and 2011 . the 2012 data included 27 200 records from 1110 hospitals and 2013 data included 17 900 records from 730 hospitals . each hospital in 20102013 contributed approximately equal numbers of randomly selected records to the mpsms . medical record abstraction was conducted at the cms clinical data abstraction center . based on 80 monthly reabstractions , the agreement between abstraction and reabstraction ranged from 94% to 99% for data elements used to identify adverse events.19 , 27 , 33 list of the 21 adverse event measures the cms mortality and readmission data for ami are available from the hospital compare website , at the individual hospital level . the data includes hospitalspecific total discharges , 30day allcause riskstandardized mortality , and readmission rates for all acute care hospitals treating medicare feeforservice patients aged 65 years or older . cms combined data from a 3year period to report mortality and readmission rates for each hospital . the 2 recent reporting periods were july 1 , 2009 through june 30 , 2012 and july 1 , 2010 through june 30 , 2013 . not all hospitals were repeated in each reporting period . to include the maximum number of hospitals from both the mpsms and cms data , we combined the two 3yearperiod data sets into a single 4yearperiod data set from july 1 , 2009 to june 30 , 2013 . if a hospital had data in both periods , we averaged its mortality and readmission rates weighted by its total discharges in each period for each outcome . to align with the cms data , we restricted mpsms data to only medicare feeforservice patients aged 65 years or older discharged for ami from july 1 , 2009 to june 30 , 2013 . patients discharged to a skilled nursing facility or other levelofcare facilities , except transferred to an acute care hospital , were included in the mpsms as well as in the cms data . patient characteristics for the cms data have been reported elsewhere.24 , 25 , 26 patient characteristics for mpsms data were obtained from medical records , including demographics ( age , sex , and race ) , common clinical comorbidities ( heart failure , obesity , coronary artery disease , renal disease , cerebrovascular disease , chronic obstructive pulmonary disease , cancer , and diabetes ) , and smoking status . hospital characteristics were obtained from the american hospital association 's 2013 annual survey database , including teaching status ( teaching vs nonteaching ) ; joint commission certification status ( yes / no ) ; geographical location ( urban vs rural ) ; ownership ( private notforprofit vs others ) ; bed size ; nursetopatient ratio ; perform cardiac catheterization and/or percutaneous coronary intervention procedures ( yes / no ) ; and perform coronary artery bypass graft surgery ( yes / no ) . our primary outcomes were the hospitalspecific riskstandardized 30day allcause mortality and allcause unplanned readmission rates . mortality was measured from the date of admission ; readmission was measured from the date of discharge for patients who were discharged alive and were not transferred to another acutecare hospital . we measured inhospital adverse events as the rate of occurrence of inhospital adverse events for which patients were at risk . the numerator for this rate is the number of adverse events that occurred , and the denominator is the number of adverse events for which patients were at risk.19 all patients were at risk for at least 2 adverse events , inhospital falls , and hospitalacquired pressure ulcers , but only a subset of patients were at risk for other events ( eg , only patients who received warfarin were at risk for a warfarinassociated adverse event ) . we reported the rate of patients with 1 or more adverse events during a hospitalization . the numerator for this rate is the number of patients who experienced 1 or more adverse events , and the denominator is the number of patients in the mpsms data ( table 2).19 among the 21 inhospital adverse event measures , no patient had a knee joint replacement and 3 had a hip joint replacement during the index ami hospitalization . we included these 3 patients because they had a principal discharge diagnosis code of ami . illustration of calculating of adverse event rates the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk . the numerator is the number of patients who experienced 1 or more adverse events and the denominator is the number of hospitalizations . using the cms riskstandardized method for profiling hospitals,34 , 35 , 36 , 37 we fitted a hierarchical generalized linear model with a poisson link function to model the occurrence of adverse events as a function of patients age , sex , and clinical comorbidities . the number of adverse events for which patients were at risk was the offset in the model . using this model we linked the hospitalspecific riskstandardized occurrence rate of adverse events data with the hospitalspecific riskstandardized mortality and readmission data at the hospital level . to ensure that each hospital had an adequate sample size , we included only hospitals with at least 30 patients in the cms data and at least 40 adverse events for which patients were at risk over the study period ( table 3 ) . the threshold of 40 was selected because the median number of adverse events for which patients were at risk per hospital was 36 for ami . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . of hospitals by outcome we identified 2530 unique hospitals that each had 30 or more patients from the cms mortality and readmission data and 921 unique hospitals that each had 40 or more adverse events for which patients were at risk from the mpsms adverse events data . the threshold of 40 was selected based on the fact that the median numbers of adverse events for which patients were at risk per hospital was 36 for ami . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . after linking the mortality and readmission data to adverse events data ami indicates acute myocardial infarction ; cms , centers for medicare & medicaid services ; mpsms , medicare patient safety monitoring system . the median numbers of adverse events for which patients were at risk was 36 for ami . to describe patient and hospital characteristics and the rates of individual adverse event measures , we classified each hospital into tertiles based on its riskstandardized occurrence rate of adverse events . we also classified each hospital into tertiles based on its number of adverse events for which patients were at risk and compared the 2 distributions . to quantify the difference in the occurrence of adverse events across hospitals , we computed betweenhospital variation in the occurrence rate of adverse events after accounting for patient characteristics using an odds ratio representing the odds of a patient experiencing an adverse event when treated in a hospital 1 sd above the overall average relative to being treated at a hospital 1 sd below the overall average.38 to evaluate the association between hospital performance on patient safety and hospital performance on mortality , we fitted 2 mixedeffects models to estimate the : ( 1 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific observed occurrence rate of adverse events and ( 2 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific riskstandardized occurrence rate of adverse events , with and without adjusting for the hospital characteristics described previously . we repeated these models for the readmission measure as well . because different states have different patient safety regulations and quality improvement initiatives that may impact hospital performance within a state , all models were fitted with statespecific random intercepts to account for withinstate and betweenstate variation and weighted by the hospitalspecific number of adverse events for which patients were at risk . we conducted a second analysis that included all hospitals regardless of their volume of possible adverse events for which patients were at risk . the institutional review board ( irb ) at solutions irb ( http://www.solutionsirb.com ) determined that the requirement for informed consent could be waived based on the nature of the study . the mpsms data , at the individual patient level , includes 21 inhospital adverse event measures19 ( table 1 ) jointly developed by federal agencies and private health care organizations.33 starting from 2009 , mpsms medical records were drawn from the cms validation sample for processofcare measures required for the hospital inpatient quality reporting program , which includes a multistage random sample of allpayer patients hospitalized for ami , heart failure , pneumonia , and major surgical conditions . all hospitals in the mpsms data were randomly selected . hospital inpatient quality reporting program validation sample also includes 200 hospitals targeted based on past data quality or performance ( selected after the random sample was drawn ) ; these were not included in mpsms data . approximately 17 500 records were randomly selected from 4000 hospitals for 2009 and 34 000 records from 1400 hospitals for 2010 and 2011 . the 2012 data included 27 200 records from 1110 hospitals and 2013 data included 17 900 records from 730 hospitals . each hospital in 20102013 contributed approximately equal numbers of randomly selected records to the mpsms . medical record abstraction was conducted at the cms clinical data abstraction center . based on 80 monthly reabstractions , the agreement between abstraction and reabstraction ranged from 94% to 99% for data elements used to identify adverse events.19 , 27 , 33 list of the 21 adverse event measures the cms mortality and readmission data for ami are available from the hospital compare website , at the individual hospital level . the data includes hospitalspecific total discharges , 30day allcause riskstandardized mortality , and readmission rates for all acute care hospitals treating medicare feeforservice patients aged 65 years or older . cms combined data from a 3year period to report mortality and readmission rates for each hospital . the 2 recent reporting periods were july 1 , 2009 through june 30 , 2012 and july 1 , 2010 through june 30 , 2013 . not all hospitals were repeated in each reporting period . to include the maximum number of hospitals from both the mpsms and cms data , we combined the two 3yearperiod data sets into a single 4yearperiod data set from july 1 , 2009 to june 30 , 2013 . if a hospital had data in both periods , we averaged its mortality and readmission rates weighted by its total discharges in each period for each outcome . to align with the cms data , we restricted mpsms data to only medicare feeforservice patients aged 65 years or older discharged for ami from july 1 , 2009 to june 30 , 2013 . patients discharged to a skilled nursing facility or other levelofcare facilities , except transferred to an acute care hospital , were included in the mpsms as well as in the cms data . patient characteristics for the cms data have been reported elsewhere.24 , 25 , 26 patient characteristics for mpsms data were obtained from medical records , including demographics ( age , sex , and race ) , common clinical comorbidities ( heart failure , obesity , coronary artery disease , renal disease , cerebrovascular disease , chronic obstructive pulmonary disease , cancer , and diabetes ) , and smoking status . hospital characteristics were obtained from the american hospital association 's 2013 annual survey database , including teaching status ( teaching vs nonteaching ) ; joint commission certification status ( yes / no ) ; geographical location ( urban vs rural ) ; ownership ( private notforprofit vs others ) ; bed size ; nursetopatient ratio ; perform cardiac catheterization and/or percutaneous coronary intervention procedures ( yes / no ) ; and perform coronary artery bypass graft surgery ( yes / no ) . our primary outcomes were the hospitalspecific riskstandardized 30day allcause mortality and allcause unplanned readmission rates . mortality was measured from the date of admission ; readmission was measured from the date of discharge for patients who were discharged alive and were not transferred to another acutecare hospital . we measured inhospital adverse events as the rate of occurrence of inhospital adverse events for which patients were at risk . the numerator for this rate is the number of adverse events that occurred , and the denominator is the number of adverse events for which patients were at risk.19 all patients were at risk for at least 2 adverse events , inhospital falls , and hospitalacquired pressure ulcers , but only a subset of patients were at risk for other events ( eg , only patients who received warfarin were at risk for a warfarinassociated adverse event ) . we reported the rate of patients with 1 or more adverse events during a hospitalization . the numerator for this rate is the number of patients who experienced 1 or more adverse events , and the denominator is the number of patients in the mpsms data ( table 2).19 among the 21 inhospital adverse event measures , no patient had a knee joint replacement and 3 had a hip joint replacement during the index ami hospitalization . we included these 3 patients because they had a principal discharge diagnosis code of ami . illustration of calculating of adverse event rates the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk . the numerator is the number of patients who experienced 1 or more adverse events and the denominator is the number of hospitalizations . using the cms riskstandardized method for profiling hospitals,34 , 35 , 36 , 37 we fitted a hierarchical generalized linear model with a poisson link function to model the occurrence of adverse events as a function of patients age , sex , and clinical comorbidities . the number of adverse events for which patients were at risk was the offset in the model . using this model we linked the hospitalspecific riskstandardized occurrence rate of adverse events data with the hospitalspecific riskstandardized mortality and readmission data at the hospital level . to ensure that each hospital had an adequate sample size , we included only hospitals with at least 30 patients in the cms data and at least 40 adverse events for which patients were at risk over the study period ( table 3 ) . the threshold of 40 was selected because the median number of adverse events for which patients were at risk per hospital was 36 for ami . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . of hospitals by outcome we identified 2530 unique hospitals that each had 30 or more patients from the cms mortality and readmission data and 921 unique hospitals that each had 40 or more adverse events for which patients were at risk from the mpsms adverse events data . the threshold of 40 was selected based on the fact that the median numbers of adverse events for which patients were at risk per hospital was 36 for ami . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . after linking the mortality and readmission data to adverse events data ami indicates acute myocardial infarction ; cms , centers for medicare & medicaid services ; mpsms , medicare patient safety monitoring system . the median numbers of adverse events for which patients were at risk was 36 for ami . to describe patient and hospital characteristics and the rates of individual adverse event measures , we classified each hospital into tertiles based on its riskstandardized occurrence rate of adverse events . we also classified each hospital into tertiles based on its number of adverse events for which patients were at risk and compared the 2 distributions . to quantify the difference in the occurrence of adverse events across hospitals , we computed betweenhospital variation in the occurrence rate of adverse events after accounting for patient characteristics using an odds ratio representing the odds of a patient experiencing an adverse event when treated in a hospital 1 sd above the overall average relative to being treated at a hospital 1 sd below the overall average.38 to evaluate the association between hospital performance on patient safety and hospital performance on mortality , we fitted 2 mixedeffects models to estimate the : ( 1 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific observed occurrence rate of adverse events and ( 2 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific riskstandardized occurrence rate of adverse events , with and without adjusting for the hospital characteristics described previously . we repeated these models for the readmission measure as well . because different states have different patient safety regulations and quality improvement initiatives that may impact hospital performance within a state , all models were fitted with statespecific random intercepts to account for withinstate and betweenstate variation and weighted by the hospitalspecific number of adverse events for which patients were at risk . we conducted a second analysis that included all hospitals regardless of their volume of possible adverse events for which patients were at risk . the institutional review board ( irb ) at solutions irb ( http://www.solutionsirb.com ) determined that the requirement for informed consent could be waived based on the nature of the study . the final sample included 793 hospitals ( table 3 ) from the linked mpsms and cms data . the hospital median ( interquartile range [ iqr ] ) number of adverse events for which patients were at risk was 59 ( 29 ) ( figure 1 , right panel ) . the cms data included 167 734 and 174 457 ami patients for the mortality and readmission outcomes , respectively . the median ( iqr ) numbers of patients per hospital for the mortality and readmission measures were 149 ( 226 ) and 158 ( 262 ) ( figure 1 , left and middle panels ) . table 4 , which is at the hospital level , shows hospital characteristics by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . teaching hospitals and hospitals with a high proportion of patients with diabetes mellitus , obesity , and cerebrovascular disease had higher occurrence rates of adverse events ( table 4 ) . box and whisker plots of the distributions of hospitalspecific patient volumes and the number of adverse events for which patients were at risk . the length of the box represents the interquartile range ( iqr ) , the horizontal line in the box interior represents the median , the whiskers represent the 1.5 iqr of the 25th quartile or 1.5 iqr of the 75th quartile , and the dots represent outliers . the median ( iqr ) numbers of patients per hospital for the mortality and unplanned readmission measures were 149 ( 226 ) and 158 ( 262 ) ; the median ( iqr ) number of adverse events for which patients were at risk was 59 ( 29 ) . hospital characteristics ami indicates acute myocardial infarction ; iqr , interquartile range ; mpsms , medicare patient safety monitoring system . hospitals ( n=793 ) had at least 30 patients for mortality and readmission measures and had at least 40 adverse events for which patients were at risk . based on mpsms abstracted data . for the occurrence of adverse events , mortality , and readmission , respectively , the ranges ( minimum to maximum ) of riskstandardized rates were 10.3% to 19.9% , 14.3% to 21.8% , and 1.4% to 19.3% . hospitalspecific riskstandardized occurrence rate of adverse events was widely dispersed across hospitals ( figure 2 , right panel ) . the mean hospitalspecific mortality and readmission rates were 14.7% and 18.0% , respectively ( figure 2 , left and middle panels ) . box and whisker plots of the distributions of hospitalspecific 30day allcause riskstandardized mortality and unplanned readmission rates and hospitalspecific riskstandardized occurrence rates of adverse events for which patients were at risk . the length of the box represents the interquartile range ( iqr ) , the horizontal line in the box interior represents the median , the whiskers represent the 1.5 iqr of the 25th quartile or 1.5 iqr of the 75th quartile , and the dots represent outliers . for mortality , unplanned readmission , and adverse events , respectively , the ranges ( minimum to maximum ) of riskstandardized rates were 10.3% to 19.9% , 14.3% to 21.8% , and 1.4% to 19.3% . table 5 , which is at the patient level , shows the number of patients and adverse event rates for each of the 21 adverse measures as well as the 2 composite adverse event rates measures by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . the number of adverse events for which patients were at risk per hospitalization varied by patient characteristics and ranged from 2 to 18 . on average , patients were at risk for 7.4 events per hospitalization . hospitals with a high riskstandardized occurrence rate of adverse events usually had more patients at risk . for the first , second , and third tertiles , respectively , patients were at risk for 6.7 , 7.3 , and 8.3 events per hospitalization ( table 5 ) . nevertheless , 32.0% of hospitals in the highest tertile of adverse events for which patients were at risk were in the lowest tertile of riskstandardized occurrence rate of adverse events . conversely , 25.8% of hospitals in the lowest tertile of patients at risk had a higher rate of adverse events ( figure 3 ) . hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk by tertiles , july 1 , 2009 to june 30 , 2013 ( aged 65 years or older , medicare feeforservice patients discharged with acute myocardial infarction ) n / a indicates not applicable . the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk . the numerator is the number of patients who experienced one or more adverse events and the denominator is the number of hospitalizations . hospitalspecific riskstandardized occurrence rates of adverse events by tertile versus hospitalspecific total number of adverse events for which patients were at risk by tertile . of the 793 hospitals , the overall occurrence rate of adverse events , in which the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk , was 3.8% . the overall rate of patients with 1 or more adverse events per hospitalization , in which the numerator is the number of patients who experienced 1 or more adverse events and the denominator is the number of patients in the mpsms data , was 20.1% ( tables 2 and 5 ) . a change of 1% point in the occurrence rate of adverse events for which patients were at risk was associated with an average change of 4.8% points ( 95% ci , 4.74.9 ) in the rate of patients with 1 or more adverse events per hospitalization ( figure 4 ) . for teaching and nonteaching hospital groups , respectively , the means ( sd ) of riskstandardized hospitalspecific rate of occurrence of adverse events were 5.3 ( 18.7 ) and 4.1 ( 19.2 ) . relationship between the observed rate of patients had 1 or more adverse events and occurrence rate of adverse events for which patients were at risk . the odds of an adverse event occurring when treated at a hospital 1 sd above the overall average relative to the odds of an adverse event occurring when treated at a hospital 1 sd below the overall average were 3.86 ( 95% ci , 3.624.12 ) . the hospital characteristicadjusted associations between the observed occurrence rate of adverse events and riskstandardized mortality and readmission rates were statistically significant ( figure 5 , top panel ) . the hospital characteristicadjusted associations between the riskstandardized occurrence rate of adverse events and riskstandardized mortality and readmission rates were also statistically significant ( figure 5 , bottom panel ) . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with an average change in the riskstandardized mortality rate of 4.86% points ( 95% ci , 0.798.94 ) and an average change in the riskstandardized readmission rate of 3.44% points ( 95% ci , 0.196.68 ) . because the overall occurrence rate of adverse events was 3.8% , a 1% point change in this rate translates to a relative change of 26.3% . the second analysis , which included all hospitals regardless of their volume of adverse events for which patients were at risk , did not change the results substantially ( figure 6 ) . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with average changes in the riskstandardized mortality rate of 5.13% points ( 95% ci , 2.238.03 ) and riskstandardized readmission rate of 2.51% points ( 95% ci , 0.214.82 ) . point estimates and 95% cis of the associations between the hospitalspecific riskstandardized 30day mortality rate and hospitalspecific riskstandardized occurrence rate of adverse events and hospitalspecific riskstandardized 30day unplanned readmission rate and hospitalspecific riskstandardized occurrence rate of adverse events . hospital characteristics included in the adjusted models were teaching status ( teaching vs nonteaching ) ; joint commission certification status ( yes / no ) ; geographical location ( urban vs rural ) ; ownership ( private notforprofit vs others ) ; bed size ; nursetopatient ratio ; perform cardiac catheterization and/or percutaneous coronary intervention procedures ( yes / no ) ; and perform coronary artery bypass graft surgery ( yes / no ) . modela models the riskstandardized mortality or unplanned readmission rate as a function of hospitalspecific observed occurrence rate of adverse events ; modelb models the riskstandardized mortality or unplanned readmission rate as a function of hospitalspecific riskstandardized occurrence rate of adverse events . point estimates and 95% cis of the associations between hospital performance on mortality and unplanned readmission rates and hospital performance on the occurrence rate of adverse events , regardless of hospitals volume of adverse events for which patients were at risk . the final sample included 793 hospitals ( table 3 ) from the linked mpsms and cms data . the hospital median ( interquartile range [ iqr ] ) number of adverse events for which patients were at risk was 59 ( 29 ) ( figure 1 , right panel ) . the cms data included 167 734 and 174 457 ami patients for the mortality and readmission outcomes , respectively . the median ( iqr ) numbers of patients per hospital for the mortality and readmission measures were 149 ( 226 ) and 158 ( 262 ) ( figure 1 , left and middle panels ) . table 4 , which is at the hospital level , shows hospital characteristics by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . teaching hospitals and hospitals with a high proportion of patients with diabetes mellitus , obesity , and cerebrovascular disease had higher occurrence rates of adverse events ( table 4 ) . box and whisker plots of the distributions of hospitalspecific patient volumes and the number of adverse events for which patients were at risk . the length of the box represents the interquartile range ( iqr ) , the horizontal line in the box interior represents the median , the whiskers represent the 1.5 iqr of the 25th quartile or 1.5 iqr of the 75th quartile , and the dots represent outliers . the median ( iqr ) numbers of patients per hospital for the mortality and unplanned readmission measures were 149 ( 226 ) and 158 ( 262 ) ; the median ( iqr ) number of adverse events for which patients were at risk was 59 ( 29 ) . hospital characteristics ami indicates acute myocardial infarction ; iqr , interquartile range ; mpsms , medicare patient safety monitoring system . hospitals ( n=793 ) had at least 30 patients for mortality and readmission measures and had at least 40 adverse events for which patients were at risk . for the occurrence of adverse events , mortality , and readmission , respectively , the ranges ( minimum to maximum ) of riskstandardized rates were 10.3% to 19.9% , 14.3% to 21.8% , and 1.4% to 19.3% . hospitalspecific riskstandardized occurrence rate of adverse events was widely dispersed across hospitals ( figure 2 , right panel ) . the mean hospitalspecific mortality and readmission rates were 14.7% and 18.0% , respectively ( figure 2 , left and middle panels ) . box and whisker plots of the distributions of hospitalspecific 30day allcause riskstandardized mortality and unplanned readmission rates and hospitalspecific riskstandardized occurrence rates of adverse events for which patients were at risk . the length of the box represents the interquartile range ( iqr ) , the horizontal line in the box interior represents the median , the whiskers represent the 1.5 iqr of the 25th quartile or 1.5 iqr of the 75th quartile , and the dots represent outliers . for mortality , unplanned readmission , and adverse events , respectively , the ranges ( minimum to maximum ) of riskstandardized rates were 10.3% to 19.9% , 14.3% to 21.8% , and 1.4% to 19.3% . table 5 , which is at the patient level , shows the number of patients and adverse event rates for each of the 21 adverse measures as well as the 2 composite adverse event rates measures by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . the number of adverse events for which patients were at risk per hospitalization varied by patient characteristics and ranged from 2 to 18 . on average , patients were at risk for 7.4 events per hospitalization . hospitals with a high riskstandardized occurrence rate of adverse events usually had more patients at risk . for the first , second , and third tertiles , respectively , patients were at risk for 6.7 , 7.3 , and 8.3 events per hospitalization ( table 5 ) . nevertheless , 32.0% of hospitals in the highest tertile of adverse events for which patients were at risk were in the lowest tertile of riskstandardized occurrence rate of adverse events . conversely , 25.8% of hospitals in the lowest tertile of patients at risk had a higher rate of adverse events ( figure 3 ) . hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk by tertiles , july 1 , 2009 to june 30 , 2013 ( aged 65 years or older , medicare feeforservice patients discharged with acute myocardial infarction ) n / a indicates not applicable . the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk . the numerator is the number of patients who experienced one or more adverse events and the denominator is the number of hospitalizations . riskstandardized occurrence rates of adverse events by tertile versus hospitalspecific total number of adverse events for which patients were at risk by tertile . of the 793 hospitals , the overall occurrence rate of adverse events , in which the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk , was 3.8% . the overall rate of patients with 1 or more adverse events per hospitalization , in which the numerator is the number of patients who experienced 1 or more adverse events and the denominator is the number of patients in the mpsms data , was 20.1% ( tables 2 and 5 ) . a change of 1% point in the occurrence rate of adverse events for which patients were at risk was associated with an average change of 4.8% points ( 95% ci , 4.74.9 ) in the rate of patients with 1 or more adverse events per hospitalization ( figure 4 ) . for teaching and nonteaching hospital groups , respectively , the means ( sd ) of riskstandardized hospitalspecific rate of occurrence of adverse events were 5.3 ( 18.7 ) and 4.1 ( 19.2 ) . relationship between the observed rate of patients had 1 or more adverse events and occurrence rate of adverse events for which patients were at risk . the odds of an adverse event occurring when treated at a hospital 1 sd above the overall average relative to the odds of an adverse event occurring when treated at a hospital 1 sd below the overall average were 3.86 ( 95% ci , 3.624.12 ) . the hospital characteristicadjusted associations between the observed occurrence rate of adverse events and riskstandardized mortality and readmission rates were statistically significant ( figure 5 , top panel ) . the hospital characteristicadjusted associations between the riskstandardized occurrence rate of adverse events and riskstandardized mortality and readmission rates were also statistically significant ( figure 5 , bottom panel ) . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with an average change in the riskstandardized mortality rate of 4.86% points ( 95% ci , 0.798.94 ) and an average change in the riskstandardized readmission rate of 3.44% points ( 95% ci , 0.196.68 ) . because the overall occurrence rate of adverse events was 3.8% , a 1% point change in this rate translates to a relative change of 26.3% . the second analysis , which included all hospitals regardless of their volume of adverse events for which patients were at risk , did not change the results substantially ( figure 6 ) . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with average changes in the riskstandardized mortality rate of 5.13% points ( 95% ci , 2.238.03 ) and riskstandardized readmission rate of 2.51% points ( 95% ci , 0.214.82 ) . point estimates and 95% cis of the associations between the hospitalspecific riskstandardized 30day mortality rate and hospitalspecific riskstandardized occurrence rate of adverse events and hospitalspecific riskstandardized 30day unplanned readmission rate and hospitalspecific riskstandardized occurrence rate of adverse events . hospital characteristics included in the adjusted models were teaching status ( teaching vs nonteaching ) ; joint commission certification status ( yes / no ) ; geographical location ( urban vs rural ) ; ownership ( private notforprofit vs others ) ; bed size ; nursetopatient ratio ; perform cardiac catheterization and/or percutaneous coronary intervention procedures ( yes / no ) ; and perform coronary artery bypass graft surgery ( yes / no ) . modela models the riskstandardized mortality or unplanned readmission rate as a function of hospitalspecific observed occurrence rate of adverse events ; modelb models the riskstandardized mortality or unplanned readmission rate as a function of hospitalspecific riskstandardized occurrence rate of adverse events . point estimates and 95% cis of the associations between hospital performance on mortality and unplanned readmission rates and hospital performance on the occurrence rate of adverse events , regardless of hospitals volume of adverse events for which patients were at risk . in this study , we found that hospital performance on patient safety , measured by the hospitalspecific riskstandardized occurrence rate of adverse events for which patients were at risk , was associated with hospitalspecific riskstandardized 30day allcause mortality and unplanned readmission rates for patients with ami . adverse event rates may be a marker of overall hospital quality , including patient safety culture , which is increasingly being recognized as associated with patient outcomes.39 , 40 , 41 , 42 , 43 it is possible that some of the interventions to reduce the risk of adverse events could also improve other patient outcomes . it is also possible that changes made at the hospital level that reduce mortality and unplanned readmissions may also improve patient safety performance , as indicated by fewer adverse events.29 , 44 , 45 furthermore , patients who experience 1 or more adverse events may be at higher risk for mortality and readmission , specifically attributed to harm caused by the adverse event.19 , 27 , 28 , 29 , 30 , 31 , 32 our study , based on medical recordabstracted patient safety information , represents a unique , large , and recent investigation of the associations between hospital performance on patient safety and mortality and readmissions for medicare patients with ami in the united states . previous studies focused on the association between adverse events and mortality or readmission rates at the patient level . this study , however , extends that association to the hospital level by linking hospital performance on patient safety to hospital performance on mortality and readmission rates . in a patientlevel analysis , for example , lyder et al.27 found that patients who developed pressure ulcers during their hospitalizations had higher 30day mortality and readmission rates . friedman et al.28 studied 1.5 million surgical care patients across 1088 hospitals and found that the 30day readmission rate was 5% higher for patients with 1 or more inhospital adverse events when compared with those with no adverse events . vorhies et al.30 reported that adverse events were associated with increased readmissions for medicare feeforservice patients with hip replacements . we chose to study the hospital level because it is the most actionable , given that improvement efforts require system changes beyond the reach of the individual practitioner and patient . although the literature has emphasized patient socioeconomic influences on readmissions , our findings suggest that hospital specific factors also play a role . getting hospitals to invest in those improvement efforts is a major undertaking . by showing an association between patient safety and mortality and readmissions for ami patients at the hospital level although our findings do not establish a causal relationship , they suggest that patient safety improvement may be afforded a place among other common strategies to reduce mortality and unplanned readmissions . further research is warranted to determine whether such improvement measurably reduces mortality and unplanned readmissions for other conditions . although we found that hospitals with a higher volume of patients at risk were more likely to have a higher adverse event rate , not all hospitals with a higher volume of patients at risk had a higher rate of adverse events . in our study , 32% of hospitals with a higher volume of adverse events for which patients were at risk had a lower riskstandardized occurrence rate of adverse events , and 26% of hospitals with a lower volume of patients at risk had a higher rate of adverse events . thus , regardless of the hospital 's volume of patients at risk , hospitals with a higher adverse event rate have room to improve their care . for example , the overall patient atrisk rate for the catheterassociated urinary tract infection indicator was 35% , but we can reduce both the number of adverse events and the number of patients at risk for this indicator by not placing a urinary catheter unless absolutely necessary . a higher volume of patients at risk , may actually indicate overuse of certain health care interventions , and not necessarily reflect casemix differences . having more opportunities to harm is not an adequate rationale to harm more , but should be the impetus to harm less . care.1 our study illustrates that variation exists in the risk of adverse events across acute care hospitals in the united states . reducing such variation by focusing on patient safety interventions may favorably impact downstream outcomes , such as mortality and unplanned readmissions . because the occurrence rate of adverse events was 4% and the rate of patients with 1 or more adverse events was 20% for medicare patients discharged with ami , an absolute 1% point decrease in the occurrence rate of adverse events is equal to a relative decline of 25% in that rate itself , or an absolute 5% point reduction in the rate of patients with 1 or more adverse events . ample opportunities for reducing inhospital adverse events exist , including promoting transparency and facilitating open discussion to prevent recurring errors,46 implementing and meaningfully using electronic health records,47 adopting and implementing evidencebased patient safety strategies,48 and , most important , enhancing patient safety culture.49 our study has several limitations . we focused on adverse events that were both detected and documented during the index hospitalization and were unable to identify events that occurred but were not documented . restricted by the sample size of the mpsms data , we were not able to determine whether some of the adverse events have stronger relationships with mortality or unplanned readmission than others . restricted by the cms mortality and readmission data , only medicare feeforservice beneficiaries were included in this study , who were older and more female than the general population of patients with ami . limited by the scope of the mpsms data , our model to calculate the riskstandardized occurrence rate of adverse events might not fully account for patient characteristics . though it is possible that some proportion of the adverse events detected in mpsms data were not preventable , each of these 21 adverse event measures is characterized as being frequently preventable with the delivery of highquality care . still , this study distinguishes itself by the breadth and standardization of events measured and its national scope . for medicare feeforservice patients discharged with ami , hospitals with poorer patient safety performance tended to have poorer performance on 30day allcause mortality and unplanned readmission rates . this strengthens the evidence that mortality and unplanned readmissions reflect the quality of hospital care . this work was supported by contract hhsa290201200003c from the agency for healthcare research and quality , us department of health and human services ( rockville , md ) . dr krumholz is partially funded by grant 1u01hl10527002 ( krumholz , center for cardiovascular outcomes research at yale university ) from the national heart , lung , and blood institute . dr normand is partially supported by a grant ( r01 gm111339 , normand ) from the national institutes of health , and dr wang is partially supported by the us environmental protection agency ( rd83490001 , dominici ) , national institutes of health ( r21 es02258501 , dominici ; r21 es024012 , zanobetti ; r01 gm111339 , normand ; r01 es024332 , zanobetti ) , and the agency for healthcare research and quality ( k18 hs021991 , dominici ) . dr krumholz is the recipient of research grants from medtronic and from johnson & johnson , through yale university , to develop methods of clinical trial data sharing , and chairs a cardiac scientific advisory board for unitedhealth . drs krumholz and normand work under contract to the centers for medicare & medicaid services to develop and maintain performance measures .

backgroundlittle is known regarding the relationship between hospital performance on adverse event rates and hospital performance on 30day mortality and unplanned readmission rates for medicare feeforservice patients hospitalized for acute myocardial infarction ( ami).methods and resultsusing 20092013 medical recordabstracted patient safety data from the agency for healthcare research and quality 's medicare patient safety monitoring system and hospital mortality and readmission data from the centers for medicare & medicaid services , we fitted a mixedeffects model , adjusting for hospital characteristics , to evaluate whether hospital performance on patient safety , as measured by the hospitalspecific riskstandardized occurrence rate of 21 common adverse event measures for which patients were at risk , is associated with hospitalspecific 30day allcause riskstandardized mortality and unplanned readmission rates for medicare patients with ami . the unit of analysis was at the hospital level . the final sample included 793 acute care hospitals that treated 30 or more medicare patients hospitalized for ami and had 40 or more adverse events for which patients were at risk . the occurrence rate of adverse events for which patients were at risk was 3.8% . a 1% point change in the riskstandardized occurrence rate of adverse events was associated with average changes in the same direction of 4.86% points ( 95% ci , 0.798.94 ) and 3.44% points ( 95% ci , 0.196.68 ) for the riskstandardized mortality and unplanned readmission rates , respectively.conclusionsfor medicare feeforservice patients discharged with ami , hospitals with poorer patient safety performance were also more likely to have poorer performance on 30day allcause mortality and on unplanned readmissions .

Introduction Methods Study Sample Patient and Hospital Characteristics Outcomes InHospital Adverse Event Rates Statistical Analysis Results Study Sample Adverse Events, Mortality, and Readmission Rates Associations Between Adverse Events and Mortality and Readmission Rates Discussion Conclusion Sources of Funding Disclosures

for over a decade , improving hospital performance on patient safety and patient outcomes have been national priorities in the united states.1 , 2 , 3 , 4 , 5 , 6 , 7 hospitals with high inhospital adverse event , mortality , or unplanned readmission rates are considered to provide poorer quality of care.7 , 8 , 9 studies estimate that the excess annual cost attributed to measurable medical errors is around $ 17 billion10 and that unplanned readmissions result in an additional $ 15 billion in annual medicare expenditures.4 although extensive nationwide efforts have focused on improving patient safety and outcomes for acute myocardial infarction ( ami ) in particular,11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 with some recent data showing that patient safety and outcomes for acute cardiovascular disease have improved,19 , 20 , 21 inhospital adverse events , shortterm mortality , and unplanned readmission rates for ami patients remain high , with considerable variation across hospitals.22 efforts to improve patient safety , reduce hospital mortality , and reduce unplanned readmission rates are largely pursued independently . their impact on one another is unclear or thought to be small.23 , 24 , 25 , 26 previous studies have shown that patients with 1 or more adverse events are more likely to have higher mortality or to be readmitted , but these studies were limited to a few measures and local data sources.27 , 28 , 29 , 30 , 31 , 32 they also focused on patientlevel analyses , rather than connecting hospitallevel performance on patient safety with other important hospitallevel outcomes . accordingly , we sought to investigate the association at the hospitallevel between a wide range of inhospital adverse event rates and both mortality and readmission rates for medicare feeforservice patients with ami , an acute condition that may be more sensitive to inhospital adverse events . to accomplish this analysis on a national scale , we used data from the agency for healthcare research and quality ( ahrq ) 's medicare patient safety monitoring system ( mpsms ) , the nation 's largest randomly selected hospital medical recordabstracted patient safety database,33 and data from the centers for medicare & medicaid services ( cms ) , which includes hospital performance on mortality and readmissions for over 4000 medicarecertified hospitals , to assess the relationship between hospital performance on patient safety and hospital performance on 30day allcause mortality and unplanned readmissions for medicare feeforservice patients discharged with ami . based on 80 monthly reabstractions , the agreement between abstraction and reabstraction ranged from 94% to 99% for data elements used to identify adverse events.19 , 27 , 33 list of the 21 adverse event measures the cms mortality and readmission data for ami are available from the hospital compare website , at the individual hospital level . the data includes hospitalspecific total discharges , 30day allcause riskstandardized mortality , and readmission rates for all acute care hospitals treating medicare feeforservice patients aged 65 years or older . illustration of calculating of adverse event rates the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk . the number of adverse events for which patients were at risk was the offset in the model . using this model we linked the hospitalspecific riskstandardized occurrence rate of adverse events data with the hospitalspecific riskstandardized mortality and readmission data at the hospital level . to ensure that each hospital had an adequate sample size , we included only hospitals with at least 30 patients in the cms data and at least 40 adverse events for which patients were at risk over the study period ( table 3 ) . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . of hospitals by outcome we identified 2530 unique hospitals that each had 30 or more patients from the cms mortality and readmission data and 921 unique hospitals that each had 40 or more adverse events for which patients were at risk from the mpsms adverse events data . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . after linking the mortality and readmission data to adverse events data ami indicates acute myocardial infarction ; cms , centers for medicare & medicaid services ; mpsms , medicare patient safety monitoring system . the median numbers of adverse events for which patients were at risk was 36 for ami . to describe patient and hospital characteristics and the rates of individual adverse event measures , we classified each hospital into tertiles based on its riskstandardized occurrence rate of adverse events . to quantify the difference in the occurrence of adverse events across hospitals , we computed betweenhospital variation in the occurrence rate of adverse events after accounting for patient characteristics using an odds ratio representing the odds of a patient experiencing an adverse event when treated in a hospital 1 sd above the overall average relative to being treated at a hospital 1 sd below the overall average.38 to evaluate the association between hospital performance on patient safety and hospital performance on mortality , we fitted 2 mixedeffects models to estimate the : ( 1 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific observed occurrence rate of adverse events and ( 2 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific riskstandardized occurrence rate of adverse events , with and without adjusting for the hospital characteristics described previously . because different states have different patient safety regulations and quality improvement initiatives that may impact hospital performance within a state , all models were fitted with statespecific random intercepts to account for withinstate and betweenstate variation and weighted by the hospitalspecific number of adverse events for which patients were at risk . based on 80 monthly reabstractions , the agreement between abstraction and reabstraction ranged from 94% to 99% for data elements used to identify adverse events.19 , 27 , 33 list of the 21 adverse event measures the cms mortality and readmission data for ami are available from the hospital compare website , at the individual hospital level . the data includes hospitalspecific total discharges , 30day allcause riskstandardized mortality , and readmission rates for all acute care hospitals treating medicare feeforservice patients aged 65 years or older . the numerator for this rate is the number of adverse events that occurred , and the denominator is the number of adverse events for which patients were at risk.19 all patients were at risk for at least 2 adverse events , inhospital falls , and hospitalacquired pressure ulcers , but only a subset of patients were at risk for other events ( eg , only patients who received warfarin were at risk for a warfarinassociated adverse event ) . illustration of calculating of adverse event rates the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk . the number of adverse events for which patients were at risk was the offset in the model . using this model we linked the hospitalspecific riskstandardized occurrence rate of adverse events data with the hospitalspecific riskstandardized mortality and readmission data at the hospital level . to ensure that each hospital had an adequate sample size , we included only hospitals with at least 30 patients in the cms data and at least 40 adverse events for which patients were at risk over the study period ( table 3 ) . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . of hospitals by outcome we identified 2530 unique hospitals that each had 30 or more patients from the cms mortality and readmission data and 921 unique hospitals that each had 40 or more adverse events for which patients were at risk from the mpsms adverse events data . the threshold of 40 was selected based on the fact that the median numbers of adverse events for which patients were at risk per hospital was 36 for ami . with this larger threshold , we reduced the likelihood that a hospital 's low adverse event rate is attributed to an insufficient number of adverse events for which patients were at risk . after linking the mortality and readmission data to adverse events data ami indicates acute myocardial infarction ; cms , centers for medicare & medicaid services ; mpsms , medicare patient safety monitoring system . the median numbers of adverse events for which patients were at risk was 36 for ami . to describe patient and hospital characteristics and the rates of individual adverse event measures , we classified each hospital into tertiles based on its riskstandardized occurrence rate of adverse events . to quantify the difference in the occurrence of adverse events across hospitals , we computed betweenhospital variation in the occurrence rate of adverse events after accounting for patient characteristics using an odds ratio representing the odds of a patient experiencing an adverse event when treated in a hospital 1 sd above the overall average relative to being treated at a hospital 1 sd below the overall average.38 to evaluate the association between hospital performance on patient safety and hospital performance on mortality , we fitted 2 mixedeffects models to estimate the : ( 1 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific observed occurrence rate of adverse events and ( 2 ) hospitalspecific riskstandardized mortality rate as a function of the hospitalspecific riskstandardized occurrence rate of adverse events , with and without adjusting for the hospital characteristics described previously . because different states have different patient safety regulations and quality improvement initiatives that may impact hospital performance within a state , all models were fitted with statespecific random intercepts to account for withinstate and betweenstate variation and weighted by the hospitalspecific number of adverse events for which patients were at risk . the hospital median ( interquartile range [ iqr ] ) number of adverse events for which patients were at risk was 59 ( 29 ) ( figure 1 , right panel ) . table 4 , which is at the hospital level , shows hospital characteristics by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . the median ( iqr ) numbers of patients per hospital for the mortality and unplanned readmission measures were 149 ( 226 ) and 158 ( 262 ) ; the median ( iqr ) number of adverse events for which patients were at risk was 59 ( 29 ) . hospitals ( n=793 ) had at least 30 patients for mortality and readmission measures and had at least 40 adverse events for which patients were at risk . box and whisker plots of the distributions of hospitalspecific 30day allcause riskstandardized mortality and unplanned readmission rates and hospitalspecific riskstandardized occurrence rates of adverse events for which patients were at risk . table 5 , which is at the patient level , shows the number of patients and adverse event rates for each of the 21 adverse measures as well as the 2 composite adverse event rates measures by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . nevertheless , 32.0% of hospitals in the highest tertile of adverse events for which patients were at risk were in the lowest tertile of riskstandardized occurrence rate of adverse events . hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk by tertiles , july 1 , 2009 to june 30 , 2013 ( aged 65 years or older , medicare feeforservice patients discharged with acute myocardial infarction ) n / a indicates not applicable . hospitalspecific riskstandardized occurrence rates of adverse events by tertile versus hospitalspecific total number of adverse events for which patients were at risk by tertile . of the 793 hospitals , the overall occurrence rate of adverse events , in which the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk , was 3.8% . a change of 1% point in the occurrence rate of adverse events for which patients were at risk was associated with an average change of 4.8% points ( 95% ci , 4.74.9 ) in the rate of patients with 1 or more adverse events per hospitalization ( figure 4 ) . relationship between the observed rate of patients had 1 or more adverse events and occurrence rate of adverse events for which patients were at risk . the hospital characteristicadjusted associations between the riskstandardized occurrence rate of adverse events and riskstandardized mortality and readmission rates were also statistically significant ( figure 5 , bottom panel ) . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with an average change in the riskstandardized mortality rate of 4.86% points ( 95% ci , 0.798.94 ) and an average change in the riskstandardized readmission rate of 3.44% points ( 95% ci , 0.196.68 ) . because the overall occurrence rate of adverse events was 3.8% , a 1% point change in this rate translates to a relative change of 26.3% . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with average changes in the riskstandardized mortality rate of 5.13% points ( 95% ci , 2.238.03 ) and riskstandardized readmission rate of 2.51% points ( 95% ci , 0.214.82 ) . point estimates and 95% cis of the associations between the hospitalspecific riskstandardized 30day mortality rate and hospitalspecific riskstandardized occurrence rate of adverse events and hospitalspecific riskstandardized 30day unplanned readmission rate and hospitalspecific riskstandardized occurrence rate of adverse events . point estimates and 95% cis of the associations between hospital performance on mortality and unplanned readmission rates and hospital performance on the occurrence rate of adverse events , regardless of hospitals volume of adverse events for which patients were at risk . the hospital median ( interquartile range [ iqr ] ) number of adverse events for which patients were at risk was 59 ( 29 ) ( figure 1 , right panel ) . table 4 , which is at the hospital level , shows hospital characteristics by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . the median ( iqr ) numbers of patients per hospital for the mortality and unplanned readmission measures were 149 ( 226 ) and 158 ( 262 ) ; the median ( iqr ) number of adverse events for which patients were at risk was 59 ( 29 ) . hospitals ( n=793 ) had at least 30 patients for mortality and readmission measures and had at least 40 adverse events for which patients were at risk . box and whisker plots of the distributions of hospitalspecific 30day allcause riskstandardized mortality and unplanned readmission rates and hospitalspecific riskstandardized occurrence rates of adverse events for which patients were at risk . table 5 , which is at the patient level , shows the number of patients and adverse event rates for each of the 21 adverse measures as well as the 2 composite adverse event rates measures by the tertiles of hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk . nevertheless , 32.0% of hospitals in the highest tertile of adverse events for which patients were at risk were in the lowest tertile of riskstandardized occurrence rate of adverse events . hospital 's riskstandardized occurrence rate of adverse events for which patients were at risk by tertiles , july 1 , 2009 to june 30 , 2013 ( aged 65 years or older , medicare feeforservice patients discharged with acute myocardial infarction ) n / a indicates not applicable . riskstandardized occurrence rates of adverse events by tertile versus hospitalspecific total number of adverse events for which patients were at risk by tertile . of the 793 hospitals , the overall occurrence rate of adverse events , in which the numerator is the number of adverse events that occurred and the denominator is the number of adverse events for which patients were at risk , was 3.8% . a change of 1% point in the occurrence rate of adverse events for which patients were at risk was associated with an average change of 4.8% points ( 95% ci , 4.74.9 ) in the rate of patients with 1 or more adverse events per hospitalization ( figure 4 ) . relationship between the observed rate of patients had 1 or more adverse events and occurrence rate of adverse events for which patients were at risk . the hospital characteristicadjusted associations between the observed occurrence rate of adverse events and riskstandardized mortality and readmission rates were statistically significant ( figure 5 , top panel ) . the hospital characteristicadjusted associations between the riskstandardized occurrence rate of adverse events and riskstandardized mortality and readmission rates were also statistically significant ( figure 5 , bottom panel ) . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with an average change in the riskstandardized mortality rate of 4.86% points ( 95% ci , 0.798.94 ) and an average change in the riskstandardized readmission rate of 3.44% points ( 95% ci , 0.196.68 ) . because the overall occurrence rate of adverse events was 3.8% , a 1% point change in this rate translates to a relative change of 26.3% . a change of 1% point in the riskstandardized occurrence rate of adverse events was associated with average changes in the riskstandardized mortality rate of 5.13% points ( 95% ci , 2.238.03 ) and riskstandardized readmission rate of 2.51% points ( 95% ci , 0.214.82 ) . point estimates and 95% cis of the associations between the hospitalspecific riskstandardized 30day mortality rate and hospitalspecific riskstandardized occurrence rate of adverse events and hospitalspecific riskstandardized 30day unplanned readmission rate and hospitalspecific riskstandardized occurrence rate of adverse events . point estimates and 95% cis of the associations between hospital performance on mortality and unplanned readmission rates and hospital performance on the occurrence rate of adverse events , regardless of hospitals volume of adverse events for which patients were at risk . in this study , we found that hospital performance on patient safety , measured by the hospitalspecific riskstandardized occurrence rate of adverse events for which patients were at risk , was associated with hospitalspecific riskstandardized 30day allcause mortality and unplanned readmission rates for patients with ami . it is also possible that changes made at the hospital level that reduce mortality and unplanned readmissions may also improve patient safety performance , as indicated by fewer adverse events.29 , 44 , 45 furthermore , patients who experience 1 or more adverse events may be at higher risk for mortality and readmission , specifically attributed to harm caused by the adverse event.19 , 27 , 28 , 29 , 30 , 31 , 32 our study , based on medical recordabstracted patient safety information , represents a unique , large , and recent investigation of the associations between hospital performance on patient safety and mortality and readmissions for medicare patients with ami in the united states . in our study , 32% of hospitals with a higher volume of adverse events for which patients were at risk had a lower riskstandardized occurrence rate of adverse events , and 26% of hospitals with a lower volume of patients at risk had a higher rate of adverse events . because the occurrence rate of adverse events was 4% and the rate of patients with 1 or more adverse events was 20% for medicare patients discharged with ami , an absolute 1% point decrease in the occurrence rate of adverse events is equal to a relative decline of 25% in that rate itself , or an absolute 5% point reduction in the rate of patients with 1 or more adverse events . for medicare feeforservice patients discharged with ami , hospitals with poorer patient safety performance tended to have poorer performance on 30day allcause mortality and unplanned readmission rates .

pediatric cancers represent 1% of all cancers and comprise cancer cases diagnosed in children younger than 14 years and adolescents and young adults aged 1519 years . more than 250,000 cases are diagnosed worldwide each year among children and adolescents younger than 20 years ( http://www.childhoodcancerinternational.org ) . although 80% of pediatric patients reach long - term remission after treatment , 20% die from recurrence of the malignancy ; therefore , more therapeutic targets are needed to improve survival rates . moreover , current treatments are based on multimodal aggressive chemotherapies , and it is well known that antineoplastic treatments have long - term side effects that could impact patients quality of life . in fact , most pediatric cancers arise from embryonal rather than epithelial cells ; consequently , the etiology of pediatric cancers is different . zhang et al . previously reported that 8%10% of pediatric cancers are associated with germline alterations leading to a cancer predisposition ( table 1 ) . the involvement of environmental factors in childhood cancer etiology is still discussed , but there is clearly a lower implication of exogenous toxic effects in children than in adults ( e.g. , smoking , alcohol consumption , sun exposure , and overweight and sedentary lifestyle ) . very few environmental factors have been related to pediatric cancers to date : ionizing radiation and electromagnetic fields are some of the effects that remain a source of controversy , whereas some chemicals such as dioxin , trichloroethane , pesticides , solvents , metals , petroleum products , boron , and pollution have been associated with specific cancer types.7 , 8 other factors such as epigenetics and immune system deregulation have also been identified as being responsible for tumorigenesis.9 , 10 vogelstein et al . highlighted that pediatric cancers usually harbor fewer genetic mutations than adult cancers . they explained that the lower mutation rate found in pediatric cancers may be due to the embryonal origin of these cancers , which did not have enough time to renew . these differences in mutation patterns could also be explained in part by the difference in cancer initiation , such as the implication of exogenous toxic effects . a possible hypothesis of their etiology could be the presence of chromosome rearrangements , which is one of the first mechanisms described to be responsible for carcinogenesis . these rearrangements can lead , in some cases , to fusion genes , which are the juxtaposition of two previously separate genes localized on the same ( intra - chromosomal ) or two different ( inter - chromosomal ) chromosomes . it has also been shown in adult cancers that some tumors with driving fusions have a much lower mutational burden compared to tumors without fusions . thus , pediatric cancers appear to be the consequence of chromosomal rearrangements rather than mutation events . we should keep in mind that a recurrence of alterations ( mutations and copy - number alterations ) of genes involved in embryogenesis and epigenetic regulation are also described in pediatric cancers but will not be discussed here . in this review we then outline the potential therapeutic utility of fusion genes , their relevance in murine models , and we describe recent findings in the clinical setting as well as challenges associated with their discovery . fusion genes were primarily discovered in leukemia and other hematological diseases . in 1962 , nowell described the first specific chromosomal rearrangement in chronic myeloid leukemia ( cml ) . in 1973 , rowley highlighted the existence of a reciprocal translocation between the long arms of chromosomes 9 and 22 , t(9q ; 22q ) , named the philadelphia chromosome . twenty years later in the early 1980s , molecular studies of the translocation revealed a fusion between the 3 part of the abl1 gene in chromosome 9 and the 5 part of the bcr1 gene in chromosome 22 . this translocation resulting in bcr - abl was one of the first specific alterations found in human neoplasm . ten years later , a tyrosine kinase inhibitor ( tki ) targeting bcr - abl , imatinib mesylate ( glivec ; novartis ) , was discovered and approved by the food and drug administration ( fda ) in 2001 as a cancer treatment for cml . this tki was one of the first targeted therapies used for cancer treatment and led to a major improvement of cml prognosis , with a remission in 80% of cases . after this time , it became clear that fusions can drive cancer development and are potential therapeutic targets in anti - cancer treatment in a very specific manner . consequently to imatinib mesylate s history , fusion genes in hematological neoplasia and sarcoma were discovered and , more recently , high - resolution sequencing technologies enabled exploration of more fusion genes in other tumors.20 , 21 it should be noted that most of the studies and discoveries to date were made among adult patients ; nevertheless , some pediatric cancers have been described to also harbor fusion genes that are involved in patients diagnosis and/or targeted treatments ( table 2 ) . however , more explorations are needed to not only identify new targets but also to understand the function of fusion genes and their correlation to tumor initiation and progression . indeed , of all of the multiple fusions identified by next - generation sequencing in each pediatric pathology ( table 2 ) , only a few of them received attention for extensive functional studies , thus precluding the investigation and discovery of new druggable targets . four different structural chromosomal rearrangements are observed in gene fusion : translocation , insertion , inversion , and deletion . according to the coding or regulatory sequences affected , the physiological consequences are different . one of the consequences of fusion genes is the abnormal expression of one of the genes involved in the fusion ( usually the 3 part of a gene ) . more precisely , the 5 part of a gene ( gene 1 ) , including the regulatory sequences and the 5 utr part , is fused to the 3 part of a second gene ( gene 2 ) , which often only includes the coding sequence and the 3 utr . the consequence of such fusion is that if gene 1 is constitutionally expressed or highly expressed and gene 2 is a proto - oncogene , then gene 2 will be upregulated by the promoter and regulatory sequences of gene 1 . in this case , the result is a strong transcriptional activation of proto - oncogene 2 , whose own regulatory elements are lost and is put under the complete control of the first gene . fusion genes leading to abnormal protein expression were first described in adult tumors . among the best - known examples are the fusions involving immunoglobulin genes and the myc proto - oncogene , found in various hematological malignancies . several translocations were found to be responsible for the fusion of myc with other genes : t(8;14 ) led to igh - myc fusion , t(2;8 ) gave igk - myc , and t(8;22 ) resulted in igl - myc . myc is an important transcription factor that binds to dna in a non - specific manner to activate genes involved in cell cycle progression and inhibition of apoptosis . in these cases , the myc gene is upregulated due to its 5 fusion with immunoglobulin regulatory sequences ( promoter and enhancers).22 , 24 indeed , the fusion results in constitutive activation of the myc oncogene , increasing cell proliferation and tumorigenicity . another often - mentioned example is the fusion tmprss2-erg found in 50% of prostate cancers , affecting chromosome 21 . this rearrangement puts erg under the complete dependence of tmprss2 regulatory sequences , upregulated by androgens present in the tissue . its translation results in a protein with functional domains derived from both of the fused genes . most of the chimeric oncoproteins characterized act as aberrant transcription factors , with a strong activation of unspecific target genes resulting in cell transformation . to date , several chimeric oncoproteins have been identified in blood malignancies but very few are described in carcinomas . one well - known example is the fusion ret - ptc found in papillary thyroid carcinomas , where ret ( a gene involved in the regulation of cell survival , growth , differentiation , and migration ) is constitutionally activated . concerning pediatric cancers , ews - fli1 fusion is found in 85% of ewing s sarcoma , which results in a chimeric oncoprotein between the amino terminus of ews and the carboxy terminus of fli , giving aberrant transcriptional activity . in rhabdomyosarcomas , pax3/7-fkhr fusions are found in 80%85% of alveolar rhabdomyosarcomas and contain the pax3/pax7 dna binding domain and the fkhr transcriptional activation domain . more recently , studies have shown a third impact of fusion genes resulting in the entire cutout of gene domains . the truncated gene is generally a tumor suppressor gene and is therefore inactivated by the truncation , leading to cell transformation . inactivation of the tumor suppressor gene can occur through different mechanisms : the fusion can either act as the second hit ( e.g. , cdkn2a or nf1 genes ) or lead to a dominant effect on the wild - type protein ( e.g. , pax5 fusions ) or even provoke haploinsufficiency of the disrupted protein ( e.g. , runx1 fusions).29 , 30 , 31 , 32 another category that does not involve chromosomal rearrangements is the occurrence of read - through transcripts . this type of modification results in transcription - induced gene fusions ( tigfs ) that are not induced by a modification in the dna sequence but are a consequence of alternative splicing . thus , the genes stay located far apart in the same chromosome or on different chromosomes , but mrna fusion transcripts are formed as a consequence of cis - tigfs or trans - tigfs . most tigfs probably represent random in vivo events with no impact on the cell s functions and some are also artifacts of deep sequencing . nevertheless , some cis - tigfs have been associated with specific organ localization , such as slc45a3-elk4 and msmb - ncoa4 detected in normal and neoplastic prostate tissue33 , 34 and scnn1a - tnfrsf1a and ctsd - ifitm10 identified in normal and neoplastic breast tissue . leukemia is the most common type of pediatric cancer ( around 30% of total cases ) and most of the recurrent identified fusions were found in this pathology , as detailed in table 2 . concerning pediatric solid tumors , the first category concerns fusions that were considered until now only as diagnostic markers , and the second category includes fusions providing new therapeutic targets . in the first category , the most described example is ewing s sarcoma . ewing s sarcoma is characterized by a reciprocal translocation t(11;22 ) ( q24;q12 ) leading to the fusion between ews and fli1 genes . different translocations have been found in patients suffering from ewing s sarcoma involving tet / ets partners and the list is still growing . another example of fusions belonging to the first category are pax3/7-fkhr fusions found in alveolar rhabdomyosarcomas due to the translocation t(2;13)(q35;q14 ) or , less commonly , t(1;13)(p36;p14 ) , leading to pax3-foxo1 ( 55%70% ) or pax7-foxo1 ( 10%22% ) fusion genes , respectively . the resulting fusion proteins harbor high transcriptional activity due to the juxtaposition of the pax protein dna binding domain and the fkhr activation domain . in another example , honeyman et al . identified the dnajb1-prkca fusion in 100% of patients with fibrolamellar hepatocellular carcinoma ( flhcc ) , a rare subset of pediatric hepatocarcinoma . although its function is not yet elucidated , this fusion provides a new and unique diagnostic biomarker for patients with flhcc . another fusion in this category is c11orf95-rela , which was identified recently in 70% of supratentorial ependymomas . rela is a gene encoding for the rela p65 subunit of the nuclear factor b ( nf-b ) complex . when fused , rela loses its upstream regulator sequences ( 5 utr ) but the coding sequence is conserved . c11orf95 has been found fused with different partners , suggesting a possible role of its zinc - finger domains . the c11orf95-rela fusion seems to arise through chromothripsis and leads to a constitutive activation of nf-b signaling . ependymomas are classified according to their location in the supratentorial , infratentorial region of the brain or the spinal cord . it is important to highlight that c11orf95-rela is detected only in supratentorial ependymomas , thus providing a new molecular diagnostic biomarker and possible new therapeutic target . finally , the reciprocal translocation t(x,18 ; p11,q11 ) , which is found in all cases of synovial sarcoma , leads to fusions involving the syt gene ( also named ssx18 ; namely , syt - ssx1 or syt - ssx2 fusions).43 , 44 the resulting chimeric oncoproteins contain the activation domain of syt and the regulatory domain of ssx1/2 genes . the newly protein appears to be a transcriptional co - regulator via protein - protein interactions and it has been described to be able to regulate swi - snf chromatin remodeling complexes . this fusion is not found in any other tumor types which makes syt - ssx an important diagnostic biomarker , and also relevant for prognostic purposes . the second category includes fusions with therapeutic outcomes , such as aspscr1-tfe3 , fgfr1-tacc1 and fgfr3-tacc3,47 , 48 , 49 kiaa1549-braf,50 , 51 npm - alk,52 , 53 and ntrk fusions ( r. nagasubramanian et al . , 2016 , j. clin . aspscr1-tfe3 [ t(x;17)(p11.2;q25 ) ] and , more generally , tfe3 fusions are found in more than 95% of patients with alveolar soft - part sarcoma and in a subset of patients with renal cell carcinoma.46 , 55 this oncoprotein acts also as an aberrant transactivator , stronger than the native tfe3 protein , and has a nuclear localization . met this may lead to sensitivity to met inhibitors in vitro , which is currently being explored in a clinical trial with crizotinib ( nct01524926 ) . the fgfr1-tacc1 and fgfr3-tacc3 fusions were first identified in glioblastoma multiforme ( gbm ; also known as grade iv astrocytoma ) . they have since been found in various cancer types , such as bladder cancer , lung adenocarcinoma , head and neck cancer , nasopharyngeal carcinoma , and esophageal squamous cell carcinoma.47 , 48 , 49 the rearrangement that fuses the tyrosine kinase domains of fibroblast growth factor receptor ( fgfr ) to the transforming acidic coiled - coil ( tacc ) harbors a constitutive kinase activity . interestingly , the inhibition of fgfr kinase by pharmacological agents counteracts the oncogenicity of the fusion in vitro and in vivo in glioblastoma . therefore , the discovery of fgfr - tacc fusions is of great interest and the role of fgfr inhibitors is currently being explored in clinical trials with multiple fgfr kinase inhibitors , such as bgj398 and azd4547 ( nct01975701 and nct02824133 ) . kiaa1549-braf also belongs to the second category of fusions and is found in 65%75% of sporadic pilocytic astrocytomas50 , 51 but is also detected in other pediatric brain tumors.59 , 60 importantly , kiaa1549-braf leads to an over - activation of mitogen - activated protein kinase ( mapk ) signaling , resulting in an oncogenic addiction to this pathway . the first experiments showing its therapeutic potential involved targeting cells transfected with the kiaa1549-braf fusion using the braf inhibitor vemurafenib ( plx4032 ) , a selective braf inhibitor , as braf mutations also result in mapk signaling activation . although the anti - tumor effects of vemurafenib in braf ( v600e)-mutated cells are well demonstrated , the treatment of cell lines expressing kiaa1549-braf with plx4032 resulted in activation of the mapk pathway , leading to increased cell proliferation in vitro and tumor growth in vivo.62 , 63 the mechanism underlying this paradoxical activation was explained by poulikos et al . , who described that plx4032 is able to bind on raf isoforms and induce interaction with ras - gtp , which leads to the pathway activation . in braf ( v600e)-mutated tumors , the level of ras may not be sufficient to transactivate wild - type braf . this ras activation is notably discussed as a mechanism responsible for the development of squamous cell carcinoma in normal skin tissue after braf- inhibiting treatment . second - generation braf inhibitors such as plx pb-3 are currently being developed , which lead to inhibition of kiaa15 - 49-braf with decreased proliferation and tumorigenicity and may not induce the paradoxical mapk activation . as a result , they could be more efficient for patients carrying kiaa1549-braf fusions ( s .- s . , 2012 , neurosurgery , abstract ) . because mek is a downstream effector of raf in the mapk signaling pathway , kiaa1549-braf - positive cells may consequently be sensitive to mek inhibitors ( e.g. , selumetinib and trametinib ) that are able to block the pathway downstream of raf and thus independently from the raf alteration underlying the pathway activation . in a pre - clinical study , selumetinib ( azd6244 ) this is being currently explored in patients carrying kiaa1549-braf fusions ( nct01089101 and nct02124772 ) . taken together , these findings demonstrate the importance of testing the suitability of a drug in a relevant setting . their effectiveness is dependent on the cellular and the molecular context of the targeted tumors , and the general use of kinase inhibitors may be considered with caution . another example is the fusion npm - alk , caused by the translocation t(2;5)(p23;q35 ) , which fuses the dimerization domain of npm with the tyrosine kinase domain of alk . alk is a tyrosine kinase receptor ; therefore , the fusion gives a chimeric oncoprotein with a constitutively activated kinase . the translocation is found in 30%50% of advanced - stage anaplastic large cell lymphoma cases in adults ; 90% of anaplastic large cell lymphoma cases among children are alk positive . this fusion can be targeted via alk inhibitors such as crizotinib or ceritinib , which have been registered for patients carrying non - small cell lung cancer ( nsclc ) with alk translocations and gave promising results with high rates of response . promising results have also been reported in pediatric patients with anaplastic large cell lymphoma ( nct00939770 and nct01742286 ) ( b. geoerger et al . , 2015 , j. clin . finally , and more recently , ntrk fusions involving either ntrk1 , 2 , or 3 located on chromosome 1 q21-q22 have raised great interest in the clinical setting , as they are found in different neoplasms in adults and children . a total of 22 different 5 partners were found to be fused with ntrk genes . ntrk codes for the family of tropomyosin receptors , which , once fused , harbor a constitutively activated kinase function . this leads to cancer progression by activating different oncogenic pathways such as the mapk or akt pathways . among childhood neoplasms harboring ntrk fusions are found soft - tissue sarcoma , congenital infantile fibrosarcoma , glioblastoma , low - grade glioma , pilocytic astrocytoma , congenital mesoblastic nephroma , acute myeloid leukemia , and various other tumor types . therefore , for these patients , targeting ntrk is of great clinical interest and a novel inhibitor ( loxo-101 ) is currently being evaluated in the clinical setting ( nct02637687 ) ( r. nagasubramanian et al . , 2016 , j. clin . all of these studies emphasize the notion that fusions can be of great clinical interest not only as new therapeutic targets but also as biomarkers . indeed , fusions specific to a cancerous disease are ideal for diagnostic purposes and for subgroup classifications ; therefore , they can also be used as prognostic biomarkers . some fusions , such as bcr - abl in cml or npm - alk in alcl , are even used to monitor the tumor load and treatment response ( as minimal residual disease markers ) . hence , even individual or non - recurrent fusions could have clinical value in an individual patient . it should be kept in mind that current treatments are at their limits of toxicity and efficacy . accordingly , specific treatments are needed to treat non - responsive patients and also to improve the quality of life of cured patients the discovery step is still in its infancy for these malignancies , probably because studies addressing the discovery of fusion genes encounter important difficulties . the first difficulty comes from the biopsy and the way in which the dna or rna was extracted . the purity and quantity of the extracted material will impact the quality of further analysis . most of the studies involved in fusion discovery use next - generation sequencing ( ngs ) techniques and new bioinformatics analysis methods . it is necessary to combine the strengths of both sequencing technologies and computational strategies , together with the discrimination of events tightly correlated with cancer development . one difficulty encountered in gene fusion detection is the ability of discrimination between existent new discoveries and computer analysis artifacts . however , too stringent selection parameters applied to avoid false positives can preclude the detection of effective oncogenic fusions . being able to cut out the artifacts from the analysis is one of the biggest challenges , since current techniques of deep sequencing are more sensitive than before and allow the detection of rare events ; however , the risk of false positives is still present . it is also important to note that each bioinformatics tool used for fusion detection and its subsequent algorithms differ in terms of sensitivity and specificity , depending on the different filters and criteria applied . therefore , combining several tools and even different types of analysis could be highly relevant.19 , 76 moreover , cancer and carcinomas especially often have highly rearranged genomes , and many of the gene fusions that are detected can represent passenger events that are caused by chromosomal instability.13 , 77 these passenger rearrangements are secondary events present in the tumor , which are different from the primary oncogenic event responsible for the development of the disease . they are particularly frequent in relapsing patients , where treatments by radiotherapy and ionization can lead to passenger fusions not involved in oncogenic processes.74 , 78 among these patients , it is more difficult to distinguish the fusion gene capable of an oncogenic effect from the passenger fusions that can be constitutively present in normal and cancerous cells . regarding pediatric cancers , the emergence of ngs and new informatics tools to detect fusions have aimed to detect recurrent fusions with expected targetable domains , such as fusions with tyrosine kinase activities . however , considering the rarity of these types of neoplasia , the probability of finding rearranged genes is much lower than for adult cohorts . in addition , the major difficulty is linked to the intratumoral heterogeneity , where subclonal fusion events can be weakly detectable , making their identification more complex.19 , 74 most in vivo studies have assessed the tumorigenic potential of a fusion gene in xenografted tumors on immunocompromised animals . despite the validity of this experimental approach , some questions ( e.g. , the role of a fusion gene in early stages of the development of a pediatric cancer ) remain difficult to answer using xenografted tumors . therefore , several mouse models were developed in order to better understand pediatric tumors ' oncogenesis . these models helped to demonstrate the importance of ( 1 ) the cell of origin , ( 2 ) the differentiation stage , and ( 3 ) a second hit in complement to the first one ( fusion oncogene ) necessary for disease development . with the first desire to provide new models to study pediatric cancers , four striking examples of ewing s sarcoma , myxoid liposarcoma , alveolar rhabdomyosarcoma , and synovial sarcoma models explored the role of ews / fli1 in ewing s sarcoma and showed that its specific expression in mesenchymal cells of the embryonic limb buds impaired the development of mouse limbs but did not induce tumors . interestingly , when tp53 was simultaneously deleted , ews - fli1 promoted tumor formation . therefore , the presence of additional mutations notably in tp53 seems to be required for the transformation process by ews - fli1 in ewing s sarcoma . in myxoid liposarcoma , previous studies demonstrated that the introduction of the fus - chop transgene into the mouse genome led to the development of liposarcomas,80 , 81 but its specific expression in differentiated ap2-expressing adipocytes did not result in tumor formation , suggesting the importance of the stage of differentiation in driving the tumors . in alveolar rhabdomyosarcoma , the expression of pax3-fkhr fusion in mouse primary mesenchymal stem cells did not lead to tumor formation , while its expression in differentiated muscle cells at a late stage of embryogenesis induced tumors but at a low frequency . the addition of tp53 or ink4a / arf disruption increased the frequency of alveolar rhabdomyosarcoma ( rms ) , emphasizing the fact that additional mutations to pax3-fhkr fusion might be necessary to generate alveolar rms . finally , in synovial sarcoma , tumors were observed in mice models only when the fusion gene syt - ssx2 was expressed in immature myoblasts . this observation again highlights the importance of both the cell of origin and the differentiation stage factors in fusion - driven tumorigenesis . despite all of the emerging challenges related to fusion detection , identifying new fusions remains one of the most important priorities of research in pediatric cancers where new therapeutic targets are needed . the difficulty in discovering new fusion genes is minimal compared to the vast clinical consequences in terms of prognosis , diagnosis , and new therapeutic opportunities and to the importance of understanding the molecular mechanisms underlying a fusion gene . characterization of genomic alterations found in pediatric cancers would clarify their relevance , help to classify patients into subgroups , and aid in the discovery of new therapeutic , prognostic , diagnostic , or predictive biomarkers . moreover , functional studies are strongly needed to better understand the relevance of the biological pathways involving fusion genes in the development of pediatric malignancies . indeed , well - characterized fusion genes could reveal new targets in pediatric cancers for which there are currently no specific therapies available . it is well known that only a small number of relevant molecular subsets have been characterized ( e.g. , the four subtypes of medulloblastoma ) ; therefore , particular attention should be given to the rare histological subgroups . these subsets could be of strong interest for diagnosis determination , understanding new mechanisms of tumor progression or resistance , and could help to bring forward new unexpected therapeutic targets . with small histological subgroups , high priority should be given to the future study of genomic alterations at relapse . in fact , several studies have reported the importance of analyzing tissues at relapse,88 , 89 and clinical trials such as mappyacts ( molecular profiling for pediatric and young adult cancer treatment stratification ) conducted in our institute will contribute to this investigation ( nct02613962 ) . the study of patients genomic patterns at relapse could lead to better knowledge of cancer - driving and resistance mechanisms but could also allow better classification of patients . moreover , knowing that pediatric cancers are rare diseases that differ from one patient to another , an isolated fusion found only in a single patient should not be discarded and case - by - case studies as well as genome - based clinical trials are required . to counteract the hurdles due to the rarity of pediatric cancers , data sharing of high - throughput sequencing analysis would offer a personalized - medicine approach based on targeting junction oncogenes . more specifically , new therapeutic technologies are emerging regarding the targeting of junction oncogenes , such as the use of small interfering rna ( sirna ) . this type of strategy offers promising opportunities for targeted therapy , since it specifically targets fusion transcripts only expressed in tumor cells , without affecting other genes . our laboratory showed previously that the inhibition of fusion oncogenes responsible for tumor progression of thyroid cancer ( ret / ptc ) or prostate cancer ( tmprss2-erg ) by the injection of vectorized sirnas led to a decrease in tumor growth.73 , 74 , 75 , 76 , 77 an increased number of new fusion genes are expected to be discovered , owing to worldwide projects ( the cancer genome atlas [ tcga ] , https://tcga-data.nci.nih.gov/docs/publications/tcga/ ? ) and to data - sharing platforms ( e.g. , the international cancer genome consortium [ icgc ] platform , https://icgc.org/ ; and the european genome - phenome archive [ ega ] , https://ega.crg.eu/ ) . this will provide new opportunities of treatments , such as therapies based on sirna technology for children carrying neoplasia .

pediatric cancers differ from adult tumors , especially by their very low mutational rate . therefore , their etiology could be explained in part by other oncogenic mechanisms such as chromosomal rearrangements , supporting the possible implication of fusion genes in the development of pediatric cancers . fusion genes result from chromosomal rearrangements leading to the juxtaposition of two genes . consequently , an abnormal activation of one or both genes is observed . the detection of fusion genes has generated great interest in basic cancer research and in the clinical setting , since these genes can lead to better comprehension of the biological mechanisms of tumorigenesis and they can also be used as therapeutic targets and diagnostic or prognostic biomarkers . in this review , we discuss the molecular mechanisms of fusion genes and their particularities in pediatric cancers , as well as their relevance in murine models and in the clinical setting . we also point out the difficulties encountered in the discovery of fusion genes . finally , we discuss future perspectives and priorities for finding new innovative therapies in childhood cancer .

Main Text

pediatric cancers represent 1% of all cancers and comprise cancer cases diagnosed in children younger than 14 years and adolescents and young adults aged 1519 years . although 80% of pediatric patients reach long - term remission after treatment , 20% die from recurrence of the malignancy ; therefore , more therapeutic targets are needed to improve survival rates . in fact , most pediatric cancers arise from embryonal rather than epithelial cells ; consequently , the etiology of pediatric cancers is different . previously reported that 8%10% of pediatric cancers are associated with germline alterations leading to a cancer predisposition ( table 1 ) . the involvement of environmental factors in childhood cancer etiology is still discussed , but there is clearly a lower implication of exogenous toxic effects in children than in adults ( e.g. very few environmental factors have been related to pediatric cancers to date : ionizing radiation and electromagnetic fields are some of the effects that remain a source of controversy , whereas some chemicals such as dioxin , trichloroethane , pesticides , solvents , metals , petroleum products , boron , and pollution have been associated with specific cancer types.7 , 8 other factors such as epigenetics and immune system deregulation have also been identified as being responsible for tumorigenesis.9 , 10 vogelstein et al . they explained that the lower mutation rate found in pediatric cancers may be due to the embryonal origin of these cancers , which did not have enough time to renew . these differences in mutation patterns could also be explained in part by the difference in cancer initiation , such as the implication of exogenous toxic effects . a possible hypothesis of their etiology could be the presence of chromosome rearrangements , which is one of the first mechanisms described to be responsible for carcinogenesis . these rearrangements can lead , in some cases , to fusion genes , which are the juxtaposition of two previously separate genes localized on the same ( intra - chromosomal ) or two different ( inter - chromosomal ) chromosomes . thus , pediatric cancers appear to be the consequence of chromosomal rearrangements rather than mutation events . we should keep in mind that a recurrence of alterations ( mutations and copy - number alterations ) of genes involved in embryogenesis and epigenetic regulation are also described in pediatric cancers but will not be discussed here . in this review we then outline the potential therapeutic utility of fusion genes , their relevance in murine models , and we describe recent findings in the clinical setting as well as challenges associated with their discovery . fusion genes were primarily discovered in leukemia and other hematological diseases . twenty years later in the early 1980s , molecular studies of the translocation revealed a fusion between the 3 part of the abl1 gene in chromosome 9 and the 5 part of the bcr1 gene in chromosome 22 . this tki was one of the first targeted therapies used for cancer treatment and led to a major improvement of cml prognosis , with a remission in 80% of cases . consequently to imatinib mesylate s history , fusion genes in hematological neoplasia and sarcoma were discovered and , more recently , high - resolution sequencing technologies enabled exploration of more fusion genes in other tumors.20 , 21 it should be noted that most of the studies and discoveries to date were made among adult patients ; nevertheless , some pediatric cancers have been described to also harbor fusion genes that are involved in patients diagnosis and/or targeted treatments ( table 2 ) . however , more explorations are needed to not only identify new targets but also to understand the function of fusion genes and their correlation to tumor initiation and progression . indeed , of all of the multiple fusions identified by next - generation sequencing in each pediatric pathology ( table 2 ) , only a few of them received attention for extensive functional studies , thus precluding the investigation and discovery of new druggable targets . four different structural chromosomal rearrangements are observed in gene fusion : translocation , insertion , inversion , and deletion . according to the coding or regulatory sequences affected , the physiological consequences are different . one of the consequences of fusion genes is the abnormal expression of one of the genes involved in the fusion ( usually the 3 part of a gene ) . more precisely , the 5 part of a gene ( gene 1 ) , including the regulatory sequences and the 5 utr part , is fused to the 3 part of a second gene ( gene 2 ) , which often only includes the coding sequence and the 3 utr . in this case , the result is a strong transcriptional activation of proto - oncogene 2 , whose own regulatory elements are lost and is put under the complete control of the first gene . fusion genes leading to abnormal protein expression were first described in adult tumors . among the best - known examples are the fusions involving immunoglobulin genes and the myc proto - oncogene , found in various hematological malignancies . in these cases , the myc gene is upregulated due to its 5 fusion with immunoglobulin regulatory sequences ( promoter and enhancers).22 , 24 indeed , the fusion results in constitutive activation of the myc oncogene , increasing cell proliferation and tumorigenicity . another often - mentioned example is the fusion tmprss2-erg found in 50% of prostate cancers , affecting chromosome 21 . this rearrangement puts erg under the complete dependence of tmprss2 regulatory sequences , upregulated by androgens present in the tissue . its translation results in a protein with functional domains derived from both of the fused genes . most of the chimeric oncoproteins characterized act as aberrant transcription factors , with a strong activation of unspecific target genes resulting in cell transformation . one well - known example is the fusion ret - ptc found in papillary thyroid carcinomas , where ret ( a gene involved in the regulation of cell survival , growth , differentiation , and migration ) is constitutionally activated . concerning pediatric cancers , ews - fli1 fusion is found in 85% of ewing s sarcoma , which results in a chimeric oncoprotein between the amino terminus of ews and the carboxy terminus of fli , giving aberrant transcriptional activity . more recently , studies have shown a third impact of fusion genes resulting in the entire cutout of gene domains . inactivation of the tumor suppressor gene can occur through different mechanisms : the fusion can either act as the second hit ( e.g. , runx1 fusions).29 , 30 , 31 , 32 another category that does not involve chromosomal rearrangements is the occurrence of read - through transcripts . nevertheless , some cis - tigfs have been associated with specific organ localization , such as slc45a3-elk4 and msmb - ncoa4 detected in normal and neoplastic prostate tissue33 , 34 and scnn1a - tnfrsf1a and ctsd - ifitm10 identified in normal and neoplastic breast tissue . leukemia is the most common type of pediatric cancer ( around 30% of total cases ) and most of the recurrent identified fusions were found in this pathology , as detailed in table 2 . concerning pediatric solid tumors , the first category concerns fusions that were considered until now only as diagnostic markers , and the second category includes fusions providing new therapeutic targets . in the first category , the most described example is ewing s sarcoma . ewing s sarcoma is characterized by a reciprocal translocation t(11;22 ) ( q24;q12 ) leading to the fusion between ews and fli1 genes . another example of fusions belonging to the first category are pax3/7-fkhr fusions found in alveolar rhabdomyosarcomas due to the translocation t(2;13)(q35;q14 ) or , less commonly , t(1;13)(p36;p14 ) , leading to pax3-foxo1 ( 55%70% ) or pax7-foxo1 ( 10%22% ) fusion genes , respectively . the resulting fusion proteins harbor high transcriptional activity due to the juxtaposition of the pax protein dna binding domain and the fkhr activation domain . identified the dnajb1-prkca fusion in 100% of patients with fibrolamellar hepatocellular carcinoma ( flhcc ) , a rare subset of pediatric hepatocarcinoma . another fusion in this category is c11orf95-rela , which was identified recently in 70% of supratentorial ependymomas . the c11orf95-rela fusion seems to arise through chromothripsis and leads to a constitutive activation of nf-b signaling . ependymomas are classified according to their location in the supratentorial , infratentorial region of the brain or the spinal cord . the second category includes fusions with therapeutic outcomes , such as aspscr1-tfe3 , fgfr1-tacc1 and fgfr3-tacc3,47 , 48 , 49 kiaa1549-braf,50 , 51 npm - alk,52 , 53 and ntrk fusions ( r. nagasubramanian et al . aspscr1-tfe3 [ t(x;17)(p11.2;q25 ) ] and , more generally , tfe3 fusions are found in more than 95% of patients with alveolar soft - part sarcoma and in a subset of patients with renal cell carcinoma.46 , 55 this oncoprotein acts also as an aberrant transactivator , stronger than the native tfe3 protein , and has a nuclear localization . met this may lead to sensitivity to met inhibitors in vitro , which is currently being explored in a clinical trial with crizotinib ( nct01524926 ) . they have since been found in various cancer types , such as bladder cancer , lung adenocarcinoma , head and neck cancer , nasopharyngeal carcinoma , and esophageal squamous cell carcinoma.47 , 48 , 49 the rearrangement that fuses the tyrosine kinase domains of fibroblast growth factor receptor ( fgfr ) to the transforming acidic coiled - coil ( tacc ) harbors a constitutive kinase activity . interestingly , the inhibition of fgfr kinase by pharmacological agents counteracts the oncogenicity of the fusion in vitro and in vivo in glioblastoma . therefore , the discovery of fgfr - tacc fusions is of great interest and the role of fgfr inhibitors is currently being explored in clinical trials with multiple fgfr kinase inhibitors , such as bgj398 and azd4547 ( nct01975701 and nct02824133 ) . kiaa1549-braf also belongs to the second category of fusions and is found in 65%75% of sporadic pilocytic astrocytomas50 , 51 but is also detected in other pediatric brain tumors.59 , 60 importantly , kiaa1549-braf leads to an over - activation of mitogen - activated protein kinase ( mapk ) signaling , resulting in an oncogenic addiction to this pathway . although the anti - tumor effects of vemurafenib in braf ( v600e)-mutated cells are well demonstrated , the treatment of cell lines expressing kiaa1549-braf with plx4032 resulted in activation of the mapk pathway , leading to increased cell proliferation in vitro and tumor growth in vivo.62 , 63 the mechanism underlying this paradoxical activation was explained by poulikos et al . , who described that plx4032 is able to bind on raf isoforms and induce interaction with ras - gtp , which leads to the pathway activation . in braf ( v600e)-mutated tumors , the level of ras may not be sufficient to transactivate wild - type braf . this ras activation is notably discussed as a mechanism responsible for the development of squamous cell carcinoma in normal skin tissue after braf- inhibiting treatment . second - generation braf inhibitors such as plx pb-3 are currently being developed , which lead to inhibition of kiaa15 - 49-braf with decreased proliferation and tumorigenicity and may not induce the paradoxical mapk activation . because mek is a downstream effector of raf in the mapk signaling pathway , kiaa1549-braf - positive cells may consequently be sensitive to mek inhibitors ( e.g. their effectiveness is dependent on the cellular and the molecular context of the targeted tumors , and the general use of kinase inhibitors may be considered with caution . alk is a tyrosine kinase receptor ; therefore , the fusion gives a chimeric oncoprotein with a constitutively activated kinase . finally , and more recently , ntrk fusions involving either ntrk1 , 2 , or 3 located on chromosome 1 q21-q22 have raised great interest in the clinical setting , as they are found in different neoplasms in adults and children . this leads to cancer progression by activating different oncogenic pathways such as the mapk or akt pathways . therefore , for these patients , targeting ntrk is of great clinical interest and a novel inhibitor ( loxo-101 ) is currently being evaluated in the clinical setting ( nct02637687 ) ( r. nagasubramanian et al . indeed , fusions specific to a cancerous disease are ideal for diagnostic purposes and for subgroup classifications ; therefore , they can also be used as prognostic biomarkers . some fusions , such as bcr - abl in cml or npm - alk in alcl , are even used to monitor the tumor load and treatment response ( as minimal residual disease markers ) . accordingly , specific treatments are needed to treat non - responsive patients and also to improve the quality of life of cured patients the discovery step is still in its infancy for these malignancies , probably because studies addressing the discovery of fusion genes encounter important difficulties . however , too stringent selection parameters applied to avoid false positives can preclude the detection of effective oncogenic fusions . being able to cut out the artifacts from the analysis is one of the biggest challenges , since current techniques of deep sequencing are more sensitive than before and allow the detection of rare events ; however , the risk of false positives is still present . therefore , combining several tools and even different types of analysis could be highly relevant.19 , 76 moreover , cancer and carcinomas especially often have highly rearranged genomes , and many of the gene fusions that are detected can represent passenger events that are caused by chromosomal instability.13 , 77 these passenger rearrangements are secondary events present in the tumor , which are different from the primary oncogenic event responsible for the development of the disease . they are particularly frequent in relapsing patients , where treatments by radiotherapy and ionization can lead to passenger fusions not involved in oncogenic processes.74 , 78 among these patients , it is more difficult to distinguish the fusion gene capable of an oncogenic effect from the passenger fusions that can be constitutively present in normal and cancerous cells . regarding pediatric cancers , the emergence of ngs and new informatics tools to detect fusions have aimed to detect recurrent fusions with expected targetable domains , such as fusions with tyrosine kinase activities . however , considering the rarity of these types of neoplasia , the probability of finding rearranged genes is much lower than for adult cohorts . , the role of a fusion gene in early stages of the development of a pediatric cancer ) remain difficult to answer using xenografted tumors . therefore , several mouse models were developed in order to better understand pediatric tumors ' oncogenesis . with the first desire to provide new models to study pediatric cancers , four striking examples of ewing s sarcoma , myxoid liposarcoma , alveolar rhabdomyosarcoma , and synovial sarcoma models explored the role of ews / fli1 in ewing s sarcoma and showed that its specific expression in mesenchymal cells of the embryonic limb buds impaired the development of mouse limbs but did not induce tumors . therefore , the presence of additional mutations notably in tp53 seems to be required for the transformation process by ews - fli1 in ewing s sarcoma . in myxoid liposarcoma , previous studies demonstrated that the introduction of the fus - chop transgene into the mouse genome led to the development of liposarcomas,80 , 81 but its specific expression in differentiated ap2-expressing adipocytes did not result in tumor formation , suggesting the importance of the stage of differentiation in driving the tumors . finally , in synovial sarcoma , tumors were observed in mice models only when the fusion gene syt - ssx2 was expressed in immature myoblasts . despite all of the emerging challenges related to fusion detection , identifying new fusions remains one of the most important priorities of research in pediatric cancers where new therapeutic targets are needed . the difficulty in discovering new fusion genes is minimal compared to the vast clinical consequences in terms of prognosis , diagnosis , and new therapeutic opportunities and to the importance of understanding the molecular mechanisms underlying a fusion gene . characterization of genomic alterations found in pediatric cancers would clarify their relevance , help to classify patients into subgroups , and aid in the discovery of new therapeutic , prognostic , diagnostic , or predictive biomarkers . moreover , functional studies are strongly needed to better understand the relevance of the biological pathways involving fusion genes in the development of pediatric malignancies . indeed , well - characterized fusion genes could reveal new targets in pediatric cancers for which there are currently no specific therapies available . , the four subtypes of medulloblastoma ) ; therefore , particular attention should be given to the rare histological subgroups . these subsets could be of strong interest for diagnosis determination , understanding new mechanisms of tumor progression or resistance , and could help to bring forward new unexpected therapeutic targets . in fact , several studies have reported the importance of analyzing tissues at relapse,88 , 89 and clinical trials such as mappyacts ( molecular profiling for pediatric and young adult cancer treatment stratification ) conducted in our institute will contribute to this investigation ( nct02613962 ) . the study of patients genomic patterns at relapse could lead to better knowledge of cancer - driving and resistance mechanisms but could also allow better classification of patients . moreover , knowing that pediatric cancers are rare diseases that differ from one patient to another , an isolated fusion found only in a single patient should not be discarded and case - by - case studies as well as genome - based clinical trials are required . to counteract the hurdles due to the rarity of pediatric cancers , data sharing of high - throughput sequencing analysis would offer a personalized - medicine approach based on targeting junction oncogenes . this type of strategy offers promising opportunities for targeted therapy , since it specifically targets fusion transcripts only expressed in tumor cells , without affecting other genes . our laboratory showed previously that the inhibition of fusion oncogenes responsible for tumor progression of thyroid cancer ( ret / ptc ) or prostate cancer ( tmprss2-erg ) by the injection of vectorized sirnas led to a decrease in tumor growth.73 , 74 , 75 , 76 , 77 an increased number of new fusion genes are expected to be discovered , owing to worldwide projects ( the cancer genome atlas [ tcga ] , https://tcga-data.nci.nih.gov/docs/publications/tcga/ ? ) this will provide new opportunities of treatments , such as therapies based on sirna technology for children carrying neoplasia .

the rapid growth of technological possibilities in health care is accompanied by a growing need to determine an emerging technology s desirability before it is introduced in the health care system . in the 1970s and 1980s health technology assessment ( hta ) was developed as a way to facilitate collective decision making on the desirability of new biomedical technologies . the birth of hta is often dated in 1975 , when the american congress asked its office for technology assessment ( ota ) which justification was necessary to introduce a new medical technology [ 4 , p. 2 ; 6 , p. 276 ] . the report , published in 1976 , defined technology assessment quite broadly as : a comprehensive form of policy research that examines the short- and long - term social consequences ( e.g. societal , economic , ethical , legal ) of the application or use of technology [ 33 , p. 45 ] . from the start hta has had a complicated relationship with ethics and politics or with normativity in general [ 6 , 36 , 38 , 44 ] . some advocates approach hta as the first step in a two - tiered process ( e.g. ) . in this conception hta researchers collect evidence about the probable consequences of a technology ( assessment ) , which is subsequently weighed by ethicists or political actors ( appraisal ) . others argue , in contrast , that hta - reports should pass judgment on the value of a technology itself ( e.g. ) . although not everyone agrees that hta should include normative conclusions as to what is or is not desirable , many authors have criticized the idea that factual assessment and normative appraisal can be clearly separated . they point out that hta methods to collect evidence on a technology s performance are inevitably imbued with values . therefore , its results are not as neutral as often purported [ 21 , 27 , 30 , 36 , 38 , 43 , 44 , 58 ] . for example , the selection of consequences to take into account , as well as the choice of specific outcome measures to assess them both have a normative impact . disability or quality of life [ 20 , 21 , 32 , 38 ] . at about the same time philosophers , sociologists and anthropologists they criticized the common idea that technology is a neutral instrument designed to perform a specific function . some argued that technology is a complex phenomenon , consisting of material , social and organizational elements [ 1 , 26 ] . others pointed out that , because of this complexity , technology does much more than it is intended to do [ 5 , 31 ] . many of these effects are indirect : technology mediates our experience of reality and the options for action we perceive [ 22 , 62 ] . such analyses imply that the effects of a novel technology are much more difficult to foresee than the instrumentalist view of technology suggests . this conceptual and empirical work on the character of technology in practice seems to suggest that tools and methods of traditional forms of hta may be problematic partly because they are based on an instrumentalist conception of technology that fits badly with the workings of technology in practice . in this paper , i will explore this hypothesis by reconstructing and discussing an early endeavor to systematically assess a technology s desirability : the assessments and deliberations preceding the introduction of breast cancer screening in the netherlands in the 1970s and 1980s . hta , the case is interesting precisely because it shows the ideal of hta in the making and set the stage for subsequent hta activities.1 in this period a model of hta emerged that was widely used , both in the netherlands and abroad , for many years ( as is visible , for example , in the special issue on the history of hta in the international journal of technology assessment in health care 25 , supplement 1 , 2009 ) . first i will reconstruct the processes preceding the decision to introduce a nation wide program for breast cancer screening in the netherlands . this reconstruction is based on a systematic survey of : ( 1 ) the relevant advisory reports and policy documents in the years 19682005 ; ( 2 ) the minutes of dutch parliament in the years 19701995 ; ( 3 ) the medical professional journals nederlands tijdschrift voor geneeskunde ( 19602005 ) and medisch contact ( 19602005 ) ; and ( 4 ) opinion articles on breast cancer screening in the dutch national newspapers ( 19602005 ) . subsequently i will reconstruct the stepwise model of hta that emerged during the process and analyze how this model was based on an instrumentalist conception of technology . these episodes illustrate how the actors during the process were already confronted with or became aware of unanticipated effects , and how this in some cases led to adjustments in the hta procedures . the historical analysis enables me , in the end , to argue how and why an instrumentalist conception of technology is not a good starting point for hta . i will conclude with some suggestions for improving the fit between hta methods and the actual character of technology in practice . the history of breast cancer screening starts in the 1950 s and 1960 s , when in france and the usa a radiological device for diagnosing breast diseases was developed . it was noticed soon that this device , called mammography , detected lesions that were not found by palpation ( the current diagnostic standard ) . mammography thus seemed to offer promising possibilities for earlier diagnosis of breast cancer , which in turn might improve curability and survival rates . when an american study showed that mammographic screening might reduce breast cancer mortality by 30% concerned scientists in the netherlands set up several pilot projects to corroborate the effectiveness of mammographic screening in a dutch setting . in 1975 projects started in nijmegen , utrecht and leiden the leiden project was meant as a pilot project offering a novel health care service and lasted only 1 year . the projects in nijmegen and utrecht had scientific goals and were designed as case control studies . in 1977 the dutch minister of health asked the health council to report on the desirability and acceptability of a national breast cancer screening program . the committee established by the health council consisted of a number of medical professionals and a sociologist ; a psychologist was added later on . the committee soon concluded that the available evidence on the effectiveness of mammographic screening was limited and decided to wait for the results of the pilot projects in utrecht and nijmegen before formulating its advice . since the dutch projects were case control studies and thus liable to bias , results from randomised clinical trials ( rct s ) in other countries were taken into account as well . the health council s committee issued two interim reports and a final report [ 1517 ] . when the second interim report cautiously judged the available evidence for effectiveness of screening as promising , the minister of health ordered an independent study of the cost effectiveness of such a screening program actually the first such a study to be ordered by dutch government . this study , written by the institute for health technology assessment of erasmus university , was published in 1990 . the study not only calculated the costs per saved life year ; it also used qaly s ( quality adjusted life years ) , a relatively new tool to take into account effects of screening on the screened individual s wellbeing . to sum up , although the label hta was not used yet , mammographic breast cancer screening was the first health care technology in the netherlands to be systematically assessed for its efficacy , safety , cost effectiveness and impact on quality of life , with the explicit aim to inform and facilitate political decision making . when the experts judged that the performance of mammographic screening on these items was acceptable , the minister proposed to gradually introduce breast cancer screening for women aged 5069 on a national scale , as the health council had advised . the dutch health insurance council was asked for advice on the financial and organizational aspects of such a screening program . it proposed to make screening free for participants and to organize it in a centralized way , closely resembling the set up of the pilot projects . the desirability of the screening program was not only discussed in scientific reports , however . both during the preparatory phase and immediately after the health council published its final report , a small group of opponents voiced criticism . they wrote pieces in newspapers and medical journals arguing that a screening program would seriously contribute to medicalization [ 8 , 41 , 4550 , 61 ] . in response , others ( often investigators of the pilot projects or members of the health council s committee ) stressed the advantages of breast cancer screening and the evidence for the positive effects of such a program [ 10 , 12 , 37 , 54 ] . the first extensive parliamentary debate took place in 1991 , when the parliamentary commission on health care discussed the proposed national breast cancer screening program with the minister of health . although no one completely opposed the screening program , several members of parliament took up criticism voiced earlier in newspapers and journals . some suggested that if women were really to decide autonomously about their participation , the invitational letter should explicitly mention the uncertainties and potentially negative effects of screening . others questioned the justification of the proposed age limits . and some argued that the program might lead to overdiagnosis , overtreatment , and thus to unnecessary medicalization . in his response , the minister first pointed out that many of the potential negative effects of screening had been taken into account in the cost effectiveness study , in particular by the use of qaly s . in a second debate between the minister and the parliamentary commission on health care , in 1993 , the minister recognized , however , that there might be a tension between voluntary participation and the need for high participation rates . he promised to guarantee ( 1 ) respect for the freedom of choice and personal life of the women involved ( by giving even handed information ) ; ( 2 ) equal access ( again by giving good information and by making participation free ) ; and ( 3 ) a high quality of care ( by installing an extensive monitoring system ) [ 53 , pp . thus , between 1989 and 1995 breast cancer screening was gradually introduced in all regions of the netherlands . since 1995 , all dutch women between 50 and 69 have been invited for breast cancer screening every 2 years . effectiveness , efficiency and quality of care of the screening program continued to be monitored by an independent scientific committee . participation rates ( and thus apparent acceptance by the target group ) slowly increased from about 75% in 1990 to 82% in 2007 . since then , the first debate took place when practice with regard to the upper age limit was changed . although the upper age limit was set at 69 , initially participants reaching the age of 70 could continue screening if they liked to . in 1992 the health insurance council announced that from now on the age limit would be strictly kept . this decision was justified mainly with budgetary reasons , but doubts about effectiveness and worries about the impact of screening on older women s lives were also mentioned [ 56 , p. 139 the decision initiated a heated debate , in which representatives of associations for the elderly claimed a right to be screened and accused the council of age discrimination and paternalism . , the upper age limit was increased to 75 years in 1998 when new evidence suggested that screening this age group could be effective after all . a second surge of debate occurred when the cochrane collaboration in 2001 published a critical metareview of the available evidence for the effectiveness of breast cancer screening . the authors concluded that this effectiveness was doubtful and that screening might even produce harm , in terms of overdiagnosis and overtreatment [ 34 , 35 ] . this led to a renewed public exchange of arguments pro and contra screening , in which the strengths and weaknesses of the scientific methods to establish effectiveness were also subjected to debate . some critics argued once more that the information given to women when invited for screening was too biased to enable autonomous decision making . the debate subsided after a health council committee , hastily asked by the minister of health to advise whether the dutch program should be adjusted or even abolished in view of the cochrane review , concluded that there was no need to do so . effectiveness might be somewhat lower than previously expected , but it was sufficient to justify a national screening programme . in effect , then , mammographic breast cancer screening was the first health care technology in the netherlands to be subjected to a more or less systematic assessment , with the aim to inform and facilitate political decision making . in the process a model of hta emerged that now seems quite familiar and self - evident because since then it has been used widely , both in the netherlands and in other countries . what is interesting about the historical case discussed here is that it shows the gradual birth of this widespread model in the dutch context.2 in reconstruction , the process consisted of four stages , each considering a specific aspect of mammographic screening : health gainscost - effectivenessorganizational conditionsethical considerations ( like justice , autonomy , screening rights , medicalization ) organizational conditions ethical considerations ( like justice , autonomy , screening rights , medicalization ) the first three stages occurred in a chronological chain : the next stage was initiated only when the results of the preceding one appeared to be sufficiently positive . the ministry of health delegated the first three stages to scientific experts : the health council , independent economic scientists and the health insurance council respectively . in contrast , the ethical considerations were ( implicitly ) left to public and political debate . they occasionally popped up during the process , with a clear increase in the final stages of decision making . this procedure seems to have been inspired by an implicit instrumentalist view of technology in several respects . such a view perceives technology as a material device that is designed to perform a specific function . it is an instrument ( just a means ) to realize a specific goal ; the instrument itself is value neutral . this view has been widespread in western thinking [ 14 , 62 ] , so it is not surprising that it influenced ideas and tools for hta . what is the task of ( h)ta from an instrumentalist perspective ? if technology is a device designed to fulfil a specific function , the first question is of course whether the proposed device is indeed capable to fulfil this function . assessments should ask , then , to what extent the device realizes the intended effect . the goal ( the intended effect ) itself is supposed to be clear and is taken for granted for the time being , as is the device s actual design . seen this way , it makes sense to separate the factual assessment of a device s performance from the normative evaluation of the intended goal . such a separation is clearly implied by the split between stages 13 and stage 4 in the mammographic screening case . whereas establishing the facts was considered a task for scientific experts , evaluating goals was assigned to the political domain . the experts involved in the mammographic screening research endorsed this view . in its final report , the health council deliberately refrained from statements about the desirability of a national screening program , because this was considered a political issue [ 17 , cover letter p. 2 ] . in a similar vein , the authors of the cost - effectiveness study stated that considerations other than cost - effectiveness could of course play a large role in the political deliberations [ 9 , p. 7 the instrumentalist view is also clearly visible in the set up of the first assessment stage . mammography was approached as a material device designed to diagnose women with asymptomatic breast cancer , in order to increase the life expectancy of these women . to decide about the desirability of this device did it diagnose sufficient asymptomatic cancers , and did this early diagnosis lead to increased survival ? the pilot projects and the report of the health council thus initially were set up to produce factual evidence on the actual magnitude of the intended effect only.3 the only additional effect that was taken into account was the potential causation of new tumours resulting from the radiation necessary to make the mammograms a side effect , that is , that would directly detract from the intended effect . in the second and third stage of the assessment the financial and organizational efforts needed to realize the intended effect were charted , to enable an informed comparison with potential alternatives . the hta procedure developed in this historical episode this is visible in particular in ( 1 ) the separation of assessment of facts by experts from the deliberation on desirability by political actors and ( 2 ) the exclusive focus on the intended effect ( i.e. mortality reduction ) . however , a closer look at the historical episode shows that the actors involved also encountered problems resulting from limitations and shortcomings of the instrumentalist view . some of these problems were diagnosed already by the actors themselves , resulting in modifications of the assessment procedure or other attempts to compensate for the procedure s weaknesses . other problems can be identified with the benefit of hindsight , suggesting a need for further modification . thus , while trying to invent a systematic procedure to assess mammographic screening , the actors involved learned important lessons about the lack of fit between an instrumentalist view of technology and the way technology works in practice . in the next part of this paper , i point out how difficulties arose with respect to three presuppositions inspired by an instrumentalist view of technology : technology is a means to realize intended effects;technology is a material device;morality may influence technology , but technology does not influence morality . technology is a means to realize intended effects ; technology is a material device ; morality may influence technology , but technology does not influence morality . as described above , the first publications on mammography present it as a tool to improve the prognosis of women with asymptomatic breast cancer . the actors involved in the assessment process are aware , however , that measuring improved prognosis is rather complicated . counting cases of early detection does not suffice ; the screening program is successful only if subsequent treatment is effective and results in prolonged healthy lives . therefore the outcome measures proposed at first are 10 years survival and mortality rates . however , because an increase of 10 years survival rates without a concurrent mortality reduction is considered undesirable , reduction of breast cancer mortality quickly becomes the central measure of effectiveness . the pilot projects in utrecht and nijmegen are explicitly set up to investigate to what extent mammographic screening reduces breast cancer mortality . the health council in its first report ( 1981 ) affirms this choice : this is the intended effect of mammographic screening and it should be the central focus of the assessment procedure . the actors gradually become aware , however , that mammographic screening does more than it was intended to do and that these additional effects may also be relevant when assessing the technology . the health council s 1984 report , for example , mentions quality of life as an additional outcome measure for the effectiveness of mammographic screening : therapeutic effectiveness in the above is translated as reduced breast cancer mortality . it is important to point at the so called breast saving surgery techniques that have gained ground in the treatment of early stage breast cancer lately . development of breast saving surgery techniques was actually spurred by the increasing number of pre - clinical tumors becoming available as a result of screening [ 13 , p. 95 ] . the novel surgery techniques were thus co - produced by mammographic screening , resulting in an unforeseen ( and up to then unintended ) effect . decreased mutilation and increased wellbeing of patients now emerged as a potential additional goal of breast cancer screening , next to mortality reduction . it is important to point at the so called breast saving surgery techniques that have gained ground in the treatment of early stage breast cancer lately . ] thus , the assessors initial focus on the intended effect only started to look too narrow . the concept of quality of life was proposed to broaden the scope of the assessment , to allow different types of presumably positive effects of screening to become visible . although such effects were not explicitly intended at the start of the projects , the health council appears to have been willing to include this novel measure in the assessment . however , the investigators of the two dutch pilot projects did not include quality of life as an additional measure of effectiveness , presumably because they did not want to change the design of their long term studies . it is only in the cost - effectiveness study ( 1990 ) , therefore , that quality of life first appears as a measure of effectiveness . the authors of this report motivate the introduction of the concept in a way that is very different from the health council s earlier suggestion : next to mortality reduction , inevitably undesirable effects will occur . an undesirable effect is , for example , anxiety in women who are referred for further diagnostics and who in the end appear not to have breast cancer ( the false positives ( ) ) . another undesirable effect is that a number of women now know they have breast cancer whereas for some of them the prognosis wo nt improve . ( ) weighing the amount of desired and undesired effects on the one hand and the expected costs and reduced expenses on the other is necessary . quality of life is not meant to measure additional positive effects of screening ; on the contrary , it is a measure for any impact ( both desirable and undesirable ) screening might have on the lives of individual participants . an undesirable effect is , for example , anxiety in women who are referred for further diagnostics and who in the end appear not to have breast cancer ( the false positives ( ) ) . another undesirable effect is that a number of women now know they have breast cancer whereas for some of them the prognosis wo nt improve . ( ) weighing the amount of desired and undesired effects on the one hand and the expected costs and reduced expenses on the other is necessary . ] thus , the actors involved in the assessment of breast cancer screening gradually realized that mammographic screening may have significant impacts besides the intended increase in life expectancy , and that these effects , moreover , need not always be positive . voices from the public debate ( on breast cancer screening , but also on other medical technologies ) may have played a role here . the introduction of quality of life was an attempt to accommodate a broader array of effects , in a way that apparently enabled a sensible comparison of these effects . this history corroborates the argument made by armstrong and caldwell , who claim that the emergence of quality of life in the 1970s and 1980s among others expressed a general discomfort regarding the human costs of technological interventions [ 2 , p. 36 ] . in the dutch case , the concept helped to weigh the various effects of mammographic screening . the desire to include patients perspectives ( often mentioned as a justification to include quality of life ) apparently hardly played a role . it is clear from the cost - effectiveness report how the researchers struggled with the operationalization of quality of life [ 9 , pp . what should or should not be included ? how to operationalize the more subjective types of impacts ? and how to compare different types of impacts ? interestingly , the questionnaires to assess quality of life impacts were not filled out by women participating in the pilot projects , because the questions were considered too complex for these participants . instead , stand ins like economists and medical professionals involved in cancer screening and care acted as respondents . the first were considered to be more used to this type of questionnaire , and the latter were supposed to have a reliable view of how breast cancer may affect women s lives . since then , struggles have continued , leading to a proliferation of instruments measuring quality of life , most of which do involve patients ( or healthy subjects ) as respondents . quality of life , in addition to mortality reduction , has become a widely accepted dimension for assessing the impact of novel medical technologies . the history of assessing breast cancer screening shows why hta should not focus on a technology s intended effect only . technology does more than it is intended to do , both directly and indirectly . as for the direct effects , screening may increase one s life expectancy and decrease wellbeing at the same time . focusing exclusively on the intended ( hoped for ) effects inevitably biases the assessment and leads to ( avoidable ) implicit normativity of the results . including a broad array of potential effects is important for even handed decision making on the desirability of a novel technology . this is even truer for indirect effects , like the improved surgery techniques resulting from the emergence of early diagnostics . good hta should nonetheless try to anticipate and include such effects , both at the start and on the way , as the dutch assessors tried to do . as described above , the reduction of breast cancer mortality by mammographic screening not only requires a well functioning mammographic device , but also an effective therapeutic follow up . breast cancer screening has , then , clear social and organizational conditions from the start . however , at first both the researchers and the political actors involved approach mammographic screening as a material device that processes input ( asymptomatic women ) to deliver a specific output ( mortality reduction ) . the pilot projects are presented as a means to find out how effective this new diagnostic technology will be when applied to asymptomatic women . practical conditions like setting age limits and frequency of screening do get some attention , but only in relation to the question how to increase the screening s effectiveness . it is interesting to see how the actors in the dutch screening assessment seem to have become gradually aware of the importance of social and organizational conditions ; an awareness that in the end led to a strict regulation of the national screening program and even to a law on population screening . when the results of the pilot projects seem sufficiently promising to warrant serious consideration of a nation - wide screening program , the researchers start pointing out that the organizational setting of screening is crucial . the positive results can be repeated only , they state , when the national program is conducted in a way similar to the pilot projects . the health council agrees . in its second report it states : the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . ( ) guarantees are needed for the quality of the work of participating centers ( diagnostics , treatment and counseling of the women involved ) . in this respect a newly instituted center of reference might serve quality control as well . [ 16 , p. 28 ] the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . ( ) guarantees are needed for the quality of the work of participating centers ( diagnostics , treatment and counseling of the women involved ) . in this respect a newly instituted center of reference might serve quality control as well . [ 16 , p. 28 ] both the scientific and the political actors thus gradually became aware that mammography is more than a material device and that breast cancer screening requires more than the distribution of mammography devices . as a result , they concluded that a national screening program should be as similar to the pilot projects as possible . the awareness that organizational conditions are crucial in realizing a certain effect motivated the health council s advice to centralize the organization of screening and make it a uniform practice . moreover , since the pilot projects were designed as a separate , autonomous service , situated between gp s and hospitals , the national program also became a separate service next to primary and secondary health care . thus , because the sociotechnical character of mammographic screening came into the picture relatively late , the organizational choices of the scientific pilot project became indicative for the organization of the national program . as one critic stated , when the committee developed a refined constellation of protocols and procedures for national screening practice , it was actually trying to approach the conditions of scientific research whether or not all actors agreed with the proposed division of roles and responsibilities was hardly discussed . the growing awareness of the social conditions for realizing the aimed for mortality reduction also influenced the approach of and the role ascribed to female participants . in its final report the health council explicitly mentions a high participation rate as a condition for attaining the aimed for effect : the committee emphasizes the importance of a maximal participation in screening for breast cancer . if the participation rate is too low , the aimed for mortality reduction on the population level may also become too low [ 17 , pp . recommendations were formulated to make screening as accessible as possible : the invitational system should function well , screening should be affordable , the location of screening centers should be accessible , counseling should be available , and education should be as persuasive as possible : careful education of the female population is necessary . research into the effectiveness of education with regard to the participation of the women involved in screening is necessary [ 17 , p. 72 ] . here , the need for a high participation rate to achieve the aimed for effects actually led to a neglect of a potentially conflicting value : the autonomy of participants . the implicit tension between effectiveness and women s autonomy came to the fore once more in 1991 , when the minister of health in a letter to the health insurance council in 1991 simply states that invitations , leaflets and campaigning material should contribute to high participation rates as well as enable considered choices by the women in the target group [ 51 , p. 11 ] . in the parliamentary debates on screening , however , mp s stressed that autonomy was the most important value . this position in the end was confirmed by the minister in a memo from 1993 , stating that information on screening should include negative aspects of screening , even if this would lead to a decrease of participation rates [ 53 , pp . this episode shows how the implicit view that technology is a material device led the assessors of mammographic screening to neglect the social and organizational elements of this technological practice for a long time . in the end they did stress the importance of social and organizational conditions , but because the outcomes of the assessment by then were quite promising , this did not result in discussion on the pros and cons of the specific design of the screening practice in the pilot studies . on the contrary , the practice investigated in the pilot projects now was put forward as the most desirable . moreover , these conditions were approached only in terms of effectiveness ; issues of feasibility and desirability did not come to the fore . these episodes show once more how the view that technology assessment is about the intended effects of a material device may lead to implicit normative effects of the assessment procedure , in this case with regard to the social and organizational conditions . it tends to limit or even close off the space to discuss the pros and cons of alternative designs of the practice at hand . moreover , it may lead to roles for both professionals and lay users that are not acceptable to them or that severely diminish the realization of alternative values ( like autonomy ) . conceiving novel technology in terms of a sociotechnical practice might help to avoid this implicit normativity by including such social and organizational aspects in the assessment procedures right from the start . the third and last presupposition of the assessment procedure for mammographic screening i want to discuss here is that the development of mammographic screening itself would not affect morality . as pointed out above , the instrumental view of technology implies that humans can first set goals and then decide whatever technology serves their goals best . in the case of mammographic screening the assessment , then , need not focus at the goal aimed for , but only at the question to what extent screening would realize this goal . the history of the hta process and the resulting screening practice show , however , that both the meaning of and the weight assigned to since this is a long term process , it is easier to discern with the benefit of hindsight . one of the first dutch professional publications on early detection of cancer pointed out that the authors explicitly asked : is it desirable indeed to ask people s attention for their own body as a source of disease time and again ? or would a healthy life rather consist in living without having to worry about one s breasts , intestinal tract or stomach ? [ 24 , p. 67 ] the issue was both conceptual ( a shift might occur in the way health is generally interpreted ) and psychological ( this conceptual shift might have impact on people s lived experience ) . typically , the authors of this report declared they were not competent to address such questions . is it desirable indeed to ask people s attention for their own body as a source of disease time and again ? or would a healthy life rather consist in living without having to worry about one s breasts , intestinal tract or stomach ? [ 24 , p. 67 ] not surprisingly , then , the issue was hardly taken into consideration in the subsequent assessment and deliberation processes . as discussed earlier the scientists first defined health exclusively in terms of survival and later also in terms of quality of life . considerations on the impact of screening on views and experiences of health incidentally reappeared in newspaper contributions arguing against screening . however , since empirical evidence for such soft impacts was absent ( and hard to investigate anyway ) , they were easily dismissed in view of the positive evidence for the effectiveness of screening . in retrospect it seems nonetheless justified to conclude that the introduction of breast cancer screening , albeit in conjunction with many other technologies for early detection of disease , did contribute to a new understanding of what it means to be healthy or diseased . the long standing view that health means absence of complaints has been exchanged for the view that disease can be a - symptomatic . as a result , most people now probably accept the view that they can have a disease without noticing anything . this is actually a generalization of the shift that mammography brought about earlier in the medical domain . the starting point of the debates on screening was , after all , the observation that this technology enabled doctors to identify lesions in women who had up to then seemed perfectly healthy , both to themselves and to their doctors . this novel view of disease and health has brought along new norms as well . on the one hand , now that it is possible to diagnose a - symptomatic disease , people are expected to subject themselves to these technologies . even though dutch women are not legally required to participate in the mammographic screening program , those who do not participate often have to justify their choice . they may even be accused of irresponsible behavior , especially if they are diagnosed with breast cancer later on . participation in screening , that is , has become a social norm . on the other hand , novel rights have been claimed . at the introduction of a nation wide program for breast cancer screening the age limits of the target group evidence for the effectiveness of screening older women was thought to be lacking ( another example of how the design of the pilot projects became normative for the national program ) . as described above , in 1992 the health insurance council ( responsible for the funding of the national program ) decided to keep a strict hand at the upper age limit , partly motivated by overspending . the council was confronted with an indignant public response from elderly women as well as medical professionals . they argued that women had a right to be screened , and that denying this right to a subgroup of women on the basis of their age amounted to age discrimination . apparently women above 70 who had been screened for some years felt that they were denied something valuable . of course one may question the legitimacy of this claim . however , just the fact that it was actually claimed shows that a substantial group of people had come to perceive screening as a thus , the screening providers and politicians were confronted with moral considerations they had not anticipated when assessing and deliberating the desirability of mammographic screening . as a result interestingly , the issue was approached in a way resembling the original assessment process : judgment on desirability was postponed pending the production of evidence on the effectiveness of screening older women . novel research was started ; in the meantime the age limits were strictly kept . in 1998 a dutch case control study claimed to have shown that reduction of mortality for 7075 years olds was possible and that screening could significantly improve the quality of life of this group . some actors argued that this evidence was not sufficiently conclusive , since it was not based on rct s and thus could be biased . however , the political decision makers thought it would be unacceptable to let older women wait any longer for the outcomes of such ( usually very long term ) studies [ 57 , p. 1098 ] . in this respect , the claim that a moral right was at stake mitigated the standard of scientific rigor used in the political decision making . in sum : although the initial assessment procedure was predicated on the assumption that mammographic screening would not affect the value of health , both common views of health and disease , as well as related obligations and rights were affected by the development and introduction of mammographic screening . to be sure , the concurrent emergence of many other technologies for early diagnosis definitely played a role as well . partly for practical reasons : if the actors involved reflect on the potential impact of technology on morality , they will be confronted with unanticipated changes in morality less often . another reason is that such changes may affect judgment on the desirability of a technology . since it is usually very difficult to withdraw or prohibit a technology already in use ( as the controversy on age limits shows ) , it makes sense to try to anticipate an emerging technology s long term impacts . this presupposes , however , that hta discards the assumption that morality is stable and independent from technology . as described above , the first publications on mammography present it as a tool to improve the prognosis of women with asymptomatic breast cancer . the actors involved in the assessment process are aware , however , that measuring improved prognosis is rather complicated . counting cases of early detection does not suffice ; the screening program is successful only if subsequent treatment is effective and results in prolonged healthy lives . therefore the outcome measures proposed at first are 10 years survival and mortality rates . however , because an increase of 10 years survival rates without a concurrent mortality reduction is considered undesirable , reduction of breast cancer mortality quickly becomes the central measure of effectiveness . the pilot projects in utrecht and nijmegen are explicitly set up to investigate to what extent mammographic screening reduces breast cancer mortality . the health council in its first report ( 1981 ) affirms this choice : this is the intended effect of mammographic screening and it should be the central focus of the assessment procedure . the actors gradually become aware , however , that mammographic screening does more than it was intended to do and that these additional effects may also be relevant when assessing the technology . the health council quality of life as an additional outcome measure for the effectiveness of mammographic screening : therapeutic effectiveness in the above is translated as reduced breast cancer mortality . it is important to point at the so called breast saving surgery techniques that have gained ground in the treatment of early stage breast cancer lately . development of breast saving surgery techniques was actually spurred by the increasing number of pre - clinical tumors becoming available as a result of screening [ 13 , p. 95 ] . the novel surgery techniques were thus co - produced by mammographic screening , resulting in an unforeseen ( and up to then unintended ) effect . decreased mutilation and increased wellbeing of patients now emerged as a potential additional goal of breast cancer screening , next to mortality reduction . it is important to point at the so called breast saving surgery techniques that have gained ground in the treatment of early stage breast cancer lately . ] thus , the assessors initial focus on the intended effect only started to look too narrow . the concept of quality of life was proposed to broaden the scope of the assessment , to allow different types of presumably positive effects of screening to become visible . although such effects were not explicitly intended at the start of the projects , the health council appears to have been willing to include this novel measure in the assessment . however , the investigators of the two dutch pilot projects did not include quality of life as an additional measure of effectiveness , presumably because they did not want to change the design of their long term studies . it is only in the cost - effectiveness study ( 1990 ) , therefore , that quality of life first appears as a measure of effectiveness . the authors of this report motivate the introduction of the concept in a way that is very different from the health council s earlier suggestion : next to mortality reduction , inevitably undesirable effects will occur . an undesirable effect is , for example , anxiety in women who are referred for further diagnostics and who in the end appear not to have breast cancer ( the false positives ( ) ) . another undesirable effect is that a number of women now know they have breast cancer whereas for some of them the prognosis wo nt improve . ( ) weighing the amount of desired and undesired effects on the one hand and the expected costs and reduced expenses on the other is necessary . quality of life is not meant to measure additional positive effects of screening ; on the contrary , it is a measure for any impact ( both desirable and undesirable ) screening might have on the lives of individual participants . an undesirable effect is , for example , anxiety in women who are referred for further diagnostics and who in the end appear not to have breast cancer ( the false positives ( ) ) . another undesirable effect is that a number of women now know they have breast cancer whereas for some of them the prognosis wo nt improve . ( ) weighing the amount of desired and undesired effects on the one hand and the expected costs and reduced expenses on the other is necessary . ] thus , the actors involved in the assessment of breast cancer screening gradually realized that mammographic screening may have significant impacts besides the intended increase in life expectancy , and that these effects , moreover , need not always be positive . voices from the public debate ( on breast cancer screening , but also on other medical technologies ) may have played a role here . the introduction of quality of life was an attempt to accommodate a broader array of effects , in a way that apparently enabled a sensible comparison of these effects . this history corroborates the argument made by armstrong and caldwell , who claim that the emergence of quality of life in the 1970s and 1980s among others expressed a general discomfort regarding the human costs of technological interventions [ 2 , p. 36 ] . in the dutch case , the desire to include patients perspectives ( often mentioned as a justification to include quality of life ) apparently hardly played a role . it is clear from the cost - effectiveness report how the researchers struggled with the operationalization of quality of life [ 9 , pp . what should or should not be included ? how to operationalize the more subjective types of impacts ? and how to compare different types of impacts ? interestingly , the questionnaires to assess quality of life impacts were not filled out by women participating in the pilot projects , because the questions were considered too complex for these participants . instead , stand ins like economists and medical professionals involved in cancer screening and care acted as respondents . the first were considered to be more used to this type of questionnaire , and the latter were supposed to have a reliable view of how breast cancer may affect women s lives . since then , struggles have continued , leading to a proliferation of instruments measuring quality of life , most of which do involve patients ( or healthy subjects ) as respondents . quality of life , in addition to mortality reduction , has become a widely accepted dimension for assessing the impact of novel medical technologies . the history of assessing breast cancer screening shows why hta should not focus on a technology s intended effect only . technology does more than it is intended to do , both directly and indirectly . as for the direct effects , screening may increase one s life expectancy and decrease wellbeing at the same time . focusing exclusively on the intended ( hoped for ) effects inevitably biases the assessment and leads to ( avoidable ) implicit normativity of the results . including a broad array of potential effects is important for even handed decision making on the desirability of a novel technology . this is even truer for indirect effects , like the improved surgery techniques resulting from the emergence of early diagnostics . good hta should nonetheless try to anticipate and include such effects , both at the start and on the way , as the dutch assessors tried to do . as described above , the reduction of breast cancer mortality by mammographic screening not only requires a well functioning mammographic device , but also an effective therapeutic follow up . breast cancer screening has , then , clear social and organizational conditions from the start . however , at first both the researchers and the political actors involved approach mammographic screening as a material device that processes input ( asymptomatic women ) to deliver a specific output ( mortality reduction ) . the pilot projects are presented as a means to find out how effective this new diagnostic technology will be when applied to asymptomatic women . practical conditions like setting age limits and frequency of screening do get some attention , but only in relation to the question how to increase the screening s effectiveness . it is interesting to see how the actors in the dutch screening assessment seem to have become gradually aware of the importance of social and organizational conditions ; an awareness that in the end led to a strict regulation of the national screening program and even to a law on population screening . when the results of the pilot projects seem sufficiently promising to warrant serious consideration of a nation - wide screening program , the researchers start pointing out that the organizational setting of screening is crucial . the positive results can be repeated only , they state , when the national program is conducted in a way similar to the pilot projects . the health council agrees . in its second report it states : the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . ( ) guarantees are needed for the quality of the work of participating centers ( diagnostics , treatment and counseling of the women involved ) . in this respect a newly instituted center of reference might serve quality control as well . [ 16 , p. 28 ] the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . ( ) guarantees are needed for the quality of the work of participating centers ( diagnostics , treatment and counseling of the women involved ) . in this respect ] both the scientific and the political actors thus gradually became aware that mammography is more than a material device and that breast cancer screening requires more than the distribution of mammography devices . as a result , they concluded that a national screening program should be as similar to the pilot projects as possible . the awareness that organizational conditions are crucial in realizing a certain effect motivated the health council s advice to centralize the organization of screening and make it a uniform practice . moreover , since the pilot projects were designed as a separate , autonomous service , situated between gp s and hospitals , the national program also became a separate service next to primary and secondary health care . thus , because the sociotechnical character of mammographic screening came into the picture relatively late , the organizational choices of the scientific pilot project became indicative for the organization of the national program . as one critic stated , when the committee developed a refined constellation of protocols and procedures for national screening practice , it was actually trying to approach the conditions of scientific research this blocked serious consideration of alternative options . even the question whether or not all actors agreed with the proposed division of roles and responsibilities was hardly discussed . the growing awareness of the social conditions for realizing the aimed for mortality reduction also influenced the approach of and the role ascribed to female participants . in its final report the health council explicitly mentions a high participation rate as a condition for attaining the aimed for effect : the committee emphasizes the importance of a maximal participation in screening for breast cancer . if the participation rate is too low , the aimed for mortality reduction on the population level may also become too low recommendations were formulated to make screening as accessible as possible : the invitational system should function well , screening should be affordable , the location of screening centers should be accessible , counseling should be available , and education should be as persuasive as possible : careful education of the female population is necessary . research into the effectiveness of education with regard to the participation of the women involved in screening is necessary [ 17 , p. 72 ] . here , the need for a high participation rate to achieve the aimed for effects actually led to a neglect of a potentially conflicting value : the autonomy of participants . the implicit tension between effectiveness and women s autonomy came to the fore once more in 1991 , when the minister of health in a letter to the health insurance council in 1991 simply states that invitations , leaflets and campaigning material should contribute to high participation rates as well as enable considered choices by the women in the target group [ 51 , p. 11 ] . in the parliamentary debates on screening , however , mp s stressed that autonomy was the most important value . this position in the end was confirmed by the minister in a memo from 1993 , stating that information on screening should include negative aspects of screening , even if this would lead to a decrease of participation rates [ 53 , pp . this episode shows how the implicit view that technology is a material device led the assessors of mammographic screening to neglect the social and organizational elements of this technological practice for a long time . in the end they did stress the importance of social and organizational conditions , but because the outcomes of the assessment by then were quite promising , this did not result in discussion on the pros and cons of the specific design of the screening practice in the pilot studies . on the contrary , the practice investigated in the pilot projects now was put forward as the most desirable . moreover , these conditions were approached only in terms of effectiveness ; issues of feasibility and desirability did not come to the fore . these episodes show once more how the view that technology assessment is about the intended effects of a material device may lead to implicit normative effects of the assessment procedure , in this case with regard to the social and organizational conditions . it tends to limit or even close off the space to discuss the pros and cons of alternative designs of the practice at hand . moreover , it may lead to roles for both professionals and lay users that are not acceptable to them or that severely diminish the realization of alternative values ( like autonomy ) . conceiving novel technology in terms of a sociotechnical practice might help to avoid this implicit normativity by including such social and organizational aspects in the assessment procedures right from the start . the third and last presupposition of the assessment procedure for mammographic screening i want to discuss here is that the development of mammographic screening itself would not affect morality . as pointed out above , the instrumental view of technology implies that humans can first set goals and then decide whatever technology serves their goals best . in the case of mammographic screening the assessment , then , need not focus at the goal aimed for , but only at the question to what extent screening would realize this goal . the history of the hta process and the resulting screening practice show , however , that both the meaning of and the weight assigned to since this is a long term process , it is easier to discern with the benefit of hindsight . one of the first dutch professional publications on early detection of cancer pointed out that the authors explicitly asked : is it desirable indeed to ask people s attention for their own body as a source of disease time and again ? or would a healthy life rather consist in living without having to worry about one s breasts , intestinal tract or stomach ? [ 24 , p. 67 ] the issue was both conceptual ( a shift might occur in the way health is generally interpreted ) and psychological ( this conceptual shift might have impact on people s lived experience ) . typically , the authors of this report declared they were not competent to address such questions . is it desirable indeed to ask people s attention for their own body as a source of disease time and again ? or would a healthy life rather consist in living without having to worry about one s breasts , intestinal tract or stomach ? [ 24 , p. 67 ] not surprisingly , then , the issue was hardly taken into consideration in the subsequent assessment and deliberation processes . as discussed earlier the scientists first defined health exclusively in terms of survival and later also in terms of quality of life . considerations on the impact of screening on views and experiences of health incidentally reappeared in newspaper contributions arguing against screening . however , since empirical evidence for such soft impacts was absent ( and hard to investigate anyway ) , they were easily dismissed in view of the positive evidence for the effectiveness of screening . in retrospect it seems nonetheless justified to conclude that the introduction of breast cancer screening , albeit in conjunction with many other technologies for early detection of disease , did contribute to a new understanding of what it means to be healthy or diseased . the long standing view that health means absence of complaints has been exchanged for the view that disease can be a - symptomatic . as a result , most people now probably accept the view that they can have a disease without noticing anything . this is actually a generalization of the shift that mammography brought about earlier in the medical domain . the starting point of the debates on screening was , after all , the observation that this technology enabled doctors to identify lesions in women who had up to then seemed perfectly healthy , both to themselves and to their doctors . now that it is possible to diagnose a - symptomatic disease , people are expected to subject themselves to these technologies . even though dutch women are not legally required to participate in the mammographic screening program , those who do not participate often have to justify their choice . they may even be accused of irresponsible behavior , especially if they are diagnosed with breast cancer later on . participation in screening , that is , has become a social norm . on the other hand , novel rights have been claimed . at the introduction of a nation wide program for breast cancer screening the age limits of the target group evidence for the effectiveness of screening older women was thought to be lacking ( another example of how the design of the pilot projects became normative for the national program ) . as described above , in 1992 the health insurance council ( responsible for the funding of the national program ) decided to keep a strict hand at the upper age limit , partly motivated by overspending . the council was confronted with an indignant public response from elderly women as well as medical professionals . they argued that women had a right to be screened , and that denying this right to a subgroup of women on the basis of their age amounted to age discrimination . apparently women above 70 who had been screened for some years felt that they were denied something valuable . however , just the fact that it was actually claimed shows that a substantial group of people had come to perceive screening as a thus , the screening providers and politicians were confronted with moral considerations they had not anticipated when assessing and deliberating the desirability of mammographic screening . as a result interestingly , the issue was approached in a way resembling the original assessment process : judgment on desirability was postponed pending the production of evidence on the effectiveness of screening older women . novel research was started ; in the meantime the age limits were strictly kept . in 1998 a dutch case control study claimed to have shown that reduction of mortality for 7075 years olds was possible and that screening could significantly improve the quality of life of this group . some actors argued that this evidence was not sufficiently conclusive , since it was not based on rct s and thus could be biased . however , the political decision makers thought it would be unacceptable to let older women wait any longer for the outcomes of such ( usually very long term ) studies [ 57 , p. 1098 the claim that a moral right was at stake mitigated the standard of scientific rigor used in the political decision making . in sum : although the initial assessment procedure was predicated on the assumption that mammographic screening would not affect the value of health , both common views of health and disease , as well as related obligations and rights were affected by the development and introduction of mammographic screening . to be sure , the concurrent emergence of many other technologies for early diagnosis definitely played a role as well . partly for practical reasons : if the actors involved reflect on the potential impact of technology on morality , they will be confronted with unanticipated changes in morality less often . another reason is that such changes may affect judgment on the desirability of a technology . since it is usually very difficult to withdraw or prohibit a technology already in use ( as the controversy on age limits shows ) , it makes sense to try to anticipate an emerging technology s long term impacts . this presupposes , however , that hta discards the assumption that morality is stable and independent from technology . to sum up : the actors involved in the assessment of mammographic screening in the netherlands not only developed a general procedure for hta . in the process of assessing mammography , they were also confronted with several limitations and weaknesses of this procedure . with the benefit of hindsight we can trace even more . my reconstruction of the case displays how at various moments a misfit emerged between the instrumentalist presuppositions of the procedures and the way technology actually works . these misfits produced implicit normative effects because important considerations were not or only marginally taken into account . this significantly reduces the moral and political legitimacy of the judgment on the desirability of mammographic screening even though mammographic screening since the historical case is interesting because it reminds us that hta procedures and tools should fit with the way technology works . too many assessments today still resemble the procedure emerging in this early example . how to tailor hta methodology to technology , then ? in this section i will summarise the lessons for hta that can be drawn from the case of mammographic screening , while briefly suggesting which tools or measures might help to avoid the weaknesses from the past . first , technology does more than it is intended to do : it has a broad array of effects . moreover , complex interactions may occur . as a result , many effects are difficult to foresee . it would be nave to suggest that all effects can be anticipated and included in hta of emerging technologies . a first step is to engage users , or more generally stakeholders , early in the assessment process [ 23 , 39 , 60 ] . including actors with different backgrounds in the assessment process will help to chart and imagine the wide variety of effects a technology might have . it also contributes to the democratic legitimacy of the deliberation process . to avoid foreclosing or steering of such an inventory , it may be advisable to start with explorative , qualitative research [ 28 , 29 ] . this would allow stakeholders to respond as much as possible from their own perspective and in their own words . stakeholders could also be asked to participate in the selection of effects to be measured and to comment on proposed outcome measures , to ensure these do justice to the rich meaning of anticipated effects . it also influences our ( to list just a few ) health care practices , roles , responsibilities and identities . because such effects usually emerge only gradually and do not lend themselves for quantitative research , they are easily neglected . earlier examples , ongoing trends and patterns can help to make us more aware of the soft impacts . in addition , they can be used to enhance our imagination of what might happen in the future ( for an example see . historical and sociological insights can be incorporated , for example , in scenarios of possible futures . the second lesson from the case discussed above , partly related to first one , is that technology should not be approached as a device , but as a sociotechnical practice . this means that the social and organizational conditions for making the material device work have to be included right from the start of the assessment . the aim here is not to assess how effective they are with regard to the intended outcome , but to explore additional impacts , for example on the division of roles and responsibilities of the actors involved . to avoid normative effects resulting from the experimental design , it might help to experiment with alternative sociotechnical designs at the same time ( for a similar suggestion see . this would enable comparisons and thus result in informed debate on the acceptability and desirability of proposed practices . finally , the third lesson is that morality is not a stable phenomenon ; it is evolving and technology is an important driver of this evolution . when assessing and debating the desirability of an emerging technology one should , then , take into account the mutual interaction of morality and technology and try to anticipate such effects . a tool to support this is , again , historical analysis , which could then be used to construct plausible scenarios that serve as input for public and political debate . as i indicated above , all this will not suffice to foresee and predict a new technology s impact . technology is a complex phenomenon and so is society , and the confrontation of the two is bound to produce events and effects no one would have been able to imagine in advance . if this is true , it makes sense to construct assessment procedures as flexible as possible . it should be possible to incorporate unexpected effects or adjust outcome measures later on . overall , these lessons and the suggested tools aim to broaden the number and type of considerations that can be put forward in deliberation and debate on the desirability of novel biomedical technologies . the underlying thought is that this both improves the quality of the assessment ( since fewer aspects will be neglected ) and enhances the democratic legitimacy of the process ( since all views can be seriously considered ) . i have hardly paid attention to the division of roles and responsibilities in the deliberation and decision making processes . much more should be said , for example , about the relation between scientific experts , clinicians , lay people and politicians , but this will have to wait for another occasion . the tools , methods and procedures for hta are the evolving products of historical learning processes . the first attempts to perform hta in the netherlands started with the endeavour to provide decision makers with scientific evidence on the effectiveness of mammographic screening . the resulting procedure was to a large extent informed by an instrumentalist view of technology . in the process , the actors performing and using the results of the assessments were confronted with the weaknesses and limitations of the instrumentalist view . the case shows how the actors involved partially responded to these surprises by adjusting their assessment methods . today , hta can pride itself on quite some history in many countries . as a result however , the history of hta is not only a source of standard models , tools and methods . hta could take advantage of these latter experiences more often , to improve the fit between methodology and technology . improving this fit

health technology assessment ( hta ) was developed in the 1970s and 1980s to facilitate decision making on the desirability of new biomedical technologies . since then , many of the standard tools and methods of hta have been criticized for their implicit normativity . at the same time research into the character of technology in practice has motivated philosophers , sociologists and anthropologists to criticize the traditional view of technology as a neutral instrument designed to perform a specific function . such research suggests that the tools and methods of more traditional forms of hta are often inspired by an instrumentalist conception of technology that does not fit the way technology actually works . this paper explores this hypothesis for a specific case : the assessments and deliberations leading to the introduction of breast cancer screening in the netherlands . after reconstructing this history of hta in the making the stepwise model of hta that emerged during the process is discussed . this model was rooted indeed in an instrumentalist conception of technology . however , a more detailed reconstruction of several episodes from this history reveals how the actors already experienced the inadequacy of some of the instrumentalist presuppositions . the historical case thus shows how an instrumentalist conception of technology may result in implicit normative effects . the paper concludes that an instrumentalist view of technology is not a good starting point for hta and briefly suggests how the fit between hta methods and the actual character of technology in practice might be improved .

Introduction Debating the Desirability of Breast Cancer Screening: HTA in the Making Emerging HTA and Its Instrumentalist Presuppositions Lesson 1: Technology Does More Than It is Intended To Do Lesson 2: Technology is More Than a Material Device Lesson 3: Technology Does Affect Morality Tailoring HTA Methodology to Technology: Some Suggestions Conclusion

in the 1970s and 1980s health technology assessment ( hta ) was developed as a way to facilitate collective decision making on the desirability of new biomedical technologies . at about the same time philosophers , sociologists and anthropologists they criticized the common idea that technology is a neutral instrument designed to perform a specific function . such analyses imply that the effects of a novel technology are much more difficult to foresee than the instrumentalist view of technology suggests . this conceptual and empirical work on the character of technology in practice seems to suggest that tools and methods of traditional forms of hta may be problematic partly because they are based on an instrumentalist conception of technology that fits badly with the workings of technology in practice . in this paper , i will explore this hypothesis by reconstructing and discussing an early endeavor to systematically assess a technology s desirability : the assessments and deliberations preceding the introduction of breast cancer screening in the netherlands in the 1970s and 1980s . hta , the case is interesting precisely because it shows the ideal of hta in the making and set the stage for subsequent hta activities.1 in this period a model of hta emerged that was widely used , both in the netherlands and abroad , for many years ( as is visible , for example , in the special issue on the history of hta in the international journal of technology assessment in health care 25 , supplement 1 , 2009 ) . first i will reconstruct the processes preceding the decision to introduce a nation wide program for breast cancer screening in the netherlands . this reconstruction is based on a systematic survey of : ( 1 ) the relevant advisory reports and policy documents in the years 19682005 ; ( 2 ) the minutes of dutch parliament in the years 19701995 ; ( 3 ) the medical professional journals nederlands tijdschrift voor geneeskunde ( 19602005 ) and medisch contact ( 19602005 ) ; and ( 4 ) opinion articles on breast cancer screening in the dutch national newspapers ( 19602005 ) . subsequently i will reconstruct the stepwise model of hta that emerged during the process and analyze how this model was based on an instrumentalist conception of technology . these episodes illustrate how the actors during the process were already confronted with or became aware of unanticipated effects , and how this in some cases led to adjustments in the hta procedures . the historical analysis enables me , in the end , to argue how and why an instrumentalist conception of technology is not a good starting point for hta . i will conclude with some suggestions for improving the fit between hta methods and the actual character of technology in practice . the history of breast cancer screening starts in the 1950 s and 1960 s , when in france and the usa a radiological device for diagnosing breast diseases was developed . when an american study showed that mammographic screening might reduce breast cancer mortality by 30% concerned scientists in the netherlands set up several pilot projects to corroborate the effectiveness of mammographic screening in a dutch setting . in 1977 the dutch minister of health asked the health council to report on the desirability and acceptability of a national breast cancer screening program . the committee soon concluded that the available evidence on the effectiveness of mammographic screening was limited and decided to wait for the results of the pilot projects in utrecht and nijmegen before formulating its advice . to sum up , although the label hta was not used yet , mammographic breast cancer screening was the first health care technology in the netherlands to be systematically assessed for its efficacy , safety , cost effectiveness and impact on quality of life , with the explicit aim to inform and facilitate political decision making . the desirability of the screening program was not only discussed in scientific reports , however . in response , others ( often investigators of the pilot projects or members of the health council s committee ) stressed the advantages of breast cancer screening and the evidence for the positive effects of such a program [ 10 , 12 , 37 , 54 ] . thus , between 1989 and 1995 breast cancer screening was gradually introduced in all regions of the netherlands . since 1995 , all dutch women between 50 and 69 have been invited for breast cancer screening every 2 years . a second surge of debate occurred when the cochrane collaboration in 2001 published a critical metareview of the available evidence for the effectiveness of breast cancer screening . in effect , then , mammographic breast cancer screening was the first health care technology in the netherlands to be subjected to a more or less systematic assessment , with the aim to inform and facilitate political decision making . in the process a model of hta emerged that now seems quite familiar and self - evident because since then it has been used widely , both in the netherlands and in other countries . what is interesting about the historical case discussed here is that it shows the gradual birth of this widespread model in the dutch context.2 in reconstruction , the process consisted of four stages , each considering a specific aspect of mammographic screening : health gainscost - effectivenessorganizational conditionsethical considerations ( like justice , autonomy , screening rights , medicalization ) organizational conditions ethical considerations ( like justice , autonomy , screening rights , medicalization ) the first three stages occurred in a chronological chain : the next stage was initiated only when the results of the preceding one appeared to be sufficiently positive . they occasionally popped up during the process , with a clear increase in the final stages of decision making . this procedure seems to have been inspired by an implicit instrumentalist view of technology in several respects . such a view perceives technology as a material device that is designed to perform a specific function . if technology is a device designed to fulfil a specific function , the first question is of course whether the proposed device is indeed capable to fulfil this function . in a similar vein , the authors of the cost - effectiveness study stated that considerations other than cost - effectiveness could of course play a large role in the political deliberations [ 9 , p. 7 the instrumentalist view is also clearly visible in the set up of the first assessment stage . the pilot projects and the report of the health council thus initially were set up to produce factual evidence on the actual magnitude of the intended effect only.3 the only additional effect that was taken into account was the potential causation of new tumours resulting from the radiation necessary to make the mammograms a side effect , that is , that would directly detract from the intended effect . however , a closer look at the historical episode shows that the actors involved also encountered problems resulting from limitations and shortcomings of the instrumentalist view . some of these problems were diagnosed already by the actors themselves , resulting in modifications of the assessment procedure or other attempts to compensate for the procedure s weaknesses . thus , while trying to invent a systematic procedure to assess mammographic screening , the actors involved learned important lessons about the lack of fit between an instrumentalist view of technology and the way technology works in practice . in the next part of this paper , i point out how difficulties arose with respect to three presuppositions inspired by an instrumentalist view of technology : technology is a means to realize intended effects;technology is a material device;morality may influence technology , but technology does not influence morality . the actors involved in the assessment process are aware , however , that measuring improved prognosis is rather complicated . decreased mutilation and increased wellbeing of patients now emerged as a potential additional goal of breast cancer screening , next to mortality reduction . although such effects were not explicitly intended at the start of the projects , the health council appears to have been willing to include this novel measure in the assessment . thus , the actors involved in the assessment of breast cancer screening gradually realized that mammographic screening may have significant impacts besides the intended increase in life expectancy , and that these effects , moreover , need not always be positive . this history corroborates the argument made by armstrong and caldwell , who claim that the emergence of quality of life in the 1970s and 1980s among others expressed a general discomfort regarding the human costs of technological interventions [ 2 , p. 36 ] . the first were considered to be more used to this type of questionnaire , and the latter were supposed to have a reliable view of how breast cancer may affect women s lives . focusing exclusively on the intended ( hoped for ) effects inevitably biases the assessment and leads to ( avoidable ) implicit normativity of the results . including a broad array of potential effects is important for even handed decision making on the desirability of a novel technology . however , at first both the researchers and the political actors involved approach mammographic screening as a material device that processes input ( asymptomatic women ) to deliver a specific output ( mortality reduction ) . it is interesting to see how the actors in the dutch screening assessment seem to have become gradually aware of the importance of social and organizational conditions ; an awareness that in the end led to a strict regulation of the national screening program and even to a law on population screening . in its second report it states : the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . [ 16 , p. 28 ] the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . [ 16 , p. 28 ] both the scientific and the political actors thus gradually became aware that mammography is more than a material device and that breast cancer screening requires more than the distribution of mammography devices . thus , because the sociotechnical character of mammographic screening came into the picture relatively late , the organizational choices of the scientific pilot project became indicative for the organization of the national program . in its final report the health council explicitly mentions a high participation rate as a condition for attaining the aimed for effect : the committee emphasizes the importance of a maximal participation in screening for breast cancer . research into the effectiveness of education with regard to the participation of the women involved in screening is necessary [ 17 , p. 72 ] . this episode shows how the implicit view that technology is a material device led the assessors of mammographic screening to neglect the social and organizational elements of this technological practice for a long time . in the end they did stress the importance of social and organizational conditions , but because the outcomes of the assessment by then were quite promising , this did not result in discussion on the pros and cons of the specific design of the screening practice in the pilot studies . these episodes show once more how the view that technology assessment is about the intended effects of a material device may lead to implicit normative effects of the assessment procedure , in this case with regard to the social and organizational conditions . conceiving novel technology in terms of a sociotechnical practice might help to avoid this implicit normativity by including such social and organizational aspects in the assessment procedures right from the start . the history of the hta process and the resulting screening practice show , however , that both the meaning of and the weight assigned to since this is a long term process , it is easier to discern with the benefit of hindsight . one of the first dutch professional publications on early detection of cancer pointed out that the authors explicitly asked : is it desirable indeed to ask people s attention for their own body as a source of disease time and again ? in retrospect it seems nonetheless justified to conclude that the introduction of breast cancer screening , albeit in conjunction with many other technologies for early detection of disease , did contribute to a new understanding of what it means to be healthy or diseased . at the introduction of a nation wide program for breast cancer screening the age limits of the target group evidence for the effectiveness of screening older women was thought to be lacking ( another example of how the design of the pilot projects became normative for the national program ) . however , just the fact that it was actually claimed shows that a substantial group of people had come to perceive screening as a thus , the screening providers and politicians were confronted with moral considerations they had not anticipated when assessing and deliberating the desirability of mammographic screening . in this respect , the claim that a moral right was at stake mitigated the standard of scientific rigor used in the political decision making . another reason is that such changes may affect judgment on the desirability of a technology . however , because an increase of 10 years survival rates without a concurrent mortality reduction is considered undesirable , reduction of breast cancer mortality quickly becomes the central measure of effectiveness . it is important to point at the so called breast saving surgery techniques that have gained ground in the treatment of early stage breast cancer lately . decreased mutilation and increased wellbeing of patients now emerged as a potential additional goal of breast cancer screening , next to mortality reduction . it is important to point at the so called breast saving surgery techniques that have gained ground in the treatment of early stage breast cancer lately . ] although such effects were not explicitly intended at the start of the projects , the health council appears to have been willing to include this novel measure in the assessment . thus , the actors involved in the assessment of breast cancer screening gradually realized that mammographic screening may have significant impacts besides the intended increase in life expectancy , and that these effects , moreover , need not always be positive . this history corroborates the argument made by armstrong and caldwell , who claim that the emergence of quality of life in the 1970s and 1980s among others expressed a general discomfort regarding the human costs of technological interventions [ 2 , p. 36 ] . as for the direct effects , screening may increase one s life expectancy and decrease wellbeing at the same time . including a broad array of potential effects is important for even handed decision making on the desirability of a novel technology . good hta should nonetheless try to anticipate and include such effects , both at the start and on the way , as the dutch assessors tried to do . however , at first both the researchers and the political actors involved approach mammographic screening as a material device that processes input ( asymptomatic women ) to deliver a specific output ( mortality reduction ) . it is interesting to see how the actors in the dutch screening assessment seem to have become gradually aware of the importance of social and organizational conditions ; an awareness that in the end led to a strict regulation of the national screening program and even to a law on population screening . in its second report it states : the committee emphasizes that a potential screening program will succeed or fail depending on the way the introduction and design are organized . thus , because the sociotechnical character of mammographic screening came into the picture relatively late , the organizational choices of the scientific pilot project became indicative for the organization of the national program . if the participation rate is too low , the aimed for mortality reduction on the population level may also become too low recommendations were formulated to make screening as accessible as possible : the invitational system should function well , screening should be affordable , the location of screening centers should be accessible , counseling should be available , and education should be as persuasive as possible : careful education of the female population is necessary . research into the effectiveness of education with regard to the participation of the women involved in screening is necessary [ 17 , p. 72 ] . this episode shows how the implicit view that technology is a material device led the assessors of mammographic screening to neglect the social and organizational elements of this technological practice for a long time . in the end they did stress the importance of social and organizational conditions , but because the outcomes of the assessment by then were quite promising , this did not result in discussion on the pros and cons of the specific design of the screening practice in the pilot studies . these episodes show once more how the view that technology assessment is about the intended effects of a material device may lead to implicit normative effects of the assessment procedure , in this case with regard to the social and organizational conditions . conceiving novel technology in terms of a sociotechnical practice might help to avoid this implicit normativity by including such social and organizational aspects in the assessment procedures right from the start . in the case of mammographic screening the assessment , then , need not focus at the goal aimed for , but only at the question to what extent screening would realize this goal . the history of the hta process and the resulting screening practice show , however , that both the meaning of and the weight assigned to since this is a long term process , it is easier to discern with the benefit of hindsight . one of the first dutch professional publications on early detection of cancer pointed out that the authors explicitly asked : is it desirable indeed to ask people s attention for their own body as a source of disease time and again ? in retrospect it seems nonetheless justified to conclude that the introduction of breast cancer screening , albeit in conjunction with many other technologies for early detection of disease , did contribute to a new understanding of what it means to be healthy or diseased . at the introduction of a nation wide program for breast cancer screening the age limits of the target group evidence for the effectiveness of screening older women was thought to be lacking ( another example of how the design of the pilot projects became normative for the national program ) . however , just the fact that it was actually claimed shows that a substantial group of people had come to perceive screening as a thus , the screening providers and politicians were confronted with moral considerations they had not anticipated when assessing and deliberating the desirability of mammographic screening . however , the political decision makers thought it would be unacceptable to let older women wait any longer for the outcomes of such ( usually very long term ) studies [ 57 , p. 1098 the claim that a moral right was at stake mitigated the standard of scientific rigor used in the political decision making . partly for practical reasons : if the actors involved reflect on the potential impact of technology on morality , they will be confronted with unanticipated changes in morality less often . to sum up : the actors involved in the assessment of mammographic screening in the netherlands not only developed a general procedure for hta . my reconstruction of the case displays how at various moments a misfit emerged between the instrumentalist presuppositions of the procedures and the way technology actually works . this significantly reduces the moral and political legitimacy of the judgment on the desirability of mammographic screening even though mammographic screening since the historical case is interesting because it reminds us that hta procedures and tools should fit with the way technology works . the aim here is not to assess how effective they are with regard to the intended outcome , but to explore additional impacts , for example on the division of roles and responsibilities of the actors involved . to avoid normative effects resulting from the experimental design , it might help to experiment with alternative sociotechnical designs at the same time ( for a similar suggestion see . this would enable comparisons and thus result in informed debate on the acceptability and desirability of proposed practices . when assessing and debating the desirability of an emerging technology one should , then , take into account the mutual interaction of morality and technology and try to anticipate such effects . technology is a complex phenomenon and so is society , and the confrontation of the two is bound to produce events and effects no one would have been able to imagine in advance . overall , these lessons and the suggested tools aim to broaden the number and type of considerations that can be put forward in deliberation and debate on the desirability of novel biomedical technologies . the underlying thought is that this both improves the quality of the assessment ( since fewer aspects will be neglected ) and enhances the democratic legitimacy of the process ( since all views can be seriously considered ) . the tools , methods and procedures for hta are the evolving products of historical learning processes . the first attempts to perform hta in the netherlands started with the endeavour to provide decision makers with scientific evidence on the effectiveness of mammographic screening . the resulting procedure was to a large extent informed by an instrumentalist view of technology . in the process , the actors performing and using the results of the assessments were confronted with the weaknesses and limitations of the instrumentalist view . as a result however , the history of hta is not only a source of standard models , tools and methods .

metal organic frameworks ( mofs ) are polymeric crystalline materials comprising of metal ions bridged via co - ordination bonds by polydentate organic linkers . mofs are typically formed by solvothermal reaction of the metal salts with the ligand , and have attracted great interest due to their potentially high internal surface area and porosity , which have great technological potential for gas storage and capture , sensing and catalysis . a wide range of organic molecules have been used as linkers for the construction of mof materials , offering the potential for structures with highly tailored properties and topologies through the systematic control of the geometry and porosity as well as the integration of chemical functionality into the structure of the organic linker . it is also possible to grow mof materials on substrates to form surface - mounted mofs , so - called surmofs . surmofs have been grown with thicknesses varying from a few monolayers up to several microns and are of particular interest since they provide a route to the integration of porous functional materials with thin film devices . for example , surmof materials are promising candidates for fabricating highly responsive gas sensors since they strongly , and in some cases selectively , adsorb various gases . there have also been recent advances in the fabrication of surmof prototype semiconductor devices , and optical sensors based on interferometry . several different approaches to the growth of surmof thin films have been reported . in the simplest method , a substrate is introduced into the solvothermal reaction solution leading , under suitable conditions , to the growth of a thin film of crystallites . it was further demonstrated that the termination of noble metal and oxide surfaces with , respectively , functionalized thiol and silane self - assembled monolayers ( sams ) can promote the growth of surmof films . in addition the crystallographic orientation of the film may be controlled through the use of sams with a specific end - group such as a cooh or oh termination . functionalization of substrates with sams may be combined with lithographic techniques to pattern the substrate surface , for example using microcontact printing ( cp ) , to locally inhibit or promote surmof growth . while representing significant progress , this approach of direct solvothermal growth onto a substrate often results in rough , polycrystalline films or , alternatively , an inhomogeneous coverage of isolated crystals . ( lbl ) technique represents an alternative approach to the growth of surmofs and has been investigated as a possible route to improving the morphology of growth , and also the formation of surmof heterostructures . in the lbl method a substrate undergoes cyclic sequences of immersion in a solution of the metal ion , followed by immersion into a solution of the organic ligand ( or vice versa ) with potential rinsing steps between immersion . variations on this implementation include exposure to metal / ligand solutions in flow reactors , and through spray deposition . importantly , the substrate is only exposed to one component ( metal or ligand ) of the framework at each step in contrast to the solvothermal method which exposes the surface to both simultaneously . it has been proposed that only one monolayer forms in each cycle of immersion in framework component solutions and the surmof is thus grown in a controllable layer - by - layer manner . the lbl method can be combined with lithographic techniques and surface chemical functionalization using sams to enhance further the degree of control of the lateral , vertical and crystallographic geometry of mof material . extensions to this idea include the growth of mof on mof structures by changing the metal or ligand used during the growth process to create a layered material , and post - synthetic modification of linkers to change the chemical functionality of the linker molecules while preserving the mof crystalline structure . the lbl method has been widely described as an epitaxial mode of growth and many of the more exotic approaches to surmof growth such as 3-component pillared structure mofs or heteroepitaxial structures implicitly assume that the growth interface is , at least approximately , parallel to the substrate , analogous to the frank van der merwe mode of thin film growth , and advances by a monolayer in each growth cycle . quartz crystal microbalance ( qcm ) measurements of the mass uptake during each step offer some support for this idealized model of growth , while images acquired using atomic force microscopy ( afm ) have confirmed that the surmof thickness increases with the number of cycles , as expected . however , the growth rate in some cases vastly exceeds that predicted by the ideal lbl model , and there is no direct evidence for the addition of precisely one monolayer in each growth cycle . indeed a recent scanning tunnelling microscopy ( stm ) study has shown no clear evidence for epitaxial features during the growth of the first few monolayers of mof growth . overall the growth of surmofs using the lbl method appears to be more complicated than that suggested by the idealized model . this has motivated the current study of the very early stages of the growth of surmofs by sequential dipping . in particular , we focus on the nucleation and subsequent growth which occurs in the first 10 growth cycles and use amplitude - modulated tapping ( ac ) mode afm to acquire images of the surface after each of the first five cycles of growth , and then again after the tenth cycle . we use a variety of oriented and polycrystalline au surfaces terminated , in some cases , by thiol layers . for most samples it is possible to return to exactly the same position on the surface allowing the repeated imaging of individual nanocrystals and subsequent characterization of their dimensions and orientation relative to the substrate at each stage of the growth process . the use of oriented gold surfaces complicates a direct comparison with other work although it is clear that our results agree with some aspects of previous work , notably the influence of thiol layers . however , under the growth conditions used here we do not observe the sequential addition of single mof layers , and we find that the growing surface which is , in general , not parallel to the substrate . hkust-1 was chosen for this study since it has been studied extensively both as a bulk material and as a surmof thin film grown on thiol or silane functionalized surfaces . in addition due to the relative lability of cu(ii ) centers , hkust-1 can be deposited under ambient conditions at room temperature from dilute ethanolic solutions of both metal salt and ligand using a sequential exposure technique . surmofs of hkust-1 were grown on au(111 ) ( 300 nm epitaxial au(111 ) layer on mica , georg albert pvd , heidelberg , germany ) . thiol sams were deposited by immersion for 30 min in 0.2 mm ethanolic solutions of either 16-mercaptanhexadecanoic acid ( mhda ) or 11-mercaptanundecanol ( muda ) to terminate the surface with , cooh and oh functional groups , respectively . this was followed by rinsing in a stream of ethanol for approximately 30 s and drying in a n2 stream . once the substrates had been prepared , a registration mark was formed in the au layer using a pair of tweezers ( cleaned by flame - annealing in a butane flame for 30s and allowed to cool ) . the substrate was loaded into an asylum research cypher - s afm and the cantilever repeatedly aligned to the registration mark using the inbuilt optics and the coarse xy movement of the sample stage . images of the substrate were acquired in repulsive ac mode using olympus ac240ts silicon cantilevers ( resonant frequency 70 khz , spring constant 2 n / m ) . surmof was grown by immersion in separate 1 mm solutions of cu(o2cch3)2 and benzene-1,3,5-tricarboxylic acid ( tma ) in ethanol ( fisher 99.95% ) ( figure 1 ) . substrates were immersed for 1 min after which the substrates were rinsed in ethanol , dried using n2 and subsequently immersed in the ligand solution for 1 min . the sample was again rinsed in ethanol to remove unreacted ligands and dried with n2 . after each cycle of immersion in metal and ligand solutions the substrates were returned to the afm for further imaging . the optics of the afm were used to align the cantilever with the registration mark to an accuracy in the range 1020 m . the region of interest was found through further alignment using the grain structure and terrace morphology on the au(111 ) surface acquired in previous scans to provide registration . films of hkust-1 on polycrystalline au were prepared ( figure 1 ) using the same preparative and sequential dipping processes on wafers of 300 nm au on si(100 ) with a 50 nm ti bonding layer ( georg albert pvd ) . in the case of the polycrystalline au , the lack of an easily identifiable grain structure and the isotropy of the au surface structure prevented the repeated imaging of the same area . figure 2 shows afm images at different stages of the growth process on various thiol - terminated au(111)/mica substrates . each row shows a sequence of images acquired for au(111 ) substrates prepared in different ways : the top row ( figure 2a ) is a clean au(111 ) surface ; in the second row ( figure 2b ) the au(111 ) surface is pretreated with mhda to give a cooh termination ; in the bottom row ( figure 2c ) the au(111 ) surface has an oh termination through pretreatment with muda . within each row afm images are presented of the surface prior to deposition of the mof in the left - hand column , followed by images acquired after , running from left to right , 1 , 2 , 3 , 4 , 5 , and 10 cycles of sequential dipping . thus , a comparison of images within a row provides a comparison of surfaces at different stages of growth , while the columns provide a comparison of images acquired after the same cycle of growth but with different surface functionalization . afm images of hkust-1 growth on ( a ) au(111 ) , ( b ) mhda on au(111 ) , and ( c ) muda on au(111 ) at different stages during the growth process ; the number of cycles of growth is shown along the horizontal axis and varies from 0 ( i ) to 10 ( vii ) . the arrows in the images a c parts i vii indicate specific surface features to provide registry between successive images . all scale bars are 1 m . the use of registration marks to obtain images of the same region of the surface is highlighted in , for example figure 2a(i , ii ) and figure 2c(i iv ) . in these images the terrace structure of the gold surface may be readily resolved and , further , the detail of this structure is immediately recognizable in successive images ( across the rows ) . as the mof layers grow in the subsequent dipping cycles , the contrast in the images is dominated by the mof crystallites when their height is greater than the variation in height of the underlying gold surface ( typically less than 2 nm over a 1 m area from afm data of clean au(111 ) surfaces ) . the terrace structure is not immediately visible in such images ( see for example figure 2a(iii vii ) ) , although it may be readily discerned in processed images . to aid the identification of the registry in figure 2 , particularly for surfaces where there has been significant growth , we include on each image an arrow identifying a specific surface feature as a reference . note that the loss of contrast of the gold terraces provides an approximate indication of the point where there has been significant surmof growth . the images therefore highlight the differing growth rates due to surface functionality with the muda surface having little growth until at least five cycles , whereas in the case of the mhda terminated surface the loss of contrast occurs after a single cycle indicating significant growth has already occurred . interestingly , we also reproducibly observe hkust-1 growth on the bare au(111 ) metal surface as shown in figure 2a . there have been several reports that mof growth on bare ( i.e. , in the absence of functionalization by thiol sams ) gold does not occur , for example in attempts to grow mof-5 under solvothermal conditions , or hkust-1 using the lbl technique on a polycrystalline au surface , but our results confirm that growth of hkust-1 is possible on au(111 ) . in figure 3 figure 3a shows the fraction of the surface covered by the growing mof film with increasing cycles of growth . for the cooh terminated surface there is already > 5% coverage after 1 cycle and this value increases monotonically over subsequent cycles , although the rate of increase drops between 5 and 10 cycles when the overall surface coverage is > 50% . on the unfunctionalized au(111 ) surface there is very little growth after one cycle and the subsequent rate of increase of surface coverage is also lower than for the cooh surface . for the oh terminated au(111 ) surface there is still a very low fraction of the surface covered even after four growth cycles ; after which the coverage increases very rapidly . these data confirm that growth occurs immediately for the cooh terminated surface , much more slowly for the oh terminated surface , while the clean au(111 ) is an intermediate case . ( a ) surface coverage , ( b ) island density , ( c ) average lateral island size , and ( d ) volume of material per unit area ( or equivalently average thickness ) deposited during the growth process plotted as a function of the number of growth cycles . there are further differences in the morphology of the growing surface , for example related to the number of nucleated islands of mof crystallites . in figure 3b , we show the number of mof islands per unit area . for the cooh terminated surface there are 80 islands per square micron after one cycle corresponding to an average center center island separation of 110 nm . this increases to 130 islands m after 4 cycles , which then decreases after further cycles indicating that islands have started to merge at this point . the island density on the clean au(111 ) the growth on the oh terminated surface differs markedly with very few islands after three cycles followed by a sharp increase in areal density . from the data in figure 3 , parts a and b , we derive the average lateral island size ( figure 3c ) . from our afm images it is also possible to estimate the total volume of material deposited and this is shown in figure 3d as a volume measured in nm adsorbed per area ( in nm ) of substrate ( equivalent to the average height of the film in nanometres ) . for the cooh terminated surface we observe a near - linear increase in both the island size and the total volume of material deposited . this implies that for this surface a near constant amount of material is added in each growth cycle , consistent with previous studies of polycrystalline gold terminated by cooh . from the gradient of the data in figure 3d ) we estimate a growth rate ( averaged across the sample ) of 1.7 nm per cycle , which is much greater than the thickness of a single cu / tma layer , 0.6 nm ( the lattice constant of the unit cell of cubic hkust-1 , which contains four layers of cu / tma is 2.6 nm ; note that in ref ( 28 ) a cycle is defined as two immersions in the metal and ligand , rather than a single immersion in each solution as adopted here , so that in ref ( 28 ) the expected growth rate within the layer - by - layer model is 1.3 nm / cycle for a half - layer comprising two layers of metal ions and two layers of molecules ) . in previous work a constant growth rate has been interpreted as supporting evidence for layer - by - layer growth in the direction perpendicular to the substrate , but it is clear that in this case a constant growth rate occurs in combination with the locally accelerated growth of nanocrystallites , rather than an extended even growth of homogeneous layers . the growth rate on the oh terminated surface is much lower compared with the case of the cooh terminated substrate and , in agreement with previous studies , more nonlinear . this results in a more inhomogeneous distribution of island sizes leading to , after 10 growth cycles , a combination of a small number of large crystallites and a large number of very small crystallites . overall our results indicate that nucleation of mof on these surfaces does not occur immediately but is likely preceded by an accumulation of physisorbed material , possibly mediated by inhomogeneities on the surface . we therefore focus our discussion on the cooh surface on which , as we show below , we can monitor the progressive growth of individual crystallites . this is also the most common choice of termination for growth of hkust-1 on surfaces . figure 4 shows images ( figure4a d ) of a single 1 m region on mhda terminated au(111 ) acquired over 4 stages of the growth process , together with selected height profiles ( figure4e h ) which are extracted from the same positions in successive afm images ( shown as colorised lines in figure 4a d ) . these images confirm unequivocally that we are able to image the same area on the substrate due to the presence of identical , growing features , for example the two bright ( topographically high ) features close to the top of each image . afm images of the same 1 m region on an mhda functionalized au(111 ) region shown in figure 2 b at ( a ) 1 , ( b ) 3 , ( c ) 5 , and ( d ) 10 cycles of growth , respectively . the blue and white boxes in part d correspond to and oriented crystallites respectively that can be traced throughout the entire growth process . ( e h ) line profiles across the features illustrated in parts a d , respectively , showing the growth of material across this region . we identify four distinct types of surface feature ; the first of these are high features with a near circular shape and no obvious faceting . examples include the islands mentioned above and also the feature through which a blue line passes ; the blue height profiles are acquired along this trajectory and show the height of these islands . surprisingly even after one growth cycle the profile indicates a height , and apparent width , of 65 nm . this feature grows so that after three cycles ( figure 4b , f ) the height is 110 nm ; the vertical size of this island is approximately constant after this cycle . it is possible that this feature corresponds to a noncrystalline material , or a three - dimensional aggregate , since there are no obvious facets as expected for a crystallite . we have investigated control samples which were exposed to only the metal , or only the ligand ; from these controls it is clear that all the features which we observe are formed only after immersion in both the metal and the ligand , ruling out the possibility that any features are due to one of these single components . we also observe linear islands ; see for example figure 4b where many linear features are observed . a comparison with the surface earlier ( figure 4a ) and later ( figure 4c ) in the growth process shows that these features grow rapidly in one lateral direction in the first few growth cycles ( they are clearly present , but shorter in figure 4a ) . however , after an initial stage of growth the sizes of these features saturate at a typical length of 150 nm and , from the profiles , a width of 50 nm and a height of 30 nm ( see green and red profiles ) . the other two types of surface features are highly faceted with either a triangular or square base . these features are seen most clearly in figure 4d where they are identified with white ( triangular ) or blue ( square ) outlines . unlike the linear and globular features , these crystallites become progressively larger through the cycles of growth ; their size increases and does not saturate . for example , the red profile in figure 4 extends over two triangular islands on either side of the linear island on which the profile is centered ; these triangular islands become higher and wider as the growth progresses . as we show below , the pyramidal features correspond to oriented islands terminated by { 111 } planes , while the triangles correspond to oriented crystallites , also terminated by { 111 } surfaces . it is possible to identify all of these features at earlier stages of growth ( for example the triangular features in figure 4c , and , more difficult to resolve on the scale of these images , figure 4b ) . to highlight the history of a particular crystallite we show in figure 5a the progressive growth of a pyramidal ( upper row ) and triangular ( lower row ) crystallite . the square and triangular bases of these structures are very clearly resolved after 10 growth cycles and it is possible to track these features back to earlier stages in their growth ; the faceted shape is apparent after 3 cycles , but is poorly resolved after 2 cycles . after 1 cycle , we can resolve the presence of a nucleated island but the shape can not be identified with confidence at this stage . ( a ) afm images of two crystallites highlighted in figure 1d ) after successive growth steps ; ( b ) plot of the height for each crystallite in part a vs the number of growth cycles with a linear fit to the data measured from line profiles across the crystallites relative to the surrounding substrate . these images allow the determination of the growth rate of individual crystallites which complements the average growth rates discussed above . in figure 6 , we show the variation of height with growth cycle for the square and triangular islands highlighted in figure 4d . although there is some variation between individual crystallites the rate of increase in height is 4.9 nm per cycle and 3.2 nm per cycle for the square and triangular islands , respectively . again , we emphasize that this is not consistent with a model in which the growing interface advances uniformly by one layer per cycle . ( a ) height as a function of growth cycles for individual oriented hkust-1 crystallites measured by afm and ( b ) for type crystallites ; ( c ) height vs width ( see inset for location of measurement ) for type crystallites and ( d ) height vs width for type crystallites . we attribute the exposed facets for both island shapes to { 111 } planes given their triangular symmetry ; in addition this is consistent with the formation of free surfaces of bulk hkust-1 crystals by { 111 } planes . the geometry of the square features is consistent with a crystallite growing with the direction oriented normal to the surface , leading to a pyramidal structure ( a square base formed by a { 100 } plane and four faces formed by { 111 } planes ; see for example ref ( 48 ) ) . the ratio of height to the half - diagonal width of the base is expected to be 1:1 for a face centered cubic crystallite with this geometry . this is consistent with observations shown in figure 6c , where this ratio is plotted for the square islands highlighted in figure 4d at different stages of their growth . the presence of a orientation is consistent with previous data for hkust-1 grown on polycrystalline au substrates functionalized with cooh end groups . we assign the triangular features to oriented crystallites terminated by a { 111 } surface . for these crystallites the growing surface is near parallel to the substrate , but we do not see a preferred in - plane orientation implying that there is no epitaxial relationship with au(111 ) surface . the presence of these crystallites is not expected from previous studies of hkust-1 on mhda terminated au . however , in these previous studies polycrystalline gold films were used as substrates , rather than the oriented gold films used here . to check whether this difference is significant we have investigated a surface grown using the protocols described above on mhda - terminated polycrystalline gold . the results , shown in figure 7 , indicate the presence of many islands with typical dimensions 540 nm which have a faceted shape similar to that of the pyramidal oriented islands discussed above , but the triangular islands are not observed ; this observation is consistent with previous work showing the selective growth of crystallites on similar surfaces and other recent afm studies . the pyramidal shape implies that these crystallites are terminated by { 111 } planes . afm image of 10 cycles of growth of hkust-1 on polycrystalline au on si coated with mhda . the afm image shows a predominantly square based pyramid nanocrystallite structure indicating a preferential orientation normal to the surface in agreement with the literature . the growth of hkust-1 on au(111)/mica islands shows significant differences to previous work using polycrystalline gold substrates . first , we observe some growth even on clean au(111)/mica , but the more interesting effects relate to growth on the mhda - terminated surface which introduces a cooh functionality . here we observe the growth of four distinct types of island of which two may be readily identified as and oriented crystallites . using afm it is possible to measure not only the average growth rate across the surface , but also the local growth rate both horizontally and vertically to the surface for individual crystallites . both these rates are greater than one layer / per cycle , while the local increase in height per cycle , measured to be 4.9 and 3.2 nm for , respectively , and type crystallites , is significantly greater than the thickness of a single cu / tma layer , 0.6 nm . this observation demonstrates that in local regions of the surface multiple ( 510 ) layers of mof are grown in each cycle . interestingly the largest crystalline features which are identified in figure 5d may be traced all the way back to the first deposition step in figure 4a . this implies that the nucleation of all these islands has already occurred after the first cycle of growth . furthermore , we can address the question of whether the growing crystallites are redissolved or significantly redistributed when the substrates are reimmersed in the relevant solutions . the reproducible appearance of growing islands and traceability of the location of structures on this highly inhomogeneous surface implies that very little reorganization or solvent - assisted recrystallization of material occurs under the conditions used here . for hkust-1 grown on polycrystalline gold , we observe significant differences ; the lateral size of individual crystallites is considerably smaller , and we do not observe triangular islands . the larger crystallite size on au(111)/mica may be due to the presence of larger , flatter terraces , as compared to the rougher evaporated gold surfaces on which we observe a higher density of nucleated islands , and thus smaller crystallites . this enhancement in nucleation density may be due to inhomogeneities in the gold film , or in the mhda termination . however , our observation of oriented growth is consistent with previous studies of thicker film growth on this surface . the termination of the faceted crystallites by { 111 } planes of hkust-1 is observed for both the and crystallites , and , in addition , for the pyramidal shapes resolved on polycrystalline gold . as stated above , the free and therefore lowest energy , surfaces of bulk hkust-1 crystals are formed by { 111 } planes , and the termination of the surmofs by these same planes is therefore expected . this implies that for growth under near - equilibrium conditions , for which the lowest energy surface will dominate , the growing interface is only parallel to the substrate surface if the crystallographic orientation is normal to the lowest energy surface plane . this is observed for the triangular oriented islands , but not for the pyramidal crystallites and implies that the growing interface of crystallites on both au(111 ) and polycrystalline gold , can not be parallel to the substrate . our afm results also show that under these conditions the rate of propagation of the growing interface is not homogeneous across a surface , and that local rates vary significantly . accordingly the incorporation of single layers of mof can not be inferred from rates averaged across a surface measured , for example , using a qcm to measure mass uptake . the results presented here highlight the very significant contribution that high resolution afm can contribute to the understanding of surmof growth and in particular to monitor the local growth of individual crystallites from the very early stages of nucleation . it is clear that this afm approach allows us to determine whether the layer - by - layer method for surmof growth is valid under the conditions used here ; we find that it is not and observe nanocrystalline growth . we hope that these results will motivate other working in the field to adopt this technique to evaluate other surmofs to provide a rigorous test of the assumptions of the layer - by - layer method . our results suggest that this approach to growth would be best suited to materials for which it is possible to select , for example using self - assembled monolayers , a crystallographic orientation that is normal to the lowest energy surface of the mof . under these circumstances ,

high resolution atomic force microscopy ( afm ) is used to resolve the evolution of crystallites of a metal organic framework ( hkust-1 ) grown on au(111 ) using a liquid - phase layer - by - layer methodology . the nucleation and faceting of individual crystallites is followed by repeatedly imaging the same submicron region after each cycle of growth and we find that the growing surface is terminated by { 111 } facets leading to the formation of pyramidal nanostructures for [ 100 ] oriented crystallites , and triangular [ 111 ] islands with typical lateral dimensions of tens of nanometres . afm images reveal that crystallites can grow by 510 layers in each cycle . the growth rate depends on crystallographic orientation and the morphology of the gold substrate , and we demonstrate that under these conditions the growth is nanocrystalline with a morphology determined by the minimum energy surface .

Introduction Methods Results and Discussion Conclusions

mofs are typically formed by solvothermal reaction of the metal salts with the ligand , and have attracted great interest due to their potentially high internal surface area and porosity , which have great technological potential for gas storage and capture , sensing and catalysis . several different approaches to the growth of surmof thin films have been reported . in the simplest method , a substrate is introduced into the solvothermal reaction solution leading , under suitable conditions , to the growth of a thin film of crystallites . it was further demonstrated that the termination of noble metal and oxide surfaces with , respectively , functionalized thiol and silane self - assembled monolayers ( sams ) can promote the growth of surmof films . in addition the crystallographic orientation of the film may be controlled through the use of sams with a specific end - group such as a cooh or oh termination . ( lbl ) technique represents an alternative approach to the growth of surmofs and has been investigated as a possible route to improving the morphology of growth , and also the formation of surmof heterostructures . in the lbl method a substrate undergoes cyclic sequences of immersion in a solution of the metal ion , followed by immersion into a solution of the organic ligand ( or vice versa ) with potential rinsing steps between immersion . it has been proposed that only one monolayer forms in each cycle of immersion in framework component solutions and the surmof is thus grown in a controllable layer - by - layer manner . extensions to this idea include the growth of mof on mof structures by changing the metal or ligand used during the growth process to create a layered material , and post - synthetic modification of linkers to change the chemical functionality of the linker molecules while preserving the mof crystalline structure . the lbl method has been widely described as an epitaxial mode of growth and many of the more exotic approaches to surmof growth such as 3-component pillared structure mofs or heteroepitaxial structures implicitly assume that the growth interface is , at least approximately , parallel to the substrate , analogous to the frank van der merwe mode of thin film growth , and advances by a monolayer in each growth cycle . quartz crystal microbalance ( qcm ) measurements of the mass uptake during each step offer some support for this idealized model of growth , while images acquired using atomic force microscopy ( afm ) have confirmed that the surmof thickness increases with the number of cycles , as expected . however , the growth rate in some cases vastly exceeds that predicted by the ideal lbl model , and there is no direct evidence for the addition of precisely one monolayer in each growth cycle . indeed a recent scanning tunnelling microscopy ( stm ) study has shown no clear evidence for epitaxial features during the growth of the first few monolayers of mof growth . overall the growth of surmofs using the lbl method appears to be more complicated than that suggested by the idealized model . this has motivated the current study of the very early stages of the growth of surmofs by sequential dipping . in particular , we focus on the nucleation and subsequent growth which occurs in the first 10 growth cycles and use amplitude - modulated tapping ( ac ) mode afm to acquire images of the surface after each of the first five cycles of growth , and then again after the tenth cycle . for most samples it is possible to return to exactly the same position on the surface allowing the repeated imaging of individual nanocrystals and subsequent characterization of their dimensions and orientation relative to the substrate at each stage of the growth process . however , under the growth conditions used here we do not observe the sequential addition of single mof layers , and we find that the growing surface which is , in general , not parallel to the substrate . in addition due to the relative lability of cu(ii ) centers , hkust-1 can be deposited under ambient conditions at room temperature from dilute ethanolic solutions of both metal salt and ligand using a sequential exposure technique . surmofs of hkust-1 were grown on au(111 ) ( 300 nm epitaxial au(111 ) layer on mica , georg albert pvd , heidelberg , germany ) . the substrate was loaded into an asylum research cypher - s afm and the cantilever repeatedly aligned to the registration mark using the inbuilt optics and the coarse xy movement of the sample stage . after each cycle of immersion in metal and ligand solutions the substrates were returned to the afm for further imaging . the optics of the afm were used to align the cantilever with the registration mark to an accuracy in the range 1020 m . films of hkust-1 on polycrystalline au were prepared ( figure 1 ) using the same preparative and sequential dipping processes on wafers of 300 nm au on si(100 ) with a 50 nm ti bonding layer ( georg albert pvd ) . in the case of the polycrystalline au , the lack of an easily identifiable grain structure and the isotropy of the au surface structure prevented the repeated imaging of the same area . figure 2 shows afm images at different stages of the growth process on various thiol - terminated au(111)/mica substrates . each row shows a sequence of images acquired for au(111 ) substrates prepared in different ways : the top row ( figure 2a ) is a clean au(111 ) surface ; in the second row ( figure 2b ) the au(111 ) surface is pretreated with mhda to give a cooh termination ; in the bottom row ( figure 2c ) the au(111 ) surface has an oh termination through pretreatment with muda . within each row afm images are presented of the surface prior to deposition of the mof in the left - hand column , followed by images acquired after , running from left to right , 1 , 2 , 3 , 4 , 5 , and 10 cycles of sequential dipping . thus , a comparison of images within a row provides a comparison of surfaces at different stages of growth , while the columns provide a comparison of images acquired after the same cycle of growth but with different surface functionalization . afm images of hkust-1 growth on ( a ) au(111 ) , ( b ) mhda on au(111 ) , and ( c ) muda on au(111 ) at different stages during the growth process ; the number of cycles of growth is shown along the horizontal axis and varies from 0 ( i ) to 10 ( vii ) . the use of registration marks to obtain images of the same region of the surface is highlighted in , for example figure 2a(i , ii ) and figure 2c(i iv ) . in these images the terrace structure of the gold surface may be readily resolved and , further , the detail of this structure is immediately recognizable in successive images ( across the rows ) . as the mof layers grow in the subsequent dipping cycles , the contrast in the images is dominated by the mof crystallites when their height is greater than the variation in height of the underlying gold surface ( typically less than 2 nm over a 1 m area from afm data of clean au(111 ) surfaces ) . note that the loss of contrast of the gold terraces provides an approximate indication of the point where there has been significant surmof growth . , in the absence of functionalization by thiol sams ) gold does not occur , for example in attempts to grow mof-5 under solvothermal conditions , or hkust-1 using the lbl technique on a polycrystalline au surface , but our results confirm that growth of hkust-1 is possible on au(111 ) . in figure 3 figure 3a shows the fraction of the surface covered by the growing mof film with increasing cycles of growth . on the unfunctionalized au(111 ) surface there is very little growth after one cycle and the subsequent rate of increase of surface coverage is also lower than for the cooh surface . for the oh terminated au(111 ) surface there is still a very low fraction of the surface covered even after four growth cycles ; after which the coverage increases very rapidly . ( a ) surface coverage , ( b ) island density , ( c ) average lateral island size , and ( d ) volume of material per unit area ( or equivalently average thickness ) deposited during the growth process plotted as a function of the number of growth cycles . there are further differences in the morphology of the growing surface , for example related to the number of nucleated islands of mof crystallites . the island density on the clean au(111 ) the growth on the oh terminated surface differs markedly with very few islands after three cycles followed by a sharp increase in areal density . from our afm images it is also possible to estimate the total volume of material deposited and this is shown in figure 3d as a volume measured in nm adsorbed per area ( in nm ) of substrate ( equivalent to the average height of the film in nanometres ) . this implies that for this surface a near constant amount of material is added in each growth cycle , consistent with previous studies of polycrystalline gold terminated by cooh . from the gradient of the data in figure 3d ) we estimate a growth rate ( averaged across the sample ) of 1.7 nm per cycle , which is much greater than the thickness of a single cu / tma layer , 0.6 nm ( the lattice constant of the unit cell of cubic hkust-1 , which contains four layers of cu / tma is 2.6 nm ; note that in ref ( 28 ) a cycle is defined as two immersions in the metal and ligand , rather than a single immersion in each solution as adopted here , so that in ref ( 28 ) the expected growth rate within the layer - by - layer model is 1.3 nm / cycle for a half - layer comprising two layers of metal ions and two layers of molecules ) . in previous work a constant growth rate has been interpreted as supporting evidence for layer - by - layer growth in the direction perpendicular to the substrate , but it is clear that in this case a constant growth rate occurs in combination with the locally accelerated growth of nanocrystallites , rather than an extended even growth of homogeneous layers . the growth rate on the oh terminated surface is much lower compared with the case of the cooh terminated substrate and , in agreement with previous studies , more nonlinear . this results in a more inhomogeneous distribution of island sizes leading to , after 10 growth cycles , a combination of a small number of large crystallites and a large number of very small crystallites . we therefore focus our discussion on the cooh surface on which , as we show below , we can monitor the progressive growth of individual crystallites . figure 4 shows images ( figure4a d ) of a single 1 m region on mhda terminated au(111 ) acquired over 4 stages of the growth process , together with selected height profiles ( figure4e h ) which are extracted from the same positions in successive afm images ( shown as colorised lines in figure 4a d ) . these images confirm unequivocally that we are able to image the same area on the substrate due to the presence of identical , growing features , for example the two bright ( topographically high ) features close to the top of each image . afm images of the same 1 m region on an mhda functionalized au(111 ) region shown in figure 2 b at ( a ) 1 , ( b ) 3 , ( c ) 5 , and ( d ) 10 cycles of growth , respectively . surprisingly even after one growth cycle the profile indicates a height , and apparent width , of 65 nm . as we show below , the pyramidal features correspond to oriented islands terminated by { 111 } planes , while the triangles correspond to oriented crystallites , also terminated by { 111 } surfaces . it is possible to identify all of these features at earlier stages of growth ( for example the triangular features in figure 4c , and , more difficult to resolve on the scale of these images , figure 4b ) . after 1 cycle , we can resolve the presence of a nucleated island but the shape can not be identified with confidence at this stage . ( a ) afm images of two crystallites highlighted in figure 1d ) after successive growth steps ; ( b ) plot of the height for each crystallite in part a vs the number of growth cycles with a linear fit to the data measured from line profiles across the crystallites relative to the surrounding substrate . these images allow the determination of the growth rate of individual crystallites which complements the average growth rates discussed above . although there is some variation between individual crystallites the rate of increase in height is 4.9 nm per cycle and 3.2 nm per cycle for the square and triangular islands , respectively . we attribute the exposed facets for both island shapes to { 111 } planes given their triangular symmetry ; in addition this is consistent with the formation of free surfaces of bulk hkust-1 crystals by { 111 } planes . the geometry of the square features is consistent with a crystallite growing with the direction oriented normal to the surface , leading to a pyramidal structure ( a square base formed by a { 100 } plane and four faces formed by { 111 } planes ; see for example ref ( 48 ) ) . the ratio of height to the half - diagonal width of the base is expected to be 1:1 for a face centered cubic crystallite with this geometry . the presence of a orientation is consistent with previous data for hkust-1 grown on polycrystalline au substrates functionalized with cooh end groups . we assign the triangular features to oriented crystallites terminated by a { 111 } surface . for these crystallites the growing surface is near parallel to the substrate , but we do not see a preferred in - plane orientation implying that there is no epitaxial relationship with au(111 ) surface . the results , shown in figure 7 , indicate the presence of many islands with typical dimensions 540 nm which have a faceted shape similar to that of the pyramidal oriented islands discussed above , but the triangular islands are not observed ; this observation is consistent with previous work showing the selective growth of crystallites on similar surfaces and other recent afm studies . the pyramidal shape implies that these crystallites are terminated by { 111 } planes . using afm it is possible to measure not only the average growth rate across the surface , but also the local growth rate both horizontally and vertically to the surface for individual crystallites . both these rates are greater than one layer / per cycle , while the local increase in height per cycle , measured to be 4.9 and 3.2 nm for , respectively , and type crystallites , is significantly greater than the thickness of a single cu / tma layer , 0.6 nm . this observation demonstrates that in local regions of the surface multiple ( 510 ) layers of mof are grown in each cycle . this implies that the nucleation of all these islands has already occurred after the first cycle of growth . for hkust-1 grown on polycrystalline gold , we observe significant differences ; the lateral size of individual crystallites is considerably smaller , and we do not observe triangular islands . the larger crystallite size on au(111)/mica may be due to the presence of larger , flatter terraces , as compared to the rougher evaporated gold surfaces on which we observe a higher density of nucleated islands , and thus smaller crystallites . the termination of the faceted crystallites by { 111 } planes of hkust-1 is observed for both the and crystallites , and , in addition , for the pyramidal shapes resolved on polycrystalline gold . as stated above , the free and therefore lowest energy , surfaces of bulk hkust-1 crystals are formed by { 111 } planes , and the termination of the surmofs by these same planes is therefore expected . this implies that for growth under near - equilibrium conditions , for which the lowest energy surface will dominate , the growing interface is only parallel to the substrate surface if the crystallographic orientation is normal to the lowest energy surface plane . this is observed for the triangular oriented islands , but not for the pyramidal crystallites and implies that the growing interface of crystallites on both au(111 ) and polycrystalline gold , can not be parallel to the substrate . our afm results also show that under these conditions the rate of propagation of the growing interface is not homogeneous across a surface , and that local rates vary significantly . the results presented here highlight the very significant contribution that high resolution afm can contribute to the understanding of surmof growth and in particular to monitor the local growth of individual crystallites from the very early stages of nucleation . it is clear that this afm approach allows us to determine whether the layer - by - layer method for surmof growth is valid under the conditions used here ; we find that it is not and observe nanocrystalline growth . we hope that these results will motivate other working in the field to adopt this technique to evaluate other surmofs to provide a rigorous test of the assumptions of the layer - by - layer method . our results suggest that this approach to growth would be best suited to materials for which it is possible to select , for example using self - assembled monolayers , a crystallographic orientation that is normal to the lowest energy surface of the mof .

globally , preterm birth ( defined as birth at < 37 weeks of gestation ) affects around 11% of deliveries ( blencowe et al . , 2013 ) and it is closely associated with cerebral palsy , cognitive impairment and neuropsychiatric disease in later life ( kerr - wilson et al . , 2012 ; moore et al . , 2012 ) . individuals born preterm are more likely to experience behavioral and emotional disturbance ( clark et al . , 2008 ; elgen et al . , 2012 ) , autistic spectrum disorder ( johnson et al . , 2010 ) , attention deficit hyperactivity disorder ( bhutta et al . , 2002 ) and psychiatric disease , and these problems persist into adulthood ( crump et al . , 2010 ; halmoy et al . , 2012 ; moster et al . , 2008 ; nosarti et al . , white matter disease is a core feature of the encephalopathy of prematurity and it is associated with reduced connectivity of neural circuitry ( ball et al . , 2013 ; boardman et al . , 2006 ; fischi - gomez et al . , 2014 ; pandit et al . , 2014 ; woodward et al antenatal magnesium sulfate ( mgso4 ) is often given to women expected to deliver preterm because it is associated with reduced rates of cerebral palsy ( doyle et al . . however , there are uncertainties about its long - term effects , and studies have not been designed or powered to investigate its influence on non - motor functions ( chollat et al . , 2014 ; doyle et al . , 2014 ) biomarkers of tissue integrity in the tracts of neural systems that sub - serve a range of functions are required to evaluate more pervasive neuroprotective effects . diffusion mri ( dmri ) and tractography enable measures of white matter tract integrity to be determined in specific tracts - of - interest ( tois ) ( basser et al . , 2000 ) . , 1999 ) or probabilistic ( behrens et al . , 2007 ) approaches to identify the major pathways , with tracts initiated at seed points and typically constrained by operator - defined waypoints segmented pathways can then be analyzed as 3-dimensional regions - of - interest and water molecule diffusion parameters extracted . neonatal dmri data present challenges to tractography algorithms because of smaller head size , resolution difficulties , wide anatomic variation , increased brain water content , and lack of reference atlas data from typically developing newborns . consequently , neonatal studies have tended to focus on a relatively small number of tois , none has provided information about tract topology , and few have included control data . an alternative technique for segmenting tois single seed point tractography is used to segment the same toi across a group of subjects by modeling how individual tracts compare with a predefined reference in terms of length and shape ( bastin et al . , 2010 ; to achieve this , candidate seed points are automatically placed in a cubic neighborhood of native space voxels , with the tract that best matches the reference tract chosen as its representation in each subject ( bastin et al . , 2010 ; clayden et al . , 2006 ; the output includes tract - averaged water diffusion tensor parameters , mean diffusivity ( d ) and fractional anisotropy ( fa ) , and the goodness - of - fit of the best - matched tract to the reference ( r ) . the approach has been useful for mapping white matter change in normal development ( clayden et al . , 2010 ) , and amyotrophic lateral sclerosis ( bastin et al . , 2013 ) . pnt overcomes the bias inherent to user - defined waypoint placement , does not require thresholding of connectivity values , and is unique in providing information about tract topology . in this paper , we generated an atlas of reference tracts for eight tois using pnt to test the hypotheses that : 1 ) tract topology , and tract - averaged d and fa , are altered by preterm birth ; 2 ) tois are separable in the newborn brain based on water diffusion parameters ; and 3 ) pnt is sensitive to detecting perinatal neuroprotective treatment effects . ethical approval was obtained from the national research ethics service ( south east scotland research ethics committee ) , and informed written parental consent was obtained . preterm infants were recruited from the royal infirmary of edinburgh between july 2012 and september 2014 . infants were eligible if they had a birth weight < 1500 g and/or were born at < 31 completed weeks ' postmenstrual age ( pma ) . exclusion criteria for the study were infants with congenital infection , major chromosomal abnormalities , cystic periventricular leucomalacia , post - hemorrhagic ventricular dilatation and porencephalic cysts . mri data from 24 healthy infants born at full term and 87 preterm infants ( mean pma at birth 29 weeks , range 2334 weeks ) were acquired at term equivalent age ( mean pma 39 weeks , range 3742 weeks ) ( table 1 ) . infants were examined in natural sleep with pulse oximetry , temperature and electrocardiography data monitoring . ear protection was used for each infant , comprising earplugs placed in the external ear and neonatal earmuffs ( minimuffs , natus medical inc . a siemens magnetom verio 3 t mri clinical scanner ( siemens ag , healthcare sector , erlangen , germany ) and 12-channel phased - array head coil were used to acquire : t1-weighted mprage volume ( ~1 mm resolution ) , t2-weighted stir ( ~0.9 mm resolution ) , t2-weighted flair ( ~1 mm resolution ) , and dmri ( 11 t2- and 64 diffusion encoding direction ( b = 750 s / mm ) single - shot spin - echo echo planar imaging ( epi ) volumes with 2 mm isotropic voxels ) data . dmri data were pre - processed using fsl tools ( fmrib , oxford , uk ; http://www.fmrib.ox.ac.uk ) . this included brain extraction , and removal of bulk infant motion and eddy current induced artifacts by registering the diffusion - weighted to the first t2-weighted epi volume for each subject . bedpostx / probtrackx algorithm was run with its default parameters : 2-fiber model per voxel , 5000 probabilistic streamlines for each tract with a fixed separation distance of 0.5 mm between successive points to generate the underlying white matter connectivity data ( behrens et al . eight reference tois were created using pnt in standard space from the tractography output produced from a training set of 20 term controls : genu and splenium of corpus callosum , left and right projections of the corticospinal tract ( cst ) , cingulum cingulate gyri ( ccg ) and inferior longitudinal fasciculi ( ilf ) . candidate tracts were generated by seeding within a 7 7 7 neighborhood of native space voxels centered around a standard space seed point located within the center of the white matter structure - of - interest and transferred to the native space of each control . these standard space seed points were defined using an age specific t2-weighted neonatal template ( http://www.brain-development.org ) ( kuklisova - murgasova et al . , 2011 ) . then , an experienced observer ( da ) selected candidate tracts from each control subject that best matched the anatomical shape of the white matter structure - of - interest . using these visually - assessed best - matching tracts , tract shape models were fitted to the tractography data using maximum likelihood ( clayden et al . , 2007 ) , thereby capturing the shape and length variation amongst the controls ' tracts . once the reference tracts and shape models had been created , pnt was re - run over a neighborhood of 3 3 3 voxels centered on the reference tract and transferred to native space for all participants ; the seed point that produced the best matching tract to the reference was then determined . all the computational analysis was implemented in tractor ( http://www.tractor-mri.org.uk ) ( clayden et al . , 2011 ) . two experienced raters ( da , meb ) visually assessed the best - matched tracts . any subject with deviated , aberrant or truncated pathways that were not anatomically plausible representations of the toi were excluded from further analysis ( table 2 ) . the tractography masks of the best match tracts were applied to the d and fa volumes , which permitted tract - averaged values of these biomarkers to be determined for each toi in every subject . finally , the goodness - of - fit of the best match tract to the reference ( r ) was determined from the log - ratio between the matching likelihood of the chosen candidate tract and the matching likelihood of the reference tract under the model . the more negative the r value , the less good the fit between the reference and best match tract . the lateral ventricles were segmented in 59 out of 101 subjects for whom high resolution t1w and t2w structural data were available ( 10 controls and 49 preterms ) . an atlas - based image segmentation approach was used that included lateral ventricles and choroid plexus , but excluded the third and fourth ventricle , cavum septum pellucidum and vergae ( serag et al . , 2012 ) . this was done so that lateral ventricular size could be added as a co - variate to account for possible partial volume effects in the model testing independent effects of antenatal mgso4 on dmri parameters , and to investigate correlation between r and ventricular size . the distributions of d , fa and r in each tract were assessed for normality using the shapiro whitney u test was used to compare r values between groups ; spearman 's rho was used to investigate correlations between r and pma at scan . because age at scan differed between the two cohorts , and water diffusion parameters are dynamic over this developmental period , glm univariate anova was used to investigate the effect of prematurity on these biomarkers in each toi with gestational age as a fixed factor and pma at scan as a covariate . type 1 error was controlled using false discovery rate ( fdr ) ( p < 0.05 ) . to investigate the effect of antenatal mgso4 exposure on tract - averaged d and fa , and r , independent of potential confounders , we first examined the association between these biomarkers with the following factors : exposure to a complete course of antenatal steroids , exposure to antenatal mgso4 , gender and the presence of bronchopulmonary dysplasia ( bpd ) . 0.05 ) were included in a multivariate linear regression model that included pma at birth . fa volumes were blurred in eight preterm infants and two controls due to motion ; these subjects were removed from further analysis leaving 79 preterm and 22 control infants in the analysis sample ( demographic shown in table 1 ) . two preterm infants required laser treatment for retinopathy of prematurity and 1 had a grade 3 intraventricular hemorrhage ( ivh ) identified on cranial ultrasound . fig . 1 displays the reference tois that were identified reliably in the control training set in standard space . across both preterm and control subjects , visual assessment of the individual segmented tracts indicated that pnt provided anatomically acceptable representations of the toi for the vast majority of tracts ( table 2 ) ; for the preterm cohort this ranged from 100% ( 79/79 ) for cst to 82% ( 65/79 ) for right ccg . there was a significant difference in r between groups , with the preterm cohort having larger negative values , and hence greater topological difference from the reference compared to controls for all tois except the splenium ( table 3 ) . 2 illustrates the range of values for r observed in the preterm cohort and how they correspond to tract shape for genu . there were no significant correlations between age at scan and r , with the exception of left cst . r correlated with ventricular volume for left ilf ( p = 0.009 ) and left cst ( p = 0.004 ) , but not with any other toi . table 2 shows tract - averaged water diffusion parameter values for each toi for all participants . in bivariate analysis , tract - averaged d was higher in all tracts in preterm infants at term equivalent age compared with term controls , while fa was lower in all tracts except the right ccg . after adjusting for pma at scan , tract - averaged d was increased in preterm infants at term equivalent age compared with controls in genu ( f = 17.57 , p < 0.001 ) , splenium ( f = 19.22 , p < 0.001 ) , left ilf ( f = 8.92 , p = 0.004 ) and right ilf ( f = 8.36 , p = 0.005 ) . tract - averaged fa was reduced in the splenium ( f = 7.16 , p = 0.009 ) and left ilf ( f = 6.73 , p = 0.01 ) in preterm infants compared with controls after adjustment for age at scan . 3 displays a scatterplot of tract - averaged d versus fa values for the eight tois . this figure indicates that cst , ccg , splenium and , to a lesser extent , ilf and genu are separable . forty ( 51% ) of the preterm cohort had been exposed to antenatal mgso4 , and this was associated with lower d in splenium ( mean difference 0.089 mm / s , 95% ci 0.161 to 0.017 , p = 0.016 ) . in multivariate analysis , antenatal mgso4 was associated with a modest but significant decrease in d in splenium independent of age at birth and bpd ( beta 115.0 , 95% ci 43.4186.5 , p = 0.002 ) . factors included in the model were : pma at birth because there was a difference between those who had [ mean pma 28 weeks ] and had not been exposed to antenatal mgso4 [ mean 29 weeks ] ; and bpd , because it influenced fa on univariate analysis ( mean difference 0.03 , 95% ci 0.05 to 0.005 , p = 0.015 ) . there was no effect of pma at scan , which did not differ between the two groups ( mean 39 weeks versus mean 40 weeks ) , or gender in univariate analysis , so these variables were not included . ventricular volumes were available for a subset of 28 of the treated group and 21 of the untreated group . there was no significant difference in ventricular volume between treated and untreated infants ( 8.4 versus 9.8 cm , p = 0.11 ) , and in a subset analysis of infants where ventricular volume was entered into the model , antenatal mgso4 exposure retained significance ( beta 100.1 , 95% ci 28.8172.3 , p = 0.007 ) . these data suggest that the observed difference in d is unlikely to be attributable to the partial volume effects from csf . we developed a single seed point tractography - based segmentation algorithm ( pnt ) that incorporates tract shape modeling for use in neonates , and have segmented reliably eight major fasciculi . the algorithm is unique in providing a metric of tract topology , r , which measures the goodness - of - fit of the best matched tract with respect to an age specific tract template , and enables the extraction of tract - averaged water diffusion parameters including d and fa . the advantages of pnt over region - of - interest and voxel - based methods for group - wise comparisons of d and fa are that it avoids the need for waypoint placement and setting of connectivity thresholds , and tracts are segmented in native space , which minimizes operator bias and takes account of individual differences in white matter structure . the method uses connectivity data produced by a probabilistic tractography algorithm , fsl 's bedpostx / probtrackx , that can model multiple fiber populations and track through regions of reduced anisotropy , which has particular utility in neonates . furthermore , by modeling the tracts of the whole study group with respect to reference tracts generated from an age specific training set , it is possible to perform group - wise analyses to study perinatal influences on r , and tract - averaged d and fa . the measurement r , which indicates how the length and shape of the best - matched tract in each subject differs from reference tract topology , was significantly altered in preterm infants at term equivalent age compared with term controls in all tracts except splenium . to our knowledge our data suggest that tract shape change occurs independently of d and fa , and is less influenced by age at scan than these measures . therefore , r may add value beyond other biomarkers that are affected by age at scan , which can be challenging to standardize in neonatal studies . a measure of tract shape is also particularly relevant given the wide anatomic variability of the brain at this stage of life , which is not captured in most tractography models . further work is required to understand the functional correlates of r and whether it alters over time , but longitudinal studies of preterm infants suggest that alterations in structure and connectivity that are apparent in the neonatal period persist through early childhood into adulthood ( fischi - gomez et al . , 2014 ; nosarti et al . , 2014 ; pandit et al . , 2014 ) . toga and thompson , 2005 ) , and the incorporation of geometric information about tracts has proven useful for studying heritability of brain integrity and connectivity inferred from dmri in adults ( jin et al . , 2011 ) . we propose that r may offer a new tool for studying genetic contributions to brain structure in the newborn , which to date has been investigated using fa and morphometry but has not included shape analysis of major fasciculi ( boardman et al . , 2014 ) . tract - averaged values of d were higher and fa values were lower in all eight tois in preterm infants at term equivalent age compared with controls in bivariate analysis . after adjustment for pma at scan , which was necessary because both metrics change rapidly during perinatal brain development ( huppi et al . , 1998 ) , d was only increased in genu and splenium , while fa was reduced in the splenium and left ilf . our findings are in broad agreement with previous region - of - interest ( counsell et al . , 2006 ; , 2002 ; rose et al . , 2007 ; skiold et al . , 2010 ) , voxel - based ( gimenez et al . , 2008 ; thompson et al . , 2014 ) , and tractography approaches ( aeby et al . , 2009 ; anjari et al . , 2007 ; thompson et al . , 2011 ) , which show that preterm infants at term equivalent age have higher d and lower fa in white matter compared with term controls , although the anatomic localization of these changes is inconsistent between studies and the role of confounding morbidities is uncertain . the relationship between water diffusion biomarkers and white matter development is complex because the maturational processes of axonal fasciculation , pre - myelination ( proliferation of glial cell bodies , extension of oligodendroglial processes , increase in macromolecules and membrane density ) , and true myelination ( ensheathment of axons ) are all anisotropic ( beaulieu , 2002 ; nossin - manor et al . , 2013 ; zanin et al . , 2011 ) , and the sequence of these processes is not uniform across neural systems . therefore , while dmri biomarkers provide sensitive measures of tissue microstructure , they are also non - specific . we found that tracts were separable based on water diffusion parameters , with the greatest difference observed between cst , ccg and splenium . this suggests that adult patterning of neural systems ( bastin et al . , 2010 ) is present very early in development , and before the onset of widespread myelination . systematic review of 5 randomized controlled trials shows that antenatal mgso4 reduces the burden of cerebral palsy and other motor impairments in the offspring of women who deliver very preterm ( doyle et al . , 2007 ) , but there is some doubt about long term benefit and none of the studies were designed to assess function in other domains as a primary outcome ( chollat et al . , 2014 ; doyle et al . , 2014 ) . magnesium is thought to act on a number of cellular targets and processes in preterm brain injury , which are not restricted to motor systems , so it is plausible that tissue effects might be apparent in other neural systems . we found a modest but significant reduction in d in the splenium of preterm infants who had been exposed to antenatal mgso4 compared with untreated infants , after adjustment for relevant confounders . the splenium connects parietal , temporal and visual cortices , so these data indicate that antenatal mgso4 exposure has beneficial tissue effects that extend beyond motor systems . detailed evaluation across all developmental domains of infants exposed to antenatal mgso4 is needed to understand fully its treatment effects . finally , our data suggest that tract - averaged water diffusion parameters may provide useful biomarkers to complement voxel - wise approaches ( such as tract - based spatial statistics ) for evaluating neuroprotective strategies in the newborn ( o'gorman et al . , 2015 ; porter et al . , 2010 first , we were unable to identify uncinate and arcuate fasciculi in the training set although these tracts can be segmented from adult data using pnt ( clayden et al . , 2007 ) ; this is likely due to lower resolution inherent to neonatal dmri or relative under - development of these tracts at term equivalent age . second , the success of pnt varied from 100% for cst to 82% for right ccg in the preterm group , which may be explained by differential susceptibility to partial volume effects for different tracts . a further potential limitation relates to multiple testing : eight simultaneous tests were carried out for the d and fa data respectively . bonferroni correction is inappropriate here because tract - averaged fa and d values are correlated across the brain ( penke et al . , 2010 ) , so we chose to control the effects of multiple testing with fdr at a threshold of 0.05 . finally , the choice of reference template is a potential source of bias : we chose a reference based on healthy controls born at term because it provided the most biologically further work would be required to determine whether a reference template drawn from a representative sample of the entire study group influences the results . we have shown that pnt , an automated single seed point tractography method , segments tois with a high degree of accuracy in the newborn brain . it is unique in providing information about tract topology , and it enables calculation of tract - averaged dmri biomarkers . tract topology is a marker of preterm birth that is relatively robust to age at scan . we found that discrete neural systems can be separated by water diffusion parameters , reflecting specific microstructural properties of tracts that are apparent in early life , and that tract - averaged dmri biomarkers provide a sensitive metric for evaluating risk factors for preterm brain injury and neuroprotective treatment effects . the approach may have utility for investigating early life determinants of long - term neurologic and psychiatric disorders .

preterm birth is associated with altered connectivity of neural circuits . we developed a tract segmentation method that provides measures of tract shape and integrity ( probabilistic neighborhood tractography , pnt ) from diffusion mri ( dmri ) data to test the hypotheses : 1 ) preterm birth is associated with alterations in tract topology ( r ) , and tract - averaged mean diffusivity ( d ) and fractional anisotropy ( fa ) ; 2 ) neural systems are separable based on tract - averaged dmri parameters ; and 3 ) pnt can detect neuroprotective treatment effects.dmri data were collected from 87 preterm infants ( mean gestational age 29 + 1 weeks , range 23 + 2 34 + 6 ) at term equivalent age and 24 controls ( mean gestational age 39 + 6 weeks ) . pnt was used to segment eight major fasciculi , characterize topology , and extract tract - averaged d and fa.tract topology was altered by preterm birth in all tracts except the splenium ( p < 0.05 , false discovery rate [ fdr ] corrected ) . after adjustment for age at scan , tract - averaged d was increased in the genu and splenium , right corticospinal tract ( cst ) and the left and right inferior longitudinal fasciculi ( ilf ) in preterm infants compared with controls ( p < 0.05 , fdr ) , while tract - averaged fa was decreased in the splenium and left ilf ( p < 0.05 , fdr ) . specific fasciculi were separable based on tract - averaged d and fa values . there was a modest decrease in tract - averaged d in the splenium of preterm infants who had been exposed to antenatal mgso4 for neuroprotection ( p = 0.002).tract topology is a biomarker of preterm brain injury . the data provide proof of concept that tract - averaged dmri parameters have utility for evaluating tissue effects of perinatal neuroprotective strategies .

Introduction Methods and materials Results Discussion Conclusions

globally , preterm birth ( defined as birth at < 37 weeks of gestation ) affects around 11% of deliveries ( blencowe et al . , 2013 ) and it is closely associated with cerebral palsy , cognitive impairment and neuropsychiatric disease in later life ( kerr - wilson et al . , 2012 ) , autistic spectrum disorder ( johnson et al . , 2010 ) , attention deficit hyperactivity disorder ( bhutta et al . , 2002 ) and psychiatric disease , and these problems persist into adulthood ( crump et al . , white matter disease is a core feature of the encephalopathy of prematurity and it is associated with reduced connectivity of neural circuitry ( ball et al . , 2014 ; woodward et al antenatal magnesium sulfate ( mgso4 ) is often given to women expected to deliver preterm because it is associated with reduced rates of cerebral palsy ( doyle et al . however , there are uncertainties about its long - term effects , and studies have not been designed or powered to investigate its influence on non - motor functions ( chollat et al . , 2014 ) biomarkers of tissue integrity in the tracts of neural systems that sub - serve a range of functions are required to evaluate more pervasive neuroprotective effects . diffusion mri ( dmri ) and tractography enable measures of white matter tract integrity to be determined in specific tracts - of - interest ( tois ) ( basser et al . neonatal dmri data present challenges to tractography algorithms because of smaller head size , resolution difficulties , wide anatomic variation , increased brain water content , and lack of reference atlas data from typically developing newborns . consequently , neonatal studies have tended to focus on a relatively small number of tois , none has provided information about tract topology , and few have included control data . an alternative technique for segmenting tois single seed point tractography is used to segment the same toi across a group of subjects by modeling how individual tracts compare with a predefined reference in terms of length and shape ( bastin et al . , 2006 ; the output includes tract - averaged water diffusion tensor parameters , mean diffusivity ( d ) and fractional anisotropy ( fa ) , and the goodness - of - fit of the best - matched tract to the reference ( r ) . , 2010 ) , and amyotrophic lateral sclerosis ( bastin et al . pnt overcomes the bias inherent to user - defined waypoint placement , does not require thresholding of connectivity values , and is unique in providing information about tract topology . in this paper , we generated an atlas of reference tracts for eight tois using pnt to test the hypotheses that : 1 ) tract topology , and tract - averaged d and fa , are altered by preterm birth ; 2 ) tois are separable in the newborn brain based on water diffusion parameters ; and 3 ) pnt is sensitive to detecting perinatal neuroprotective treatment effects . ethical approval was obtained from the national research ethics service ( south east scotland research ethics committee ) , and informed written parental consent was obtained . preterm infants were recruited from the royal infirmary of edinburgh between july 2012 and september 2014 . mri data from 24 healthy infants born at full term and 87 preterm infants ( mean pma at birth 29 weeks , range 2334 weeks ) were acquired at term equivalent age ( mean pma 39 weeks , range 3742 weeks ) ( table 1 ) . ear protection was used for each infant , comprising earplugs placed in the external ear and neonatal earmuffs ( minimuffs , natus medical inc . a siemens magnetom verio 3 t mri clinical scanner ( siemens ag , healthcare sector , erlangen , germany ) and 12-channel phased - array head coil were used to acquire : t1-weighted mprage volume ( ~1 mm resolution ) , t2-weighted stir ( ~0.9 mm resolution ) , t2-weighted flair ( ~1 mm resolution ) , and dmri ( 11 t2- and 64 diffusion encoding direction ( b = 750 s / mm ) single - shot spin - echo echo planar imaging ( epi ) volumes with 2 mm isotropic voxels ) data . dmri data were pre - processed using fsl tools ( fmrib , oxford , uk ; http://www.fmrib.ox.ac.uk ) . this included brain extraction , and removal of bulk infant motion and eddy current induced artifacts by registering the diffusion - weighted to the first t2-weighted epi volume for each subject . eight reference tois were created using pnt in standard space from the tractography output produced from a training set of 20 term controls : genu and splenium of corpus callosum , left and right projections of the corticospinal tract ( cst ) , cingulum cingulate gyri ( ccg ) and inferior longitudinal fasciculi ( ilf ) . using these visually - assessed best - matching tracts , tract shape models were fitted to the tractography data using maximum likelihood ( clayden et al . , 2007 ) , thereby capturing the shape and length variation amongst the controls ' tracts . once the reference tracts and shape models had been created , pnt was re - run over a neighborhood of 3 3 3 voxels centered on the reference tract and transferred to native space for all participants ; the seed point that produced the best matching tract to the reference was then determined . any subject with deviated , aberrant or truncated pathways that were not anatomically plausible representations of the toi were excluded from further analysis ( table 2 ) . the tractography masks of the best match tracts were applied to the d and fa volumes , which permitted tract - averaged values of these biomarkers to be determined for each toi in every subject . finally , the goodness - of - fit of the best match tract to the reference ( r ) was determined from the log - ratio between the matching likelihood of the chosen candidate tract and the matching likelihood of the reference tract under the model . the lateral ventricles were segmented in 59 out of 101 subjects for whom high resolution t1w and t2w structural data were available ( 10 controls and 49 preterms ) . an atlas - based image segmentation approach was used that included lateral ventricles and choroid plexus , but excluded the third and fourth ventricle , cavum septum pellucidum and vergae ( serag et al . this was done so that lateral ventricular size could be added as a co - variate to account for possible partial volume effects in the model testing independent effects of antenatal mgso4 on dmri parameters , and to investigate correlation between r and ventricular size . the distributions of d , fa and r in each tract were assessed for normality using the shapiro whitney u test was used to compare r values between groups ; spearman 's rho was used to investigate correlations between r and pma at scan . because age at scan differed between the two cohorts , and water diffusion parameters are dynamic over this developmental period , glm univariate anova was used to investigate the effect of prematurity on these biomarkers in each toi with gestational age as a fixed factor and pma at scan as a covariate . type 1 error was controlled using false discovery rate ( fdr ) ( p < 0.05 ) . to investigate the effect of antenatal mgso4 exposure on tract - averaged d and fa , and r , independent of potential confounders , we first examined the association between these biomarkers with the following factors : exposure to a complete course of antenatal steroids , exposure to antenatal mgso4 , gender and the presence of bronchopulmonary dysplasia ( bpd ) . 0.05 ) were included in a multivariate linear regression model that included pma at birth . fa volumes were blurred in eight preterm infants and two controls due to motion ; these subjects were removed from further analysis leaving 79 preterm and 22 control infants in the analysis sample ( demographic shown in table 1 ) . two preterm infants required laser treatment for retinopathy of prematurity and 1 had a grade 3 intraventricular hemorrhage ( ivh ) identified on cranial ultrasound . 1 displays the reference tois that were identified reliably in the control training set in standard space . across both preterm and control subjects , visual assessment of the individual segmented tracts indicated that pnt provided anatomically acceptable representations of the toi for the vast majority of tracts ( table 2 ) ; for the preterm cohort this ranged from 100% ( 79/79 ) for cst to 82% ( 65/79 ) for right ccg . there was a significant difference in r between groups , with the preterm cohort having larger negative values , and hence greater topological difference from the reference compared to controls for all tois except the splenium ( table 3 ) . 2 illustrates the range of values for r observed in the preterm cohort and how they correspond to tract shape for genu . there were no significant correlations between age at scan and r , with the exception of left cst . r correlated with ventricular volume for left ilf ( p = 0.009 ) and left cst ( p = 0.004 ) , but not with any other toi . table 2 shows tract - averaged water diffusion parameter values for each toi for all participants . in bivariate analysis , tract - averaged d was higher in all tracts in preterm infants at term equivalent age compared with term controls , while fa was lower in all tracts except the right ccg . after adjusting for pma at scan , tract - averaged d was increased in preterm infants at term equivalent age compared with controls in genu ( f = 17.57 , p < 0.001 ) , splenium ( f = 19.22 , p < 0.001 ) , left ilf ( f = 8.92 , p = 0.004 ) and right ilf ( f = 8.36 , p = 0.005 ) . tract - averaged fa was reduced in the splenium ( f = 7.16 , p = 0.009 ) and left ilf ( f = 6.73 , p = 0.01 ) in preterm infants compared with controls after adjustment for age at scan . 3 displays a scatterplot of tract - averaged d versus fa values for the eight tois . this figure indicates that cst , ccg , splenium and , to a lesser extent , ilf and genu are separable . forty ( 51% ) of the preterm cohort had been exposed to antenatal mgso4 , and this was associated with lower d in splenium ( mean difference 0.089 mm / s , 95% ci 0.161 to 0.017 , p = 0.016 ) . in multivariate analysis , antenatal mgso4 was associated with a modest but significant decrease in d in splenium independent of age at birth and bpd ( beta 115.0 , 95% ci 43.4186.5 , p = 0.002 ) . factors included in the model were : pma at birth because there was a difference between those who had [ mean pma 28 weeks ] and had not been exposed to antenatal mgso4 [ mean 29 weeks ] ; and bpd , because it influenced fa on univariate analysis ( mean difference 0.03 , 95% ci 0.05 to 0.005 , p = 0.015 ) . there was no effect of pma at scan , which did not differ between the two groups ( mean 39 weeks versus mean 40 weeks ) , or gender in univariate analysis , so these variables were not included . there was no significant difference in ventricular volume between treated and untreated infants ( 8.4 versus 9.8 cm , p = 0.11 ) , and in a subset analysis of infants where ventricular volume was entered into the model , antenatal mgso4 exposure retained significance ( beta 100.1 , 95% ci 28.8172.3 , p = 0.007 ) . we developed a single seed point tractography - based segmentation algorithm ( pnt ) that incorporates tract shape modeling for use in neonates , and have segmented reliably eight major fasciculi . the algorithm is unique in providing a metric of tract topology , r , which measures the goodness - of - fit of the best matched tract with respect to an age specific tract template , and enables the extraction of tract - averaged water diffusion parameters including d and fa . the advantages of pnt over region - of - interest and voxel - based methods for group - wise comparisons of d and fa are that it avoids the need for waypoint placement and setting of connectivity thresholds , and tracts are segmented in native space , which minimizes operator bias and takes account of individual differences in white matter structure . furthermore , by modeling the tracts of the whole study group with respect to reference tracts generated from an age specific training set , it is possible to perform group - wise analyses to study perinatal influences on r , and tract - averaged d and fa . the measurement r , which indicates how the length and shape of the best - matched tract in each subject differs from reference tract topology , was significantly altered in preterm infants at term equivalent age compared with term controls in all tracts except splenium . to our knowledge our data suggest that tract shape change occurs independently of d and fa , and is less influenced by age at scan than these measures . therefore , r may add value beyond other biomarkers that are affected by age at scan , which can be challenging to standardize in neonatal studies . a measure of tract shape is also particularly relevant given the wide anatomic variability of the brain at this stage of life , which is not captured in most tractography models . further work is required to understand the functional correlates of r and whether it alters over time , but longitudinal studies of preterm infants suggest that alterations in structure and connectivity that are apparent in the neonatal period persist through early childhood into adulthood ( fischi - gomez et al . toga and thompson , 2005 ) , and the incorporation of geometric information about tracts has proven useful for studying heritability of brain integrity and connectivity inferred from dmri in adults ( jin et al . we propose that r may offer a new tool for studying genetic contributions to brain structure in the newborn , which to date has been investigated using fa and morphometry but has not included shape analysis of major fasciculi ( boardman et al . tract - averaged values of d were higher and fa values were lower in all eight tois in preterm infants at term equivalent age compared with controls in bivariate analysis . after adjustment for pma at scan , which was necessary because both metrics change rapidly during perinatal brain development ( huppi et al . , 1998 ) , d was only increased in genu and splenium , while fa was reduced in the splenium and left ilf . , 2010 ) , voxel - based ( gimenez et al . , 2014 ) , and tractography approaches ( aeby et al . , 2011 ) , which show that preterm infants at term equivalent age have higher d and lower fa in white matter compared with term controls , although the anatomic localization of these changes is inconsistent between studies and the role of confounding morbidities is uncertain . the relationship between water diffusion biomarkers and white matter development is complex because the maturational processes of axonal fasciculation , pre - myelination ( proliferation of glial cell bodies , extension of oligodendroglial processes , increase in macromolecules and membrane density ) , and true myelination ( ensheathment of axons ) are all anisotropic ( beaulieu , 2002 ; nossin - manor et al . , 2011 ) , and the sequence of these processes is not uniform across neural systems . therefore , while dmri biomarkers provide sensitive measures of tissue microstructure , they are also non - specific . we found that tracts were separable based on water diffusion parameters , with the greatest difference observed between cst , ccg and splenium . this suggests that adult patterning of neural systems ( bastin et al . systematic review of 5 randomized controlled trials shows that antenatal mgso4 reduces the burden of cerebral palsy and other motor impairments in the offspring of women who deliver very preterm ( doyle et al . magnesium is thought to act on a number of cellular targets and processes in preterm brain injury , which are not restricted to motor systems , so it is plausible that tissue effects might be apparent in other neural systems . we found a modest but significant reduction in d in the splenium of preterm infants who had been exposed to antenatal mgso4 compared with untreated infants , after adjustment for relevant confounders . the splenium connects parietal , temporal and visual cortices , so these data indicate that antenatal mgso4 exposure has beneficial tissue effects that extend beyond motor systems . detailed evaluation across all developmental domains of infants exposed to antenatal mgso4 is needed to understand fully its treatment effects . finally , our data suggest that tract - averaged water diffusion parameters may provide useful biomarkers to complement voxel - wise approaches ( such as tract - based spatial statistics ) for evaluating neuroprotective strategies in the newborn ( o'gorman et al . , 2010 first , we were unable to identify uncinate and arcuate fasciculi in the training set although these tracts can be segmented from adult data using pnt ( clayden et al . , 2007 ) ; this is likely due to lower resolution inherent to neonatal dmri or relative under - development of these tracts at term equivalent age . second , the success of pnt varied from 100% for cst to 82% for right ccg in the preterm group , which may be explained by differential susceptibility to partial volume effects for different tracts . a further potential limitation relates to multiple testing : eight simultaneous tests were carried out for the d and fa data respectively . bonferroni correction is inappropriate here because tract - averaged fa and d values are correlated across the brain ( penke et al . , 2010 ) , so we chose to control the effects of multiple testing with fdr at a threshold of 0.05 . finally , the choice of reference template is a potential source of bias : we chose a reference based on healthy controls born at term because it provided the most biologically further work would be required to determine whether a reference template drawn from a representative sample of the entire study group influences the results . we have shown that pnt , an automated single seed point tractography method , segments tois with a high degree of accuracy in the newborn brain . it is unique in providing information about tract topology , and it enables calculation of tract - averaged dmri biomarkers . tract topology is a marker of preterm birth that is relatively robust to age at scan . we found that discrete neural systems can be separated by water diffusion parameters , reflecting specific microstructural properties of tracts that are apparent in early life , and that tract - averaged dmri biomarkers provide a sensitive metric for evaluating risk factors for preterm brain injury and neuroprotective treatment effects . the approach may have utility for investigating early life determinants of long - term neurologic and psychiatric disorders .

carbamazepine ( cbz ) is an effective antiepileptic drug and a potent inducer of microsomal enzymes in liver . epoxidation and hydroxylation are the main metabolic pathways , though conjugation reactions may also have a role . a transient and asymptomatic elevation of liver enzymes occurs in 25 - 61% of patients receiving cbz . the most important metabolic product of cbz is 10,11-cbz epoxide , which has been shown to be pharmacologically active . cbz - associated hepatotoxicity manifests as granulomatous hepatitis , abnormal liver function tests , hepatocellular necrosis , lymphadenopathy and biliary tract infection . hepatotoxic reactions of cbz usually occur within 3 - 4 weeks after the initiation of therapy and are independent of serum cbz levels . symptoms usually resolve if the drug is discontinued , however , fatal hepatotoxicity can occur even after early intervention and discontinuation of the drug . cbz - induced acute hepatitis and hepatotoxicity is proved to be fatal with parenchymal collapse and bile duct proliferation . cbz showed pathological liver function tests in children aged 1 year or above . in adults , cbz showed granulomatous hepatitis , eosinophilic infiltrates , portal inflammation , hepatocellular necrosis , bile duct proliferation , cholangitis and cholestasis . cbz is a potent enzyme inducer causing increased liver microsomal enzymes , thereby altering the metabolism of lipids , bile acids and bilirubin . cbz - provoked hepatotoxicity may be due to tnf--activated apoptosis , neoantigen formation and genetic or acquired mitochondrial abnormalities . cbz is found to produce hepatotoxicity shortly after initiating the treatment , usually in 4 weeks , with a range of 1 - 16 weeks . the mechanism responsible for aromatic antiepileptic drug ( aaed)-induced hepatotoxicity has been attributed to the accumulation of arene oxides , due to defective detoxification by epoxide hydrolase , immune - mediated reactions and by direct toxicity . despite the mode of cell death ( apoptosis , necrosis or autophagy ) the idiosyncratic drug - induced hepatotoxicity may be mediated , at least in part , by oxidative stress , characterized by enhanced levels of ros ( e.g. hydroxyl radical , superoxide anion and hydrogen peroxide ) , due to reduced elimination and/or increased production of these species . in the present study , the oxidative stress as a potential mechanism the pathogen - free adult male albino wistar rats weighing 150 - 200 g were used . the rats were housed in polypropylene cages at room temperature ( 25 3c ) with 12/12 hours light and dark cycle and the animals were fed with a balanced diet and tap water ad libitum . the first group served as control and received drinking water orally daily by gavage for 45 days . the second group received 50 mg / kg cbz dissolved in water daily by oral gavage for 45 days between 11.00 hrs and 12 hrs . third , fourth and fifth groups received 50 , 100 and 200 mg / kg ( p.o ) of nac in 0.2% cmc , respectively 1 h prior to administration of 50 mg / kg cbz for 45 days between 11.00 hours and 12.00 hours . on 45 day of drug administration , the animals were anesthetized under light ether anesthesia and the blood samples were collected from retroorbital plexus for estimation of biochemical parameters such as sgot , sgpt , alp , total bilirubin , total protein and albumin . serum was separated by centrifuging blood at 2500 rpm for 10 min and the levels of sgot , sgpt , alp , bilirubin , albumin and total protein were analyzed by using a commercially available enzymatic kit ( agappe , india ) and an autoanalyser ( chemistry analyser ( ca 2005 ) , b4b diagnostic division , china ) . the animals were then killed , liver tissues were isolated and rinsed with cold phosphate buffer ( pb , 100 mm , ph 7.4 ) , weighed , sliced for histopathological studies and stored at -40c . the stored tissues were homogenized and the homogenate was centrifuged at 10,000 g for 10 min at 4c . the supernatant was stored at -40c for further biochemical estimations of endogenous activities of antioxidants such as sod , catalase and gsh and lipid peroxidation . the histopathological study in liver tissue was conducted according to the method of li et al . , ( 1998 ) . a block was prepared in block preparation unit ( shandon histocenter-2 ) and coronal sections ( 10 m ) were cut with the help of a microtome ( leica rm 2255 , lab india ) and picked up on poly - l - lysine - coated slides and were stained with hematoxylin and eosin ( he ) . statistical analysis was performed using one - way analysis of variance ( anova ) with tukey 's post hoc statistical tests using instat . the pathogen - free adult male albino wistar rats weighing 150 - 200 g were used . the rats were housed in polypropylene cages at room temperature ( 25 3c ) with 12/12 hours light and dark cycle and the animals were fed with a balanced diet and tap water ad libitum . the first group served as control and received drinking water orally daily by gavage for 45 days . the second group received 50 mg / kg cbz dissolved in water daily by oral gavage for 45 days between 11.00 hrs and 12 hrs . third , fourth and fifth groups received 50 , 100 and 200 mg / kg ( p.o ) of nac in 0.2% cmc , respectively 1 h prior to administration of 50 mg / kg cbz for 45 days between 11.00 hours and 12.00 hours . on 45 day of drug administration , the animals were anesthetized under light ether anesthesia and the blood samples were collected from retroorbital plexus for estimation of biochemical parameters such as sgot , sgpt , alp , total bilirubin , total protein and albumin . serum was separated by centrifuging blood at 2500 rpm for 10 min and the levels of sgot , sgpt , alp , bilirubin , albumin and total protein were analyzed by using a commercially available enzymatic kit ( agappe , india ) and an autoanalyser ( chemistry analyser ( ca 2005 ) , b4b diagnostic division , china ) . the animals were then killed , liver tissues were isolated and rinsed with cold phosphate buffer ( pb , 100 mm , ph 7.4 ) , weighed , sliced for histopathological studies and stored at -40c . the stored tissues were homogenized and the homogenate was centrifuged at 10,000 g for 10 min at 4c . the supernatant was stored at -40c for further biochemical estimations of endogenous activities of antioxidants such as sod , catalase and gsh and lipid peroxidation . the histopathological study in liver tissue was conducted according to the method of li et al . , ( 1998 ) . a block was prepared in block preparation unit ( shandon histocenter-2 ) and coronal sections ( 10 m ) were cut with the help of a microtome ( leica rm 2255 , lab india ) and picked up on poly - l - lysine - coated slides and were stained with hematoxylin and eosin ( he ) . statistical analysis was performed using one - way analysis of variance ( anova ) with tukey 's post hoc statistical tests using instat . the pathogen - free adult male albino wistar rats weighing 150 - 200 g were used . the rats were housed in polypropylene cages at room temperature ( 25 3c ) with 12/12 hours light and dark cycle and the animals were fed with a balanced diet and tap water ad libitum . the first group served as control and received drinking water orally daily by gavage for 45 days . the second group received 50 mg / kg cbz dissolved in water daily by oral gavage for 45 days between 11.00 hrs and 12 hrs . third , fourth and fifth groups received 50 , 100 and 200 mg / kg ( p.o ) of nac in 0.2% cmc , respectively 1 h prior to administration of 50 mg / kg cbz for 45 days between 11.00 hours and 12.00 hours . on 45 day of drug administration , the animals were anesthetized under light ether anesthesia and the blood samples were collected from retroorbital plexus for estimation of biochemical parameters such as sgot , sgpt , alp , total bilirubin , total protein and albumin . serum was separated by centrifuging blood at 2500 rpm for 10 min and the levels of sgot , sgpt , alp , bilirubin , albumin and total protein were analyzed by using a commercially available enzymatic kit ( agappe , india ) and an autoanalyser ( chemistry analyser ( ca 2005 ) , b4b diagnostic division , china ) . the animals were then killed , liver tissues were isolated and rinsed with cold phosphate buffer ( pb , 100 mm , ph 7.4 ) , weighed , sliced for histopathological studies and stored at -40c . the stored tissues were homogenized and the homogenate was centrifuged at 10,000 g for 10 min at 4c . the supernatant was stored at -40c for further biochemical estimations of endogenous activities of antioxidants such as sod , catalase and gsh and lipid peroxidation . the histopathological study in liver tissue was conducted according to the method of li et al . , ( 1998 ) . a block was prepared in block preparation unit ( shandon histocenter-2 ) and coronal sections ( 10 m ) were cut with the help of a microtome ( leica rm 2255 , lab india ) and picked up on poly - l - lysine - coated slides and were stained with hematoxylin and eosin ( he ) . statistical analysis was performed using one - way analysis of variance ( anova ) with tukey 's post hoc statistical tests using instat . sgot , sgpt , alp , total bilirubin , total protein and albumin are indicators of hepatic function . the cbz - treated group showed significantly elevated levels of sgot , sgpt , alp and total bilirubin and reduced levels of total protein and albumin ( p < 0.05 ) as compared to the control group . administration of cbz along with nac 50 mg / kg does not show significant difference in the liver parameters . administration of cbz along with nac 100 mg / kg showed mild improvement in the liver parameters . administration of cbz along with nac 200 mg / kg showed significant difference in the liver parameters . administration of cbz along with nac significantly reversed the elevated levels of sgot , sgpt , alp and total bilirubin and decreased levels of total protein and albumin ( p < 0.05 ) [ table 1 ] . effect of chronic treatment of carbamazepine and carbamazepine+n - acetyl cysteine on liver enzymes , bilirubin , albumin , total protein and liver lipid peroxidation the cbz treatment significantly increased the liver lipid peroxidation as compared to the control group . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation [ table 1 ] . table 2 shows the effect of chronic treatment of cbz and cbz along with nac on sod , gsh and catalase . chronic cbz treatment significantly decreased the sod , gsh and catalase levels when compared to control animals . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase [ table 2 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on superoxide dismutase , catalase and glutathione the end of 45 days treatment with cbz , there was a significant decrease in body weight and an increase in the absolute and relative liver weights when compared to the control group ( p < 0.05 ) . nac at the dose of 50 , 100 and 200 mg / kg reversed the cbz decreased body weight and cbz increased the absolute and relative liver weights compared with the cbz group ( p < 0.05 ) [ table 3 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on body weight , absolute and relative liver weight figure 1a reveals normal hepatic architecture , which radiates from the central vein to the lobular periphery in the control group . in the cbz - treated group some of the hepatocytes showed hemorrhage , centrilobular and sinusoidal congestion revealing hepatic damage [ figure 1b ] . cbz + nac at a dose of 50 mg / kg showed interlobular fibrosis [ figure 1c ] . cbz + 100 mg / kg nac showed sinusoids with mild congestion [ figure 1d ] . cbz + 200 mg / kg nac showed a normal periportal area and appeared similar to the control liver [ figure 1e ] . effect of carbamazepine on liver histopathology a - control b - cbz effect of n - acetyl cysteine on carbamazepine - induced alterations in liver histopathology c - cbz + nac ( 50 mg / kg ) d - cbz + nac ( 100 mg / kg ) e - cbz + nac ( 200 mg / kg ) sgot , sgpt , alp , total bilirubin , total protein and albumin are indicators of hepatic function . the cbz - treated group showed significantly elevated levels of sgot , sgpt , alp and total bilirubin and reduced levels of total protein and albumin ( p < 0.05 ) as compared to the control group . administration of cbz along with nac 50 mg / kg does not show significant difference in the liver parameters . administration of cbz along with nac 100 mg / kg showed mild improvement in the liver parameters . administration of cbz along with nac 200 mg / kg showed significant difference in the liver parameters . administration of cbz along with nac significantly reversed the elevated levels of sgot , sgpt , alp and total bilirubin and decreased levels of total protein and albumin ( p < 0.05 ) [ table 1 ] . effect of chronic treatment of carbamazepine and carbamazepine+n - acetyl cysteine on liver enzymes , bilirubin , albumin , total protein and liver lipid peroxidation the cbz treatment significantly increased the liver lipid peroxidation as compared to the control group . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation [ table 1 ] . table 2 shows the effect of chronic treatment of cbz and cbz along with nac on sod , gsh and catalase . chronic cbz treatment significantly decreased the sod , gsh and catalase levels when compared to control animals . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase [ table 2 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on superoxide dismutase , catalase and glutathione the end of 45 days treatment with cbz , there was a significant decrease in body weight and an increase in the absolute and relative liver weights when compared to the control group ( p < 0.05 ) . nac at the dose of 50 , 100 and 200 mg / kg reversed the cbz decreased body weight and cbz increased the absolute and relative liver weights compared with the cbz group ( p < 0.05 ) [ table 3 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on body weight , absolute and relative liver weight figure 1a reveals normal hepatic architecture , which radiates from the central vein to the lobular periphery in the control group . in the cbz - treated group some of the hepatocytes showed hemorrhage , centrilobular and sinusoidal congestion revealing hepatic damage [ figure 1b ] . cbz + nac at a dose of 50 mg / kg showed interlobular fibrosis [ figure 1c ] . cbz + 100 mg / kg nac showed sinusoids with mild congestion [ figure 1d ] . cbz + 200 mg / kg nac showed a normal periportal area and appeared similar to the control liver [ figure 1e ] . effect of carbamazepine on liver histopathology a - control b - cbz effect of n - acetyl cysteine on carbamazepine - induced alterations in liver histopathology c - cbz + nac ( 50 mg / kg ) d - cbz + nac ( 100 mg / kg ) e - cbz + nac ( 200 mg / kg ) sgot , sgpt , alp , total bilirubin , total protein and albumin are indicators of hepatic function . the cbz - treated group showed significantly elevated levels of sgot , sgpt , alp and total bilirubin and reduced levels of total protein and albumin ( p < 0.05 ) as compared to the control group . administration of cbz along with nac 50 mg / kg does not show significant difference in the liver parameters . administration of cbz along with nac 100 mg / kg showed mild improvement in the liver parameters . administration of cbz along with nac 200 mg / kg showed significant difference in the liver parameters . administration of cbz along with nac significantly reversed the elevated levels of sgot , sgpt , alp and total bilirubin and decreased levels of total protein and albumin ( p < 0.05 ) [ table 1 ] . effect of chronic treatment of carbamazepine and carbamazepine+n - acetyl cysteine on liver enzymes , bilirubin , albumin , total protein and liver lipid peroxidation the cbz treatment significantly increased the liver lipid peroxidation as compared to the control group . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation [ table 1 ] . table 2 shows the effect of chronic treatment of cbz and cbz along with nac on sod , gsh and catalase . chronic cbz treatment significantly decreased the sod , gsh and catalase levels when compared to control animals . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase [ table 2 ] . the end of 45 days treatment with cbz , there was a significant decrease in body weight and an increase in the absolute and relative liver weights when compared to the control group ( p < 0.05 ) . nac at the dose of 50 , 100 and 200 mg / kg reversed the cbz decreased body weight and cbz increased the absolute and relative liver weights compared with the cbz group ( p < 0.05 ) [ table 3 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on body weight , absolute and relative liver weight figure 1a reveals normal hepatic architecture , which radiates from the central vein to the lobular periphery in the control group . in the cbz - treated group some of the hepatocytes showed hemorrhage , centrilobular and sinusoidal congestion revealing hepatic damage [ figure 1b ] . cbz + nac at a dose of 50 mg / kg showed interlobular fibrosis [ figure 1c ] . cbz + 100 mg / kg nac showed sinusoids with mild congestion [ figure 1d ] . cbz + 200 mg / kg nac showed a normal periportal area and appeared similar to the control liver [ figure 1e ] . effect of carbamazepine on liver histopathology a - control b - cbz effect of n - acetyl cysteine on carbamazepine - induced alterations in liver histopathology c - cbz + nac ( 50 mg / kg ) d - cbz + nac ( 100 mg / kg ) e - cbz + nac ( 200 mg / kg ) a study by eghba et al . showed that cbz induced oxidative stress , increased ros formation and lipid peroxidation products and also decreased mitochondrial membrane potential . cbz caused a decrease in cellular gsh content and an elevation in oxidized gsh levels . their investigation showed that taurine could alleviate oxidative damages induced by cbz and melatonin acts as a good antioxidant to protect hepatocytes against cytotoxicity induced by cbz . it was concluded that taurine and melatonin are effective antioxidants to prevent cbz - induced hepatotoxicity . adikwu and deo concluded that vitamin c has a hepatoprotective effect , owing to its antioxidant property . vitamin c was reported to normalize the levels of serum alanine aminotransferase , aspartate aminotransferase , gamma glutamine , alp , lactate dehydrogenase ( ldh ) and malondialdehyde ( mda ) and serum bilirubin in intoxicated animals . so , nac also possess the same property as antioxidant and exerts hepatoprotection and reduces lipid peroxidation due to its antioxidant activity . in our previous study , it was concluded that the administration of vitamin c exerted hepatoprotective activity against cbz - induced hepatotoxicity . in the present study , it was observed that the cbz treatment significantly increased the levels of sgot , sgpt and bilirubin , the markers of hepatotoxicity and decreased the levels of albumin and total protein . supplementation with nac decreased the markers of hepatotoxicity and exerted significant protection against cbz - induced toxicity by its ability to decrease the lipid peroxidation and thus oxidative stress through its free radical - scavenging activity , which improved the levels of antioxidant defense system . nac at 50 and 100 mg / kg showed interlobular fibrosis and mild sinusoidal congestion . diazinon is an organophosphate compound that is widely used in agriculture ; nac ( 160 mg / kg , i.p . ) significantly reversed the diazion ( 100 mg / kg)-induced hepatotoxicity as observed by reversal of elevated markers of hepatotoxicity . found that in hepatoma - derived cells , acetaminophen caused cytotoxic effects leading to oxidative stress , mitochondrial dysfunction , alterations of membrane permeability and apoptosis . in the course of acetaminophen cytotoxicity , the generation of ros appears as an early event , which is preceded by ldh leakage , gsh depletion and apoptosis . nac pretreatment partially decreased the covalent binding of ccl4-reactive metabolites at 1 and 3 hour of poisoning and to a small extent , the concentration of ccl4 reaching the liver at 3 hour . the results suggested that early and late protective effects of nac might be attributable to its prior conversion to cysteine and gsh . ethanol treatment resulted in steatosis , inflammatory infiltrates , occasional foci of necrosis , elevated alt , accumulation of mda and hydroxynonenal protein adducts , decreased antioxidant capacity , and increased antibody titers toward serum hydroxyethyl radical , mda and hydroxynonenal adducts . nac treatment increased cytosolic antioxidant capacity , abolished ethanol - induced lipid peroxidation , and inhibited the formation of antibodies toward hydroxynonenal and hydroxyethyl radical adducts . n - acetyl cysteine ( nac ) decreased the mortality rate , partly prevented the paracetamol - induced liver damage and partly restored enzyme activities . wong et al . investigated the protective effect of nac against ccl4 and trichloroethylene - induced hepatotoxicity in rats . rats administered with higher dose of nac caused significant reduction in sgpt and sgot levels . they concluded that nac effectively protects against ccl4 and trichloroethylene - induced hepatotoxicity in rats . azathioprine ( aza ) decreased the viability of cultured hepatocytes , depleted intracellular gsh , reduced metabolic activity , mitochondrial swelling , increased oxygen consumption of intact mitochondria and ldh release . aza ( 15 mg / kg , i.p . ) increased the activity of hepatic aminotransferases , liver lipid peroxides , and reduced the gsh contents in liver . the protective effect of aminoguanidine against the enzyme leakage seems to be through the liver cell membrane permeability restoration . pretreatment with nac attenuated the ischemia and reperfusion - induced liver injury by scavenging the radicals and by increasing the sod and catalase levels . in the present study nac decreased the lipid peroxidation by restoring the depleted cat , sod and gsh levels , whereby prevented the hepatotoxicity as it is a precursor for gsh . according to our study results , it could be concluded that nac acts as an effective antioxidant in the prevention of cbz - induced hepatotoxicity . we recommend further clinical trial studies on the antioxidant effect of nac in patients receiving cbz .

objectives : the present study evaluates the hepatoprotective activity of n - acetyl cysteine ( nac ) against carbamazepine ( cbz)-induced hepatotoxicity.materials and methods : rats were treated with cbz ( 50 mg / kg p.o . ) and cbz supplemented with nac 50 , 100 and 200 mg / kg for 45 days , after which blood samples were collected and subjected to liver function tests . animals were killed , liver was separated , weighed and the levels of antioxidants and liver enzymes were estimated . in addition , histopathological investigation was also performed.results:serum glutamate pyruvate transaminase ( sgpt ) , serum glutamate oxaloacetate ( sgot ) transaminase , alkaline phosphatase ( alp ) , bilirubin , lipid peroxidation , absolute and relative liver weights were significantly ( p < 0.05 ) elevated , whereas serum levels of albumin , total protein and body weight were decreased in the cbz - treated animals . cbz also produced vacuolar degeneration , centrilobular congestion and hepatic necrosis as evidenced from histopathological report . nac significantly reduced the levels of serum transaminase , alp , bilirubin and liver weight and increased the levels of total protein , albumin and body weight.conclusion:it was observed that nac increased the glutathione ( gsh ) content , reduced lipid peroxidation and reversed the cbz - induced histopathological abnormalities . cbz - induced hepatotoxicity may be due its toxic epoxide metabolite - induced oxidative stress .

Introduction Materials and Methods None Animals Study Protocol Histopathological studies Statistical analysis Results None Effect Of Chronic Treatment Of Cbz And Cbz + Nac On Liver Parameters Effect Of Nac On Cbz-Induced Alterations On Liver Lipid Peroxidation Effect Of Chronic Treatment Of Cbz And Cbz Along With Nac On Sod, Gsh And Catalase Effect Of Cbz And Cbz Supplemented Nac On Body Weight, Absolute And Relative Liver Weight Histopathological Changes Discussion Conclusion

cbz - associated hepatotoxicity manifests as granulomatous hepatitis , abnormal liver function tests , hepatocellular necrosis , lymphadenopathy and biliary tract infection . cbz showed pathological liver function tests in children aged 1 year or above . cbz - provoked hepatotoxicity may be due to tnf--activated apoptosis , neoantigen formation and genetic or acquired mitochondrial abnormalities . despite the mode of cell death ( apoptosis , necrosis or autophagy ) the idiosyncratic drug - induced hepatotoxicity may be mediated , at least in part , by oxidative stress , characterized by enhanced levels of ros ( e.g. in the present study , the oxidative stress as a potential mechanism the pathogen - free adult male albino wistar rats weighing 150 - 200 g were used . the rats were housed in polypropylene cages at room temperature ( 25 3c ) with 12/12 hours light and dark cycle and the animals were fed with a balanced diet and tap water ad libitum . the second group received 50 mg / kg cbz dissolved in water daily by oral gavage for 45 days between 11.00 hrs and 12 hrs . third , fourth and fifth groups received 50 , 100 and 200 mg / kg ( p.o ) of nac in 0.2% cmc , respectively 1 h prior to administration of 50 mg / kg cbz for 45 days between 11.00 hours and 12.00 hours . on 45 day of drug administration , the animals were anesthetized under light ether anesthesia and the blood samples were collected from retroorbital plexus for estimation of biochemical parameters such as sgot , sgpt , alp , total bilirubin , total protein and albumin . serum was separated by centrifuging blood at 2500 rpm for 10 min and the levels of sgot , sgpt , alp , bilirubin , albumin and total protein were analyzed by using a commercially available enzymatic kit ( agappe , india ) and an autoanalyser ( chemistry analyser ( ca 2005 ) , b4b diagnostic division , china ) . the animals were then killed , liver tissues were isolated and rinsed with cold phosphate buffer ( pb , 100 mm , ph 7.4 ) , weighed , sliced for histopathological studies and stored at -40c . the supernatant was stored at -40c for further biochemical estimations of endogenous activities of antioxidants such as sod , catalase and gsh and lipid peroxidation . the rats were housed in polypropylene cages at room temperature ( 25 3c ) with 12/12 hours light and dark cycle and the animals were fed with a balanced diet and tap water ad libitum . the first group served as control and received drinking water orally daily by gavage for 45 days . the second group received 50 mg / kg cbz dissolved in water daily by oral gavage for 45 days between 11.00 hrs and 12 hrs . third , fourth and fifth groups received 50 , 100 and 200 mg / kg ( p.o ) of nac in 0.2% cmc , respectively 1 h prior to administration of 50 mg / kg cbz for 45 days between 11.00 hours and 12.00 hours . on 45 day of drug administration , the animals were anesthetized under light ether anesthesia and the blood samples were collected from retroorbital plexus for estimation of biochemical parameters such as sgot , sgpt , alp , total bilirubin , total protein and albumin . serum was separated by centrifuging blood at 2500 rpm for 10 min and the levels of sgot , sgpt , alp , bilirubin , albumin and total protein were analyzed by using a commercially available enzymatic kit ( agappe , india ) and an autoanalyser ( chemistry analyser ( ca 2005 ) , b4b diagnostic division , china ) . the animals were then killed , liver tissues were isolated and rinsed with cold phosphate buffer ( pb , 100 mm , ph 7.4 ) , weighed , sliced for histopathological studies and stored at -40c . the rats were housed in polypropylene cages at room temperature ( 25 3c ) with 12/12 hours light and dark cycle and the animals were fed with a balanced diet and tap water ad libitum . the second group received 50 mg / kg cbz dissolved in water daily by oral gavage for 45 days between 11.00 hrs and 12 hrs . third , fourth and fifth groups received 50 , 100 and 200 mg / kg ( p.o ) of nac in 0.2% cmc , respectively 1 h prior to administration of 50 mg / kg cbz for 45 days between 11.00 hours and 12.00 hours . on 45 day of drug administration , the animals were anesthetized under light ether anesthesia and the blood samples were collected from retroorbital plexus for estimation of biochemical parameters such as sgot , sgpt , alp , total bilirubin , total protein and albumin . serum was separated by centrifuging blood at 2500 rpm for 10 min and the levels of sgot , sgpt , alp , bilirubin , albumin and total protein were analyzed by using a commercially available enzymatic kit ( agappe , india ) and an autoanalyser ( chemistry analyser ( ca 2005 ) , b4b diagnostic division , china ) . the animals were then killed , liver tissues were isolated and rinsed with cold phosphate buffer ( pb , 100 mm , ph 7.4 ) , weighed , sliced for histopathological studies and stored at -40c . sgot , sgpt , alp , total bilirubin , total protein and albumin are indicators of hepatic function . the cbz - treated group showed significantly elevated levels of sgot , sgpt , alp and total bilirubin and reduced levels of total protein and albumin ( p < 0.05 ) as compared to the control group . administration of cbz along with nac 50 mg / kg does not show significant difference in the liver parameters . administration of cbz along with nac 100 mg / kg showed mild improvement in the liver parameters . administration of cbz along with nac 200 mg / kg showed significant difference in the liver parameters . administration of cbz along with nac significantly reversed the elevated levels of sgot , sgpt , alp and total bilirubin and decreased levels of total protein and albumin ( p < 0.05 ) [ table 1 ] . effect of chronic treatment of carbamazepine and carbamazepine+n - acetyl cysteine on liver enzymes , bilirubin , albumin , total protein and liver lipid peroxidation the cbz treatment significantly increased the liver lipid peroxidation as compared to the control group . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation [ table 1 ] . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase [ table 2 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on superoxide dismutase , catalase and glutathione the end of 45 days treatment with cbz , there was a significant decrease in body weight and an increase in the absolute and relative liver weights when compared to the control group ( p < 0.05 ) . nac at the dose of 50 , 100 and 200 mg / kg reversed the cbz decreased body weight and cbz increased the absolute and relative liver weights compared with the cbz group ( p < 0.05 ) [ table 3 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on body weight , absolute and relative liver weight figure 1a reveals normal hepatic architecture , which radiates from the central vein to the lobular periphery in the control group . in the cbz - treated group some of the hepatocytes showed hemorrhage , centrilobular and sinusoidal congestion revealing hepatic damage [ figure 1b ] . cbz + nac at a dose of 50 mg / kg showed interlobular fibrosis [ figure 1c ] . cbz + 200 mg / kg nac showed a normal periportal area and appeared similar to the control liver [ figure 1e ] . effect of carbamazepine on liver histopathology a - control b - cbz effect of n - acetyl cysteine on carbamazepine - induced alterations in liver histopathology c - cbz + nac ( 50 mg / kg ) d - cbz + nac ( 100 mg / kg ) e - cbz + nac ( 200 mg / kg ) sgot , sgpt , alp , total bilirubin , total protein and albumin are indicators of hepatic function . the cbz - treated group showed significantly elevated levels of sgot , sgpt , alp and total bilirubin and reduced levels of total protein and albumin ( p < 0.05 ) as compared to the control group . administration of cbz along with nac 50 mg / kg does not show significant difference in the liver parameters . administration of cbz along with nac 100 mg / kg showed mild improvement in the liver parameters . administration of cbz along with nac 200 mg / kg showed significant difference in the liver parameters . administration of cbz along with nac significantly reversed the elevated levels of sgot , sgpt , alp and total bilirubin and decreased levels of total protein and albumin ( p < 0.05 ) [ table 1 ] . effect of chronic treatment of carbamazepine and carbamazepine+n - acetyl cysteine on liver enzymes , bilirubin , albumin , total protein and liver lipid peroxidation the cbz treatment significantly increased the liver lipid peroxidation as compared to the control group . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation [ table 1 ] . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase [ table 2 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on superoxide dismutase , catalase and glutathione the end of 45 days treatment with cbz , there was a significant decrease in body weight and an increase in the absolute and relative liver weights when compared to the control group ( p < 0.05 ) . nac at the dose of 50 , 100 and 200 mg / kg reversed the cbz decreased body weight and cbz increased the absolute and relative liver weights compared with the cbz group ( p < 0.05 ) [ table 3 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on body weight , absolute and relative liver weight figure 1a reveals normal hepatic architecture , which radiates from the central vein to the lobular periphery in the control group . in the cbz - treated group some of the hepatocytes showed hemorrhage , centrilobular and sinusoidal congestion revealing hepatic damage [ figure 1b ] . cbz + nac at a dose of 50 mg / kg showed interlobular fibrosis [ figure 1c ] . cbz + 200 mg / kg nac showed a normal periportal area and appeared similar to the control liver [ figure 1e ] . effect of carbamazepine on liver histopathology a - control b - cbz effect of n - acetyl cysteine on carbamazepine - induced alterations in liver histopathology c - cbz + nac ( 50 mg / kg ) d - cbz + nac ( 100 mg / kg ) e - cbz + nac ( 200 mg / kg ) sgot , sgpt , alp , total bilirubin , total protein and albumin are indicators of hepatic function . the cbz - treated group showed significantly elevated levels of sgot , sgpt , alp and total bilirubin and reduced levels of total protein and albumin ( p < 0.05 ) as compared to the control group . administration of cbz along with nac 50 mg / kg does not show significant difference in the liver parameters . administration of cbz along with nac 100 mg / kg showed mild improvement in the liver parameters . administration of cbz along with nac 200 mg / kg showed significant difference in the liver parameters . administration of cbz along with nac significantly reversed the elevated levels of sgot , sgpt , alp and total bilirubin and decreased levels of total protein and albumin ( p < 0.05 ) [ table 1 ] . effect of chronic treatment of carbamazepine and carbamazepine+n - acetyl cysteine on liver enzymes , bilirubin , albumin , total protein and liver lipid peroxidation the cbz treatment significantly increased the liver lipid peroxidation as compared to the control group . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated lipid peroxidation [ table 1 ] . cbz along with nac 50 and 100 mg / kg does not showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase . cbz along with nac ( 200 mg / kg ) showed dose - dependent reversal ( p < 0.05 ) in the levels of cbz - induced elevated sod , gsh and catalase [ table 2 ] . the end of 45 days treatment with cbz , there was a significant decrease in body weight and an increase in the absolute and relative liver weights when compared to the control group ( p < 0.05 ) . nac at the dose of 50 , 100 and 200 mg / kg reversed the cbz decreased body weight and cbz increased the absolute and relative liver weights compared with the cbz group ( p < 0.05 ) [ table 3 ] . effect of carbamazepine and carbamazepine+n - acetyl cysteine on body weight , absolute and relative liver weight figure 1a reveals normal hepatic architecture , which radiates from the central vein to the lobular periphery in the control group . in the cbz - treated group some of the hepatocytes showed hemorrhage , centrilobular and sinusoidal congestion revealing hepatic damage [ figure 1b ] . cbz + nac at a dose of 50 mg / kg showed interlobular fibrosis [ figure 1c ] . cbz + 100 mg / kg nac showed sinusoids with mild congestion [ figure 1d ] . cbz + 200 mg / kg nac showed a normal periportal area and appeared similar to the control liver [ figure 1e ] . effect of carbamazepine on liver histopathology a - control b - cbz effect of n - acetyl cysteine on carbamazepine - induced alterations in liver histopathology c - cbz + nac ( 50 mg / kg ) d - cbz + nac ( 100 mg / kg ) e - cbz + nac ( 200 mg / kg ) a study by eghba et al . showed that cbz induced oxidative stress , increased ros formation and lipid peroxidation products and also decreased mitochondrial membrane potential . it was concluded that taurine and melatonin are effective antioxidants to prevent cbz - induced hepatotoxicity . vitamin c was reported to normalize the levels of serum alanine aminotransferase , aspartate aminotransferase , gamma glutamine , alp , lactate dehydrogenase ( ldh ) and malondialdehyde ( mda ) and serum bilirubin in intoxicated animals . in our previous study , it was concluded that the administration of vitamin c exerted hepatoprotective activity against cbz - induced hepatotoxicity . in the present study , it was observed that the cbz treatment significantly increased the levels of sgot , sgpt and bilirubin , the markers of hepatotoxicity and decreased the levels of albumin and total protein . supplementation with nac decreased the markers of hepatotoxicity and exerted significant protection against cbz - induced toxicity by its ability to decrease the lipid peroxidation and thus oxidative stress through its free radical - scavenging activity , which improved the levels of antioxidant defense system . nac at 50 and 100 mg / kg showed interlobular fibrosis and mild sinusoidal congestion . significantly reversed the diazion ( 100 mg / kg)-induced hepatotoxicity as observed by reversal of elevated markers of hepatotoxicity . nac treatment increased cytosolic antioxidant capacity , abolished ethanol - induced lipid peroxidation , and inhibited the formation of antibodies toward hydroxynonenal and hydroxyethyl radical adducts . n - acetyl cysteine ( nac ) decreased the mortality rate , partly prevented the paracetamol - induced liver damage and partly restored enzyme activities . they concluded that nac effectively protects against ccl4 and trichloroethylene - induced hepatotoxicity in rats . increased the activity of hepatic aminotransferases , liver lipid peroxides , and reduced the gsh contents in liver . pretreatment with nac attenuated the ischemia and reperfusion - induced liver injury by scavenging the radicals and by increasing the sod and catalase levels . in the present study nac decreased the lipid peroxidation by restoring the depleted cat , sod and gsh levels , whereby prevented the hepatotoxicity as it is a precursor for gsh . according to our study results , it could be concluded that nac acts as an effective antioxidant in the prevention of cbz - induced hepatotoxicity .

despite the progress achieved in the past three decades , human immunodeficiency virus/ human immunodeficiency virus infection ( hiv / aids ) is still considered a serious public health issue ( 1 ) . this infection , in some subgroups in iran which are at higher risk for hiv transmission , exists in form of an epidemic and is extensively spreading in most parts of the world . these subgroups include female sex workers , drug users , and spouses who are hiv positive and act as a vehicle to infect the other people in the community ( 2 ) . it is estimated that around 92000 people in iran are infected with hiv / aids ( 1 ) . in iran , the primary means of hiv / aids transmission among women is through heterosexual sex and there are serious concerns regarding the spread of this disease in the country ( 3 ) . in addition to biological factors , social factors also play a role in women s hiv risks ( 4 ) . in the first quarter of 2011 , sexual transmission has accounted for 21.1% of the total individuals infected with hiv / aids ( 5 ) . access to reliable and accurate information regarding the behaviors of subgroups that are at risk of hiv / aids is important to keep track of the epidemic and program planning , and to form the development of future prevention programs ( 6 ) . therefore , as the first step to assess the predictors of protective sexual behaviors of women at risk for hiv / aids , it is necessary to identify the constructs , important , and influencing concepts for these behaviors . in the next stage of the research , appropriate tools to assess protective sexual behaviors can be designed . currently , most of the questionnaires and their constructs to predict and assess protective sexual behaviors are patterned after the ones in the west and are used in the community and in different groups of the population ; whereas , these models are not very successful in predicting preventive interventions for hiv ( 7 , 8) . the available scales developed to explore hiv related sexual behavior , considered individual concepts such as knowledge ( 9 ) , attitude to the condom ( 10 ) , self - efficacy to condom use ( 11 ) , and the perceived risks ( 12 ) that are not the entire variables influencing hiv related behaviors . considering the different social and economic contexts of these behaviors in different countries , a valid and appropriate instrument inherent to the social and cultural needs of the country must be provided , especially on the sensitive issues such as sexual behavior . as there are no gender sensitive scales in iranian population , the current study was the first and aimed to design a valid and reliable questionnaire within the ecological framework that conforms to the cultural and social conditions of the iranian population in order to predict protective sexual behaviors of women at risk of hiv . the present cross - sectional and methodological study aimed to develop a valid and reliable instrument and was conducted in tehran , iran , in 2012 ( 13 ) . the first step in the process of developing the questionnaire is a deep understanding of the concepts ( 14 ) . structures and relevant factors associated with the protective sexual behaviors of women at risk were identified through in - depth interviews based on a semi - structured interview guide with 25 women at risk aged 21 - 49 years . participants were selected from those who came to the voluntary counseling and testing ( vct ) centers of the shahid behashti university of medical sciences and non - governmental organization ( ngo ) drop - in - centers ( dics ) in iran . constructs and variables identified from the qualitative study were as follows : skills to negotiate condom and control sexual excitement , situational factors , social support , ability to use condom under the influence of drug use , male dominance in sexual decision making , attitudes towards condoms , and prioritization of male sexual needs . in the next step , items for the predictors of protective sexual behaviors of women at risk of hiv / aids questionnaire were determined based on the qualitative study and a review of the literature ( 9 - 12 ) . construct on knowledge was designed as dichotomy . in the next phase , in order to verify the validity of the questionnaire , qualitative face validity , and content and construct validity were assessed . in terms of assessing fluency in writing and appropriate phrasing , the questionnaire was analyzed through a face - to - face method with the participation of 12 individuals , and then modifications were implemented in order to achieve qualitative validity . in order to evaluate the content validity of the questionnaire quantitatively ; the content validity ratio ( cvr ) and content validity index ( cvi ) were used . to validate a questionnaire , the cvr is an item statistic that is useful to reject or retain a specific item . the ratio of content validity was calculated by summing up the comments of the 14 experts from different disciplines including ; health education , reproductive health , midwifery , nursing , epidemiology and researchers and experts from center for aids control in the ministry of health , and also experts from the national aids research centre , then the necessary modifications were implemented . for this purpose , these experts were asked to assess each item based on lawshe scale ( 15 ) . the following formula was devised for the content validity ratio ( cvr ) : in which the ne is the number of panelists indicating essential , and n is the total number of panelists . based on the lawshe s table , when total number of the panelists is 14 , as in the present study , a minimum value of cvr 51 is required to satisfy the five percent level ( 15 ) . for cvi , the 12 experts used a 4-point likert scale to assess the questionnaire in order to identify the following criteria : relevance , simplicity and clarity of statements for each item ( 16 ) . participants of the present study included women who were at risk of hiv , including drug injecting married wives , wives with hiv positive husbands , wives with husbands who were taking narcotics and psychotropic substances , and wives of husbands who had multiple sexual partners . these women were referred to the voluntary counseling and testing ( vct ) centers of the shahid beheshti university of medical sciences ( three centers ) , and four ngo drop - in - centers ( dics ) in tehran , aged 18 years and above , were farsi speakers residing in tehran and had sexual intercourse experience in the past three months . women with severe psychological disorders such as severe mental disorders or psychosis based on medical examination ( two cases ) , transgender ( o case ) , hiv positive ( 1 ) and women who were not interested to continue to participate in the study and the corresponding data were incomplete ( six cases ) were excluded . in the current study , based on the qualitative study that revealed seven concepts concerning protective sexual behaviors of women at risk of hiv , and the rule that 30 samples should be selected per construct ( 17 ) , the sample size was determined 210 . in order to analyze the exploratory factors , the principal component analysis was used and factors were extracted by the oblique factors approach . therefore , 200 women at risk of hiv aged 19 to 49 years participated in the current study . the cut - off point of 0.35 was considered as a minimum load factor required to maintain each item ( 18 ) . in the next phase , the reliability of the questionnaire was assessed by internal consistency ( cronbach s alpha with kuder - richardson 20 ) and stability ( test - retest ) . cronbach s alpha coefficient was calculated for each factor , and the questionnaire as a whole , for 30 samples . also , kuder - richardson 20 was calculated to construct knowledge by 30 samples . according to the existing sources , the coefficient alpha for standard reliability of a tool was set at 0.7 or higher ; however , for descriptive studies , the acceptable alpha value was greater than 0.6 ( 19 ) . in order to determine the reliability of the questionnaire , the test - retest was applied to the 20 participants and was repeated after two weeks . convenience sampling was used . after obtaining the necessary permits , the research was initiated and after obtaining informed consent from the participants , the data collection was implemented . the current study was approved by ethics committee of shahid beheshti university of medical sciences on june 2012 . the present cross - sectional and methodological study aimed to develop a valid and reliable instrument and was conducted in tehran , iran , in 2012 ( 13 ) . the first step in the process of developing the questionnaire is a deep understanding of the concepts ( 14 ) . structures and relevant factors associated with the protective sexual behaviors of women at risk were identified through in - depth interviews based on a semi - structured interview guide with 25 women at risk aged 21 - 49 years . participants were selected from those who came to the voluntary counseling and testing ( vct ) centers of the shahid behashti university of medical sciences and non - governmental organization ( ngo ) drop - in - centers ( dics ) in iran . constructs and variables identified from the qualitative study were as follows : skills to negotiate condom and control sexual excitement , situational factors , social support , ability to use condom under the influence of drug use , male dominance in sexual decision making , attitudes towards condoms , and prioritization of male sexual needs . in the next step , items for the predictors of protective sexual behaviors of women at risk of hiv / aids questionnaire were determined based on the qualitative study and a review of the literature ( 9 - 12 ) . the initial pool of the items was composed of 81 items . construct on knowledge was designed as dichotomy . in the next phase , in order to verify the validity of the questionnaire , qualitative face validity , and content and construct validity were assessed . in terms of assessing fluency in writing and appropriate phrasing , the questionnaire was analyzed through a face - to - face method with the participation of 12 individuals , and then modifications were implemented in order to achieve qualitative validity . in order to evaluate the content validity of the questionnaire quantitatively ; the content validity ratio ( cvr ) and content validity index ( cvi ) were used . to validate a questionnaire , the cvr is an item statistic that is useful to reject or retain a specific item . the ratio of content validity was calculated by summing up the comments of the 14 experts from different disciplines including ; health education , reproductive health , midwifery , nursing , epidemiology and researchers and experts from center for aids control in the ministry of health , and also experts from the national aids research centre , then the necessary modifications were implemented . for this purpose , these experts were asked to assess each item based on lawshe scale ( 15 ) . the following formula was devised for the content validity ratio ( cvr ) : in which the ne is the number of panelists indicating essential , and n is the total number of panelists . based on the lawshe s table , when total number of the panelists is 14 , as in the present study , a minimum value of cvr 51 is required to satisfy the five percent level ( 15 ) . for cvi , the 12 experts used a 4-point likert scale to assess the questionnaire in order to identify the following criteria : relevance , simplicity and clarity of statements for each item ( 16 ) . participants of the present study included women who were at risk of hiv , including drug injecting married wives , wives with hiv positive husbands , wives with husbands who were taking narcotics and psychotropic substances , and wives of husbands who had multiple sexual partners . these women were referred to the voluntary counseling and testing ( vct ) centers of the shahid beheshti university of medical sciences ( three centers ) , and four ngo drop - in - centers ( dics ) in tehran , aged 18 years and above , were farsi speakers residing in tehran and had sexual intercourse experience in the past three months . women with severe psychological disorders such as severe mental disorders or psychosis based on medical examination ( two cases ) , transgender ( o case ) , hiv positive ( 1 ) and women who were not interested to continue to participate in the study and the corresponding data were incomplete ( six cases ) were excluded . in the current study , based on the qualitative study that revealed seven concepts concerning protective sexual behaviors of women at risk of hiv , and the rule that 30 samples should be selected per construct ( 17 ) , the sample size was determined 210 . in order to analyze the exploratory factors , the principal component analysis was used and factors were extracted by the oblique factors approach . therefore , 200 women at risk of hiv aged 19 to 49 years participated in the current study . the cut - off point of 0.35 was considered as a minimum load factor required to maintain each item ( 18 ) . in the next phase , the reliability of the questionnaire was assessed by internal consistency ( cronbach s alpha with kuder - richardson 20 ) and stability ( test - retest ) . cronbach s alpha coefficient was calculated for each factor , and the questionnaire as a whole , for 30 samples . also , kuder - richardson 20 was calculated to construct knowledge by 30 samples . according to the existing sources , the coefficient alpha for standard reliability of a tool was set at 0.7 or higher ; however , for descriptive studies , the acceptable alpha value was greater than 0.6 ( 19 ) . in order to determine the reliability of the questionnaire , the test - retest was applied to the 20 participants and was repeated after two weeks . convenience sampling was used . after obtaining the necessary permits , the research was initiated and after obtaining informed consent from the participants , the data collection was implemented . the current study was approved by ethics committee of shahid beheshti university of medical sciences on june 2012 . two hundred women at risk of hiv , aged 19 - 49 years participated in the current study . participants none of them had a monthly income more than $ 500 , and 38.5% of these women had six or more sexual partners in their lifetime . only 29% of the participants used condoms in their last sexual intercourse and 37.5% of them expressed that they abstained from using condom due to their partner s disapproval . only 13% of the participants had always used male condoms in their sexual relationships for the last three months . nearly 50% of the participants had transactional sexual relationships . assessing the questionnaire for cvr showed that 31 items must be deleted , since they had not met the necessary scores . reduction phase , principal components analysis ( pca ) was used as the extraction method oblique ( direct - oblimin ) rotation ( 18 ) . decision making on the number of factors to retain for rotation was based on both the kaiser criteria ( eigenvalues more than one ) and the scree plot test ( 18 ) . constructs were maintained with an eigen value of more than one . since the kaiser - meyer - olkin ( kmo ) value greater than 0.85 was obtained , the quality of factor analysis in kmo was considered adequate . in this sequence , to predict the model , 48 items in the form of seven factors with the variance of 51.9% were detected . the number of items for each construct had the following sequence : skills to negotiate condoms and control excitement ( 16 items ) , situational factors ( four items ) , social support ( six items ) , ability to use condom despite the influence of drugs ( four items ) , male dominance in sexual decision making ( seven items ) , attitudes towards condom ( four items ) , and sexual needs of male in priority ( three items ) . four items had been loaded on more than one factor ; therefore , due to their complexity ( 20 ) were deleted . table 3 shows cronbach s alpha coefficient for the constructs . since rating the knowledge on hiv construct had two values , the kuder - richardson 20 was used to assess the internal consistency and the extracted value was 0.79 . correlation and cronbach 's alpha coefficient on the questionnaire constructs are shown in table 3 . assessing the questionnaire for cvr showed that 31 items must be deleted , since they had not met the necessary scores . in data reduction phase , principal components analysis ( pca ) was used as the extraction method oblique ( direct - oblimin ) rotation ( 18 ) . decision making on the number of factors to retain for rotation was based on both the kaiser criteria ( eigenvalues more than one ) and the scree plot test ( 18 ) . constructs were maintained with an eigen value of more than one . since the kaiser - meyer - olkin ( kmo ) value greater than 0.85 was obtained , the quality of factor analysis in kmo was considered adequate . in this sequence , to predict the model , 48 items in the form of seven factors with the variance of 51.9% were detected . the number of items for each construct had the following sequence : skills to negotiate condoms and control excitement ( 16 items ) , situational factors ( four items ) , social support ( six items ) , ability to use condom despite the influence of drugs ( four items ) , male dominance in sexual decision making ( seven items ) , attitudes towards condom ( four items ) , and sexual needs of male in priority ( three items ) . four items had been loaded on more than one factor ; therefore , due to their complexity ( 20 ) were deleted . table 3 shows cronbach s alpha coefficient for the constructs . since rating the knowledge on hiv construct had two values , the kuder - richardson 20 was used to assess the internal consistency and the extracted value was 0.79 . correlation and cronbach 's alpha coefficient on the questionnaire constructs are shown in table 3 . although various scales are designed worldwide to assess sexual protective behaviors in the communities , to date , there has been no scale to examine these constructs while conforming to the social and cultural conditions of the iranian population . the appropriateness of the developed questionnaire relies on the fact that it enables the risk to be monitored within a population at risk of hiv by taking into account many important factors influencing hiv prevention . since the goal of most hiv programs and preventive interventions is the use of condoms as a protective sexual behavior ( 4 ) , the present questionnaire would be able to identify the women at risk and also monitor and evaluate hiv preventive programs . the existing models in the context of hiv prevention is mainly focused on cognitive and individual structures , and often the role of environmental and structural factors are overlooked ( 21 ) , while factors such as stereotypical schema and partner acceptance of condom use had important roles ( 21 , 22 ) . therefore available instruments are designed using an individual framework . the instrument designed in the current study was within the ecological framework and its construct was formulated based on the concepts explored in the qualitative study . findings of the current study with mixed methodology showed important constructs related to protective behaviors . it was determined that , although variables such as attitudes toward condoms , and perceived risk regarding hiv , are of great importance in order to adopt this protective sexual behavior , it is not sufficient for women at risk of hiv ( 23 - 26 ) . other concepts such as social support in the area of hiv prevention , and also stereotypical gender roles which are considered as environmental and structural factors are considered to play a significant role for women at risk of hiv . validity of the questionnaire was evaluated and confirmed by qualitative face validity , cvr , cvi , and construct validity . in this evaluation , more than 33 items which did not meet the needed requirements were excluded ; all these excluded items were taken from the review of articles . this point can indicate the importance and the urgency of developing indigenous tools that conform to the culture of the community in order to evaluate sensitive topics such as issues on sexual behavior . using exploratory factor analysis , construct validity showed that the protective sexual behavior of women at risk of hiv in the present study had seven factors . all the existing constructs and variables in the questionnaire regarding the use of condoms as a sexual protective behavior showed a meaningful statistical relationship which was another reason to confirm the construct validity . although the alpha coefficient of the constructs , attitudes towards condoms , ability to use condoms despite the influence of drugs and the priority of male sexual satisfaction got a weak rating in the evaluation , the limited number of items in this construct ( three and four items ) as the total item correlation was from 0.3 to 0.4 in the constructs ; therefore , the reliability of these constructs are within the acceptable range . the predictive factors of protective sexual behaviors of women at risk for hiv questionnaire designed in the current study was composed of seven constructs and 44 items and required 15 minutes to complete . despite being the only available questionnaire used for the socially vulnerable women , its complexity , and lengthiness , and being composed of a large volume of questions resulted in decreasing the accuracy of data and loss of concentration the participants , but this questionnaire had its advantages . in addition , the presence of diverse groups of women at risk in the present study was considered as another strong point to this questionnaire . since it was difficult to access the risky women in the community , the current study relies on convenience sampling method . many studies revealed that women tended to under - report their sexual behaviors especially in conservative countries , but the current research did its best to lessen the stress of the participants to build a safe environment to obtain more accurate data on sexual behaviors . however , considering the importance of the present questionnaire and the fact that the current study was a preliminary study ; therefore , more studies are needed in order to improve it . evidently , further studies with larger sample sizes and also randomized sampling could help with a better presentation of the relationships between the existing constructs in the questionnaire and a step towards improving it . in general , psychometric properties of each construct including internal consistency and construct validity for the questionnaire were satisfactory and results of the current study revealed that the current questionnaire ( pspb ) was a valid and a reliable tool to assess at risk women in the iranian community .

background : one much needed tool to assist with the monitoring and evaluation of human immunodeficiency virus ( hiv ) prevention programs is to provide a valid instrument to measure protective sexual behavior and related factors.objectives:the current study aimed to design a valid and reliable instrument to predict the protective sexual behaviors of women at risk of hiv in iran.patients and methods : the current study was a sequential mixed cross - sectional and methodological research . initially , via a qualitative research , constructs and factors associated with sexual protective behavior of women at risk were identified through 25 in - depth interviews . the questionnaire on predictors of protective sexual behaviors in women at risk of hiv ( pspb ) was designed based on a qualitative study , and then its qualitative validity , content , and construct validity were evaluated . exploratory factor analysis was performed and 200 women at risk participated.results:seven concepts emerged after exploratory factor analysis of the 48 items . the content validity ratio ( cvr ) of the questionnaire constructs were 0.55 to 0.76 , and content validity index ( cvi ) structure was 0.86 to 0.95 . cronbach 's alpha coefficient for the entire questionnaire was 0.78 , and correlation coefficient of the test - retest reliability for the constructs was from 0.73 to 0.89.conclusions:the current study proved the capability of the predictors of sexual protective behavior in women at risk for hiv questionnaire as a valid and reliable instrument for the iranian community .

1. Background 2. Objectives 3. Patients and Methods 3.1. Qualitative Study 3.2. Initial Pool and Psychometric Characteristics 4. Results 4.1. CVR and CVI 4.2. Construct Validity 5. Discussion

despite the progress achieved in the past three decades , human immunodeficiency virus/ human immunodeficiency virus infection ( hiv / aids ) is still considered a serious public health issue ( 1 ) . this infection , in some subgroups in iran which are at higher risk for hiv transmission , exists in form of an epidemic and is extensively spreading in most parts of the world . these subgroups include female sex workers , drug users , and spouses who are hiv positive and act as a vehicle to infect the other people in the community ( 2 ) . it is estimated that around 92000 people in iran are infected with hiv / aids ( 1 ) . in iran , the primary means of hiv / aids transmission among women is through heterosexual sex and there are serious concerns regarding the spread of this disease in the country ( 3 ) . in addition to biological factors , social factors also play a role in women s hiv risks ( 4 ) . in the first quarter of 2011 , sexual transmission has accounted for 21.1% of the total individuals infected with hiv / aids ( 5 ) . access to reliable and accurate information regarding the behaviors of subgroups that are at risk of hiv / aids is important to keep track of the epidemic and program planning , and to form the development of future prevention programs ( 6 ) . therefore , as the first step to assess the predictors of protective sexual behaviors of women at risk for hiv / aids , it is necessary to identify the constructs , important , and influencing concepts for these behaviors . in the next stage of the research , appropriate tools to assess protective sexual behaviors can be designed . currently , most of the questionnaires and their constructs to predict and assess protective sexual behaviors are patterned after the ones in the west and are used in the community and in different groups of the population ; whereas , these models are not very successful in predicting preventive interventions for hiv ( 7 , 8) . the available scales developed to explore hiv related sexual behavior , considered individual concepts such as knowledge ( 9 ) , attitude to the condom ( 10 ) , self - efficacy to condom use ( 11 ) , and the perceived risks ( 12 ) that are not the entire variables influencing hiv related behaviors . considering the different social and economic contexts of these behaviors in different countries , a valid and appropriate instrument inherent to the social and cultural needs of the country must be provided , especially on the sensitive issues such as sexual behavior . as there are no gender sensitive scales in iranian population , the current study was the first and aimed to design a valid and reliable questionnaire within the ecological framework that conforms to the cultural and social conditions of the iranian population in order to predict protective sexual behaviors of women at risk of hiv . the present cross - sectional and methodological study aimed to develop a valid and reliable instrument and was conducted in tehran , iran , in 2012 ( 13 ) . the first step in the process of developing the questionnaire is a deep understanding of the concepts ( 14 ) . structures and relevant factors associated with the protective sexual behaviors of women at risk were identified through in - depth interviews based on a semi - structured interview guide with 25 women at risk aged 21 - 49 years . participants were selected from those who came to the voluntary counseling and testing ( vct ) centers of the shahid behashti university of medical sciences and non - governmental organization ( ngo ) drop - in - centers ( dics ) in iran . constructs and variables identified from the qualitative study were as follows : skills to negotiate condom and control sexual excitement , situational factors , social support , ability to use condom under the influence of drug use , male dominance in sexual decision making , attitudes towards condoms , and prioritization of male sexual needs . in the next step , items for the predictors of protective sexual behaviors of women at risk of hiv / aids questionnaire were determined based on the qualitative study and a review of the literature ( 9 - 12 ) . construct on knowledge was designed as dichotomy . in the next phase , in order to verify the validity of the questionnaire , qualitative face validity , and content and construct validity were assessed . in terms of assessing fluency in writing and appropriate phrasing , the questionnaire was analyzed through a face - to - face method with the participation of 12 individuals , and then modifications were implemented in order to achieve qualitative validity . in order to evaluate the content validity of the questionnaire quantitatively ; the content validity ratio ( cvr ) and content validity index ( cvi ) were used . the ratio of content validity was calculated by summing up the comments of the 14 experts from different disciplines including ; health education , reproductive health , midwifery , nursing , epidemiology and researchers and experts from center for aids control in the ministry of health , and also experts from the national aids research centre , then the necessary modifications were implemented . for this purpose , these experts were asked to assess each item based on lawshe scale ( 15 ) . the following formula was devised for the content validity ratio ( cvr ) : in which the ne is the number of panelists indicating essential , and n is the total number of panelists . based on the lawshe s table , when total number of the panelists is 14 , as in the present study , a minimum value of cvr 51 is required to satisfy the five percent level ( 15 ) . for cvi , the 12 experts used a 4-point likert scale to assess the questionnaire in order to identify the following criteria : relevance , simplicity and clarity of statements for each item ( 16 ) . participants of the present study included women who were at risk of hiv , including drug injecting married wives , wives with hiv positive husbands , wives with husbands who were taking narcotics and psychotropic substances , and wives of husbands who had multiple sexual partners . these women were referred to the voluntary counseling and testing ( vct ) centers of the shahid beheshti university of medical sciences ( three centers ) , and four ngo drop - in - centers ( dics ) in tehran , aged 18 years and above , were farsi speakers residing in tehran and had sexual intercourse experience in the past three months . in the current study , based on the qualitative study that revealed seven concepts concerning protective sexual behaviors of women at risk of hiv , and the rule that 30 samples should be selected per construct ( 17 ) , the sample size was determined 210 . in order to analyze the exploratory factors , the principal component analysis was used and factors were extracted by the oblique factors approach . therefore , 200 women at risk of hiv aged 19 to 49 years participated in the current study . the cut - off point of 0.35 was considered as a minimum load factor required to maintain each item ( 18 ) . in the next phase , the reliability of the questionnaire was assessed by internal consistency ( cronbach s alpha with kuder - richardson 20 ) and stability ( test - retest ) . cronbach s alpha coefficient was calculated for each factor , and the questionnaire as a whole , for 30 samples . in order to determine the reliability of the questionnaire , the test - retest was applied to the 20 participants and was repeated after two weeks . the current study was approved by ethics committee of shahid beheshti university of medical sciences on june 2012 . the present cross - sectional and methodological study aimed to develop a valid and reliable instrument and was conducted in tehran , iran , in 2012 ( 13 ) . the first step in the process of developing the questionnaire is a deep understanding of the concepts ( 14 ) . structures and relevant factors associated with the protective sexual behaviors of women at risk were identified through in - depth interviews based on a semi - structured interview guide with 25 women at risk aged 21 - 49 years . participants were selected from those who came to the voluntary counseling and testing ( vct ) centers of the shahid behashti university of medical sciences and non - governmental organization ( ngo ) drop - in - centers ( dics ) in iran . constructs and variables identified from the qualitative study were as follows : skills to negotiate condom and control sexual excitement , situational factors , social support , ability to use condom under the influence of drug use , male dominance in sexual decision making , attitudes towards condoms , and prioritization of male sexual needs . in the next step , items for the predictors of protective sexual behaviors of women at risk of hiv / aids questionnaire were determined based on the qualitative study and a review of the literature ( 9 - 12 ) . the initial pool of the items was composed of 81 items . construct on knowledge was designed as dichotomy . in the next phase , in order to verify the validity of the questionnaire , qualitative face validity , and content and construct validity were assessed . in terms of assessing fluency in writing and appropriate phrasing , the questionnaire was analyzed through a face - to - face method with the participation of 12 individuals , and then modifications were implemented in order to achieve qualitative validity . in order to evaluate the content validity of the questionnaire quantitatively ; the content validity ratio ( cvr ) and content validity index ( cvi ) were used . the ratio of content validity was calculated by summing up the comments of the 14 experts from different disciplines including ; health education , reproductive health , midwifery , nursing , epidemiology and researchers and experts from center for aids control in the ministry of health , and also experts from the national aids research centre , then the necessary modifications were implemented . for this purpose , these experts were asked to assess each item based on lawshe scale ( 15 ) . the following formula was devised for the content validity ratio ( cvr ) : in which the ne is the number of panelists indicating essential , and n is the total number of panelists . based on the lawshe s table , when total number of the panelists is 14 , as in the present study , a minimum value of cvr 51 is required to satisfy the five percent level ( 15 ) . for cvi , the 12 experts used a 4-point likert scale to assess the questionnaire in order to identify the following criteria : relevance , simplicity and clarity of statements for each item ( 16 ) . participants of the present study included women who were at risk of hiv , including drug injecting married wives , wives with hiv positive husbands , wives with husbands who were taking narcotics and psychotropic substances , and wives of husbands who had multiple sexual partners . these women were referred to the voluntary counseling and testing ( vct ) centers of the shahid beheshti university of medical sciences ( three centers ) , and four ngo drop - in - centers ( dics ) in tehran , aged 18 years and above , were farsi speakers residing in tehran and had sexual intercourse experience in the past three months . women with severe psychological disorders such as severe mental disorders or psychosis based on medical examination ( two cases ) , transgender ( o case ) , hiv positive ( 1 ) and women who were not interested to continue to participate in the study and the corresponding data were incomplete ( six cases ) were excluded . in the current study , based on the qualitative study that revealed seven concepts concerning protective sexual behaviors of women at risk of hiv , and the rule that 30 samples should be selected per construct ( 17 ) , the sample size was determined 210 . in order to analyze the exploratory factors , the principal component analysis was used and factors were extracted by the oblique factors approach . therefore , 200 women at risk of hiv aged 19 to 49 years participated in the current study . the cut - off point of 0.35 was considered as a minimum load factor required to maintain each item ( 18 ) . in the next phase , the reliability of the questionnaire was assessed by internal consistency ( cronbach s alpha with kuder - richardson 20 ) and stability ( test - retest ) . cronbach s alpha coefficient was calculated for each factor , and the questionnaire as a whole , for 30 samples . in order to determine the reliability of the questionnaire , the test - retest was applied to the 20 participants and was repeated after two weeks . the current study was approved by ethics committee of shahid beheshti university of medical sciences on june 2012 . two hundred women at risk of hiv , aged 19 - 49 years participated in the current study . participants none of them had a monthly income more than $ 500 , and 38.5% of these women had six or more sexual partners in their lifetime . only 29% of the participants used condoms in their last sexual intercourse and 37.5% of them expressed that they abstained from using condom due to their partner s disapproval . only 13% of the participants had always used male condoms in their sexual relationships for the last three months . nearly 50% of the participants had transactional sexual relationships . assessing the questionnaire for cvr showed that 31 items must be deleted , since they had not met the necessary scores . reduction phase , principal components analysis ( pca ) was used as the extraction method oblique ( direct - oblimin ) rotation ( 18 ) . decision making on the number of factors to retain for rotation was based on both the kaiser criteria ( eigenvalues more than one ) and the scree plot test ( 18 ) . constructs were maintained with an eigen value of more than one . since the kaiser - meyer - olkin ( kmo ) value greater than 0.85 was obtained , the quality of factor analysis in kmo was considered adequate . in this sequence , to predict the model , 48 items in the form of seven factors with the variance of 51.9% were detected . the number of items for each construct had the following sequence : skills to negotiate condoms and control excitement ( 16 items ) , situational factors ( four items ) , social support ( six items ) , ability to use condom despite the influence of drugs ( four items ) , male dominance in sexual decision making ( seven items ) , attitudes towards condom ( four items ) , and sexual needs of male in priority ( three items ) . table 3 shows cronbach s alpha coefficient for the constructs . since rating the knowledge on hiv construct had two values , the kuder - richardson 20 was used to assess the internal consistency and the extracted value was 0.79 . correlation and cronbach 's alpha coefficient on the questionnaire constructs are shown in table 3 . assessing the questionnaire for cvr showed that 31 items must be deleted , since they had not met the necessary scores . in data reduction phase , principal components analysis ( pca ) was used as the extraction method oblique ( direct - oblimin ) rotation ( 18 ) . decision making on the number of factors to retain for rotation was based on both the kaiser criteria ( eigenvalues more than one ) and the scree plot test ( 18 ) . constructs were maintained with an eigen value of more than one . since the kaiser - meyer - olkin ( kmo ) value greater than 0.85 was obtained , the quality of factor analysis in kmo was considered adequate . in this sequence , to predict the model , 48 items in the form of seven factors with the variance of 51.9% were detected . the number of items for each construct had the following sequence : skills to negotiate condoms and control excitement ( 16 items ) , situational factors ( four items ) , social support ( six items ) , ability to use condom despite the influence of drugs ( four items ) , male dominance in sexual decision making ( seven items ) , attitudes towards condom ( four items ) , and sexual needs of male in priority ( three items ) . table 3 shows cronbach s alpha coefficient for the constructs . since rating the knowledge on hiv construct had two values , the kuder - richardson 20 was used to assess the internal consistency and the extracted value was 0.79 . correlation and cronbach 's alpha coefficient on the questionnaire constructs are shown in table 3 . although various scales are designed worldwide to assess sexual protective behaviors in the communities , to date , there has been no scale to examine these constructs while conforming to the social and cultural conditions of the iranian population . the appropriateness of the developed questionnaire relies on the fact that it enables the risk to be monitored within a population at risk of hiv by taking into account many important factors influencing hiv prevention . since the goal of most hiv programs and preventive interventions is the use of condoms as a protective sexual behavior ( 4 ) , the present questionnaire would be able to identify the women at risk and also monitor and evaluate hiv preventive programs . the existing models in the context of hiv prevention is mainly focused on cognitive and individual structures , and often the role of environmental and structural factors are overlooked ( 21 ) , while factors such as stereotypical schema and partner acceptance of condom use had important roles ( 21 , 22 ) . the instrument designed in the current study was within the ecological framework and its construct was formulated based on the concepts explored in the qualitative study . findings of the current study with mixed methodology showed important constructs related to protective behaviors . it was determined that , although variables such as attitudes toward condoms , and perceived risk regarding hiv , are of great importance in order to adopt this protective sexual behavior , it is not sufficient for women at risk of hiv ( 23 - 26 ) . other concepts such as social support in the area of hiv prevention , and also stereotypical gender roles which are considered as environmental and structural factors are considered to play a significant role for women at risk of hiv . validity of the questionnaire was evaluated and confirmed by qualitative face validity , cvr , cvi , and construct validity . this point can indicate the importance and the urgency of developing indigenous tools that conform to the culture of the community in order to evaluate sensitive topics such as issues on sexual behavior . using exploratory factor analysis , construct validity showed that the protective sexual behavior of women at risk of hiv in the present study had seven factors . all the existing constructs and variables in the questionnaire regarding the use of condoms as a sexual protective behavior showed a meaningful statistical relationship which was another reason to confirm the construct validity . although the alpha coefficient of the constructs , attitudes towards condoms , ability to use condoms despite the influence of drugs and the priority of male sexual satisfaction got a weak rating in the evaluation , the limited number of items in this construct ( three and four items ) as the total item correlation was from 0.3 to 0.4 in the constructs ; therefore , the reliability of these constructs are within the acceptable range . the predictive factors of protective sexual behaviors of women at risk for hiv questionnaire designed in the current study was composed of seven constructs and 44 items and required 15 minutes to complete . despite being the only available questionnaire used for the socially vulnerable women , its complexity , and lengthiness , and being composed of a large volume of questions resulted in decreasing the accuracy of data and loss of concentration the participants , but this questionnaire had its advantages . in addition , the presence of diverse groups of women at risk in the present study was considered as another strong point to this questionnaire . since it was difficult to access the risky women in the community , the current study relies on convenience sampling method . many studies revealed that women tended to under - report their sexual behaviors especially in conservative countries , but the current research did its best to lessen the stress of the participants to build a safe environment to obtain more accurate data on sexual behaviors . however , considering the importance of the present questionnaire and the fact that the current study was a preliminary study ; therefore , more studies are needed in order to improve it . evidently , further studies with larger sample sizes and also randomized sampling could help with a better presentation of the relationships between the existing constructs in the questionnaire and a step towards improving it . in general , psychometric properties of each construct including internal consistency and construct validity for the questionnaire were satisfactory and results of the current study revealed that the current questionnaire ( pspb ) was a valid and a reliable tool to assess at risk women in the iranian community .

it has been firmly shown that cardiac resynchronization therapy ( crt ) is an established treatment option for a subgroup of appropriately selected patients with diverse heart failure ( hf ) severity to improve left ventricular ( lv ) reverse remodeling , reduce clinical symptoms and decrease cardiovascular mortality and morbidity . despite the indubitable advantages of crt , the response to crt appears to vary substantially and up to approximately 30% patients fail to benefit from crt , leading to a desire to identify of so - called non - responders prior to crt implantation . high sensitivity c - reactive protein ( hscrp ) is synthesized and secreted by hepatocytes in response to proinflammatory cytokines , namely il-6 , and it seems to be the most reliable biomarkers to assess inflammatory processes in clinical practice . plasma hscrp levels are commonly elevated in patients with depressed lv function and have been regarded as an independent and powerful predictor of adverse cardiac events in advanced hf patients. however , data regarding the association of hscrp levels with lv reverse remodeling as well as long - term outcome in the subset of patients with hf receiving crt are limited , and the value of hscrp levels in predicting mortality or morbidity in crt recipients currently remains unclear . therefore , aims of the present study were to : ( 1 ) investigate the relation between baseline hscrp levels and long - term outcome after crt ; ( 2 ) evaluate the impact of crt on the concentrations of hscrp during 6-month follow - up ; and ( 3 ) explore the prognostic value of follow - up hscrp assessment following crt . a total of 232 consecutive chronic hf patients who underwent successful implantation of a crt system between january 1999 and december 2013 at fuwai hospital were included . all subjects received crt therapy according to the accepted criteria of new york heart association ( nyha ) class iii or iv despite optimal medical therapy , lv ejection fraction ( lvef ) 35% , qrs width 120 ms and left ventricular end - diastolic diameter ( lvedd ) 55 mm . , data including demographics , echocardiographic parameters , laboratory values , and medications at the initial evaluation were retrospectively obtained from the electronic medical record . long - term follow - up evaluations after device implantation were performed through the chart review , device interrogation or a telephone interview . all of the patients signed informed consent forms , and the study complied with the declaration of helsinki and was approved by the research ethics board of fuwai hospital . venous blood samples were obtained from the entire patient cohort before crt implantation for measurement of hscrp . we used the recommended conventional cut - off value for hscrp of 3.0 mg / l provided by consensus conference of the centers of disease control ( cdc ) and the american heart association ( aha ) on the use of hscrp in clinical practice , and patients at baseline were dichotomized into high ( > 3.0 mg / l ) and low hscrp groups ( 3.0 mg / l ) according to enrollment hscrp levels . during 6-month follow - up , there was a subset of 198 ( 85.3% ) patients with routinely acquired blood samples for hscrp assessment . follow - up changes in hscrp levels from baseline to 6-month were categorized as high / high ( baseline/6-month ) , high / low , low / high , and low / low . patients were classified as responders to crt in terms of improvement in nyha class by 1 combined with an absolute increase 5% in lvef during 6-month follow - up . patients who died , underwent heart transplantation or hospitalized for hf within 6-month were regarded as non - responders . primary endpoint events were defined as all - cause mortality ( due to hf , sudden cardiac death , or non - cardiac cause ) , cardiac transplantation and hospitalizations for decompensated hf . echocardiograms were obtained at baseline for all patients and at 6-month were available for 215 ( 92.7% ) patients , respectively . echocardiographic parameters including the left atrium diameters ( lad ) and lvedd were measured using a commercially available system ( ie33 ; philips medical systems ) equipped with a 3.5-mhz transducer according with the recommendations of the american society of echocardiography protocols . the severity of mitral regurgitation ( mr ) was assessed as the average area of the regurgitant jet area to left atrium using the color - flow doppler images at the parasternal long - axis . a coronary sinus venogram was obtained using a balloon catheter , and the lv pacing lead was inserted through the coronary sinus with the help of an 8-f or 9-f guiding catheter and positioned as far as possible in the venous system , preferably in the lateral or posterolateral vein . the atrial and right ventricular leads were placed routinely in the right atrial appendage and the right ventricular apex . the decision to use a crt device with defibrillator function ( crt - d ) was based on primary or secondary prevention for episodes of sustained ventricular tachycardia or inducible ventricular arrhythmia . continuous data are presented as mean sd , and dichotomous data are expressed as numbers and percentages . comparison of data between patient groups was performed using the independent - samples t test for continuous data . the fisher 's exact tests or tests survival of patients was evaluated with the kaplan - meier method and log rank test was utilized to compare the survival curves . variables significant in univariate analysis were entered in a multivariate cox proportional hazards model to determine the independent predictors of event - free survival using a backward stepwise selection . at each step , the least significant variable was discarded from the model until all variables in the model reached a p value below 0.25 . the analyses were conducted using the spss 16.0 software ( spss , chicago , il , usa ) . a total of 232 consecutive chronic hf patients who underwent successful implantation of a crt system between january 1999 and december 2013 at fuwai hospital were included . all subjects received crt therapy according to the accepted criteria of new york heart association ( nyha ) class iii or iv despite optimal medical therapy , lv ejection fraction ( lvef ) 35% , qrs width 120 ms and left ventricular end - diastolic diameter ( lvedd ) 55 mm . , data including demographics , echocardiographic parameters , laboratory values , and medications at the initial evaluation were retrospectively obtained from the electronic medical record . long - term follow - up evaluations after device implantation were performed through the chart review , device interrogation or a telephone interview . all of the patients signed informed consent forms , and the study complied with the declaration of helsinki and was approved by the research ethics board of fuwai hospital . venous blood samples were obtained from the entire patient cohort before crt implantation for measurement of hscrp . we used the recommended conventional cut - off value for hscrp of 3.0 mg / l provided by consensus conference of the centers of disease control ( cdc ) and the american heart association ( aha ) on the use of hscrp in clinical practice , and patients at baseline were dichotomized into high ( > 3.0 mg / l ) and low hscrp groups ( 3.0 mg / l ) according to enrollment hscrp levels . during 6-month follow - up , there was a subset of 198 ( 85.3% ) patients with routinely acquired blood samples for hscrp assessment . follow - up changes in hscrp levels from baseline to 6-month were categorized as high / high ( baseline/6-month ) , high / low , low / high , and low / low . patients were classified as responders to crt in terms of improvement in nyha class by 1 combined with an absolute increase 5% in lvef during 6-month follow - up . patients who died , underwent heart transplantation or hospitalized for hf within 6-month were regarded as non - responders . primary endpoint events were defined as all - cause mortality ( due to hf , sudden cardiac death , or non - cardiac cause ) , cardiac transplantation and hospitalizations for decompensated hf . echocardiograms were obtained at baseline for all patients and at 6-month were available for 215 ( 92.7% ) patients , respectively . echocardiographic parameters including the left atrium diameters ( lad ) and lvedd were measured using a commercially available system ( ie33 ; philips medical systems ) equipped with a 3.5-mhz transducer according with the recommendations of the american society of echocardiography protocols . the severity of mitral regurgitation ( mr ) was assessed as the average area of the regurgitant jet area to left atrium using the color - flow doppler images at the parasternal long - axis . a coronary sinus venogram was obtained using a balloon catheter , and the lv pacing lead was inserted through the coronary sinus with the help of an 8-f or 9-f guiding catheter and positioned as far as possible in the venous system , preferably in the lateral or posterolateral vein . the atrial and right ventricular leads were placed routinely in the right atrial appendage and the right ventricular apex . the decision to use a crt device with defibrillator function ( crt - d ) was based on primary or secondary prevention for episodes of sustained ventricular tachycardia or inducible ventricular arrhythmia . continuous data are presented as mean sd , and dichotomous data are expressed as numbers and percentages . comparison of data between patient groups was performed using the independent - samples t test for continuous data . survival of patients was evaluated with the kaplan - meier method and log rank test was utilized to compare the survival curves . variables significant in univariate analysis were entered in a multivariate cox proportional hazards model to determine the independent predictors of event - free survival using a backward stepwise selection . at each step , the least significant variable was discarded from the model until all variables in the model reached a p value below 0.25 . the analyses were conducted using the spss 16.0 software ( spss , chicago , il , usa ) . table 1 shows the baseline demographics and clinical characteristics of 232 patients according to the initial hscrp levels . patients were subdivided into high ( > 3 mg / l ) and low ( 3 mg / l ) hscrp groups , with 105 patients in the high hscrp group ( 7.8 4.3 mg / l ) and 127 subjects in the low hscrp group ( 1.3 1.6 mg / l ) . the mean age of study population was 59.7 10.4 years , and 154 ( 66.4% ) patients were male . crt recipients showed depressed lv systolic function ( mean lvef , 27.7% 6.7% ) with qrs prolongation ( 158.1 20.0 ms ) . compared to those with low hscrp , patients with high hscrp tended to be much older and more symptomatic , and presented less left bundle branch block ( lbbb ) and larger lad during their initial evaluation . in addition , the concentrations of biochemical markers including serum creatinine , blood urea nitrogen ( bun ) and n - terminal pro - brain natriuretic peptide ( nt - probnp ) were significantly higher in patients with elevated hscrp than in those with low hscrp . no differences regarding the comorbidities and treatment strategies between elevated hscrp group and low hscrp group existed at baseline . the data are presented as the n ( % ) or the means sd . acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; bun : blood urea nitrogen ; crt - d : cardiac resynchronization therapy - defibrillator ; hscrp : high - sensitivity c - reactive protein ; lad : left atrial diameter ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; mr : mitral regurgitation ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class ; pah : pulmonary arterial hypertension ; rvedd : right ventricular end - diastolic diameter . in our study , the mean follow - up duration was 31.3 31.5 months . thirty - two patients ( 13.8% ) experienced all - cause mortality including 24 patients with hf - related death , five patients with sudden death and three patients with non - cardiac death . four patients ( 1.7% ) underwent heart transplantation and 56 patients ( 24.1% ) were hospitalized for worsening hf . a total of 59 patients ( 25.4% ) experienced the combined endpoint of death or hf hospitalizations . over the long - term follow - up , patients with hscrp > 3 mg / l had a significantly lower probability of surviving without suffering the combined endpoint of death or hf hospitalizations as compared to those with hscrp 3 mg mg / l ) at baseline were shown to independently predict adverse clinical response ( hr : 2.39 , 95%ci : 1.284.47 , p = 0.006 , table 2 ) . when assessed as a continuous variable , each 1 mg / l increase in hscrp was associated with a corresponding 8.3% increase ( p = 0.005 ) in the risk of the combined endpoint of death or hf hospitalizations . crt : cardiac resynchronization therapy ; hf : heart failure ; hscrp : high sensitivity c - reactive protein . during 6-month follow - up , a total of 170 of 232 ( 73.3% ) patients were classified as responders . baseline hscrp values as well as 6-month hscrp levels were significantly lower in patients who responded to crt than in those who did not ( p = 0.001 and p < 0.001 , respectively ) , with a mean 0.5 4.1 mg / l reduction from baseline for patients who were responders and 1.7 6.1 mg / l increase from baseline for those who were non - responders ( p = 0.018 ) . both baseline and 6-month hscrp values for responders and non - responders to crt are displayed in figure 2 . arb : angiotensin receptor blocker ; bun : blood urea nitrogen ; crt - d : cardiac resynchronization therapy - defibrillator ; hscrp : high - sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; mr : mitral regurgitation ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class . absolute changes in hscrp were calculated as the difference between 6 month and baseline hscrp levels , among the 198 patients with available paired baseline and 6-month hscrp assessment . the survival curve for all - cause mortality or hf hospitalizations among patients with for each of the baseline to 6-month hscrp change group is shown in figure 3 . during long - term follow - up , compared with those with low / high baseline and low 6-month hscrp values , patients with low / high baseline and high 6-month hscrp values displayed higher rate of the combined endpoint of death or hf hospitalizations ( log - rank p < 0.01 ) . after multivariate adjustment , compared with those with 6-month hscrp values which stayed low , patients with 6-month hscrp levels that remained high experienced the highest rate of death or hf hospitalizations ( hr : 7.6 , p < 0.001 ) , and patients whose levels increased from low baseline to high 6-month hscrp values suffered secondary risk ( hr : 2.9 , p = 0.079 ) , and patients whose levels declined from a high baseline to low 6-month hscrp values underwent similar risk ( hr : 1.4 , p = 0.576 , table 3 ) . when assessed as a continuous variable , each 1 mg / l increase in 6-month hscrp levels was related to a corresponding 9.6% increase ( p = 0.001 ) in the risk of death or hf hospitalizations . bl : baseline ; 6 : 6 months ; hf : heart failure ; hscrp : high sensitivity c - reactive protein ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 mg / l ) . the relationship between the pattern of hscrp change and lv reverse at 6-month among the 198 patients with paired echocardiograms and hscrp values is shown in figure 4 . there were no significant differences in baseline lvedd and lvef between the low and high baseline hscrp groups . similar to the association of clinical outcome with hscrp change , patients with low 6-month hscrp values displayed the largest concurrent mean reductions in lvedd and increases in lvef ( 6.4 7.7 mm reduction in lvedd and 11.0% 8.6 % increase in lvef among those with low baseline hscrp , and 7.1 8.9 mm reduction in lvedd and 11.1% 8.5 % increase in lvef among those with high baseline hscrp ) . in contrast , patients with high 6-month hscrp values showed significantly lower reductions in lvedd and increases in lvef ( 3.9 5.9 mm reduction in lvedd and 6.2% 8.3% increase in lvef among those with low baseline hscrp and 1.5 9.8 mm reduction in lvedd and 5.8% 9.4% increase in lvef among those with high baseline hscrp ; p < 0.01 for the overall difference ) . no significant differences in lvedd reduction or lvef increases were noted between the two groups with low ( low / low and high / low ) and high ( low / high and high / high ) 6-month hscrp values . the multivariable cox regression analysis controls for age , gender , ischaemic cardiomyopathy , lbbb , nyha class , lvedd , lvef , bun , serum creatinine , nt - probnp , -blocker , and acei / arb . acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; bl : baseline ; crt : cardiac resynchronization therapy ; hscrp : high sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class ; 6-mo : 6-month . absolute changes in lvef and lvedd were calculated as the difference between 6-month and baseline values . for comparison across the four groups , there were significant differences in lvedd and lvef changes ( p = 0.003 and p = 0.002 , respectively ) . bl : baseline ; hscrp : high sensitivity c - reactive protein ; lvedd : left ventricular end diastolic dimension ; lvef : left ventricular ejection fraction ; 6 : 6 months ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 table 1 shows the baseline demographics and clinical characteristics of 232 patients according to the initial hscrp levels . patients were subdivided into high ( > 3 mg / l ) and low ( 3 mg / l ) hscrp groups , with 105 patients in the high hscrp group ( 7.8 4.3 mg / l ) and 127 subjects in the low hscrp group ( 1.3 1.6 mg / l ) . the mean age of study population was 59.7 10.4 years , and 154 ( 66.4% ) patients were male . crt recipients showed depressed lv systolic function ( mean lvef , 27.7% 6.7% ) with qrs prolongation ( 158.1 20.0 ms ) . compared to those with low hscrp , patients with high hscrp tended to be much older and more symptomatic , and presented less left bundle branch block ( lbbb ) and larger lad during their initial evaluation . in addition , the concentrations of biochemical markers including serum creatinine , blood urea nitrogen ( bun ) and n - terminal pro - brain natriuretic peptide ( nt - probnp ) were significantly higher in patients with elevated hscrp than in those with low hscrp . no differences regarding the comorbidities and treatment strategies between elevated hscrp group and low hscrp group existed at baseline . the data are presented as the n ( % ) or the means sd . acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; bun : blood urea nitrogen ; crt - d : cardiac resynchronization therapy - defibrillator ; hscrp : high - sensitivity c - reactive protein ; lad : left atrial diameter ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; mr : mitral regurgitation ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class ; pah : pulmonary arterial hypertension ; rvedd : right ventricular end - diastolic diameter . in our study , the mean follow - up duration was 31.3 31.5 months . thirty - two patients ( 13.8% ) experienced all - cause mortality including 24 patients with hf - related death , five patients with sudden death and three patients with non - cardiac death . four patients ( 1.7% ) underwent heart transplantation and 56 patients ( 24.1% ) were hospitalized for worsening hf . a total of 59 patients ( 25.4% ) experienced the combined endpoint of death or hf hospitalizations . over the long - term follow - up , patients with hscrp > 3 mg / l had a significantly lower probability of surviving without suffering the combined endpoint of death or hf hospitalizations as compared to those with hscrp 3 mg / l ( log - rank p < 0.01 , figure 1 ) . after controlling for confounding factors , elevated hscrp levels ( > 3 mg / l ) at baseline were shown to independently predict adverse clinical response ( hr : 2.39 , 95%ci : 1.284.47 , p = 0.006 , table 2 ) . when assessed as a continuous variable , each 1 mg / l increase in hscrp was associated with a corresponding 8.3% increase ( p = 0.005 ) in the risk of the combined endpoint of death or hf hospitalizations . crt : cardiac resynchronization therapy ; hf : heart failure ; hscrp : high sensitivity c - reactive protein . during 6-month follow - up , a total of 170 of 232 ( 73.3% ) patients were classified as responders . baseline hscrp values as well as 6-month hscrp levels were significantly lower in patients who responded to crt than in those who did not ( p = 0.001 and p < 0.001 , respectively ) , with a mean 0.5 4.1 mg / l reduction from baseline for patients who were responders and 1.7 6.1 mg / l increase from baseline for those who were non - responders ( p = 0.018 ) . both baseline and 6-month hscrp values for responders and non - responders to crt are displayed in figure 2 . arb : angiotensin receptor blocker ; bun : blood urea nitrogen ; crt - d : cardiac resynchronization therapy - defibrillator ; hscrp : high - sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; mr : mitral regurgitation ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class . absolute changes in hscrp were calculated as the difference between 6 month and baseline hscrp levels , among the 198 patients with available paired baseline and 6-month hscrp assessment . the survival curve for all - cause mortality or hf hospitalizations among patients with for each of the baseline to 6-month hscrp change group is shown in figure 3 . during long - term follow - up , compared with those with low / high baseline and low 6-month hscrp values , patients with low / high baseline and high 6-month hscrp values displayed higher rate of the combined endpoint of death or hf hospitalizations ( log - rank p < 0.01 ) . after multivariate adjustment , compared with those with 6-month hscrp values which stayed low , patients with 6-month hscrp levels that remained high experienced the highest rate of death or hf hospitalizations ( hr : 7.6 , p < 0.001 ) , and patients whose levels increased from low baseline to high 6-month hscrp values suffered secondary risk ( hr : 2.9 , p = 0.079 ) , and patients whose levels declined from a high baseline to low 6-month hscrp values underwent similar risk ( hr : 1.4 , p = 0.576 , table 3 ) . when assessed as a continuous variable , each 1 mg / l increase in 6-month hscrp levels was related to a corresponding 9.6% increase ( p = 0.001 ) in the risk of death or hf hospitalizations . bl : baseline ; 6 : 6 months ; hf : heart failure ; hscrp : high sensitivity c - reactive protein ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 mg / l ) . the relationship between the pattern of hscrp change and lv reverse at 6-month among the 198 patients with paired echocardiograms and hscrp values is shown in figure 4 . there were no significant differences in baseline lvedd and lvef between the low and high baseline hscrp groups . similar to the association of clinical outcome with hscrp change , patients with low 6-month hscrp values displayed the largest concurrent mean reductions in lvedd and increases in lvef ( 6.4 7.7 mm reduction in lvedd and 11.0% 8.6 % increase in lvef among those with low baseline hscrp , and 7.1 8.9 mm reduction in lvedd and 11.1% 8.5 % increase in lvef among those with high baseline hscrp ) . in contrast , patients with high 6-month hscrp values showed significantly lower reductions in lvedd and increases in lvef ( 3.9 5.9 mm reduction in lvedd and 6.2% 8.3% increase in lvef among those with low baseline hscrp and 1.5 9.8 mm reduction in lvedd and 5.8% 9.4% increase in lvef among those with high baseline hscrp ; p < 0.01 for the overall difference ) . no significant differences in lvedd reduction or lvef increases were noted between the two groups with low ( low / low and high / low ) and high ( low / high and high / high ) 6-month hscrp values . the multivariable cox regression analysis controls for age , gender , ischaemic cardiomyopathy , lbbb , nyha class , lvedd , lvef , bun , serum creatinine , nt - probnp , -blocker , and acei / arb . acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; bl : baseline ; crt : cardiac resynchronization therapy ; hscrp : high sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class ; 6-mo : 6-month . absolute changes in lvef and lvedd were calculated as the difference between 6-month and baseline values . for comparison across the four groups , there were significant differences in lvedd and lvef changes ( p = 0.003 and p = 0.002 , respectively ) . bl : baseline ; hscrp : high sensitivity c - reactive protein ; lvedd : left ventricular end diastolic dimension ; lvef : left ventricular ejection fraction ; 6 : 6 months ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 mg / l ) . the current analysis explored several significant implications on the prognostic value of hscrp levels for severe hf patients receiving crt . first , elevated hscrp prior to crt device implantation was identified as an independent determinant of adverse survival and more hf hospitalizations . secondly , during 6-month follow - up , plasma levels of hscrp were reduced or remained low in patients who were responders to crt , yet a similar improvement was not observed among patients who were non - responders . finally , the pattern of hscrp changes after six months of crt was significantly associated with the lv reverse parameters and subsequent clinical outcome . to date , there have been relatively limited available studies regarding the relationship between baseline hscrp levels and long - term outcome after crt . kamioka , et al . found , in a small cohort of 65 patients , that hscrp levels before crt implantation were identified as the strongest predictive factor for cardiac death irrespective of other prognostic factors and that being in the high hscrp group ( > 3 this finding is similar to our results that hscrp levels pre - crt implant independently predict all - cause mortality or hospitalizations for hf following crt after adjusting other established risk factors . what the exact potential mechanisms are for the observed the heightened risk of mortality and morbidity in patients with high hscrp levels before crt implantation currently remain unresolved . several prior reports have demonstrated that elevated levels of hscrp , as a biomarker of inflammation , was a powerful independent predictor of prognosis in chronic hf, and a recent study has shown that increased hscrp can directly predict progressive regional myocardial functional deterioration independent of confounding cardiovascular events . the similar effect of hscrp levels on the long - term outcome may also occur in crt recipients , this hypothesis , however , can not be directly confirmed by the current or prior results . moreover , a few previous studies have evaluated the association of changes in hscrp levels after crt and the subsequent clinical and echocardiographic response . in 140 patients undergoing crt , michelucci , et al . reported that crt reduced hscrp status only in patients who showed lv reverse and reduction of hscrp levels were associated with the absence of adverse cardiac events . on the contrary , glick , et al . reported that , although hscrp decreased significantly within two weeks after crt implantation , levels of hscrp or their drop after two weeks of crt did not correlate with clinical outcome . this discrepancy may possibly be due to the small sample size ( 32 patients ) as well as a relatively short follow - up period . another small observational study of 27 patients with advanced hf showed similar results to ours , in that hscrp levels at 6-month follow up decreased in responders while such improvements were not observed in non - responders . however , the authors did not investigate whether changes in hscrp levels were associated with clinical outcome after crt . the findings of these small retrospective studies were mostly congruent with our results that patients who showed significant lv reverse remodeling experienced significant reductions or maintained low 6-month hscrp values , whereas patients who do not exhibit obvious cardiac reverse remodeling experienced a slight increase or remained high with regard to their 6-month hscrp levels after crt . additionally , when patients were further stratified into four subgroups according to hscrp change , we found that the pattern of hscrp change from baseline to 6-month was associated with a lack of favorable lv reverse remodeling parameters with respect to lv dimensions and lvef , and independently predicted subsequent all - cause mortality or hf hospitalizations . the pathophysiological mechanisms responsible for improvement of lv systolic function resulting in reduction or maintenance in hscrp levels restoration of synchronization induced by crt may lead to attenuated sympathetic and neurohormonal over - activation , reduced lv wall stress as well endothelial oxidative stress , which consequently exerts beneficial effects on the observed reduction in concentration of proinflammatory cytokines such as hscrp. in turn , the alleviation of inflammatory status may also result in improved clinical prognosis after crt . further studies are needed to elucidate the mechanisms underlying the changes in hscrp levels and lv reverse remodeling as well as prognosis in patients undergoing crt . firstly , this study is a nonrandomized retrospective analysis , and it involved a relatively small sample size , which may lead to statistical limitations and subsequently affect our findings . moreover , the present study was only designed to examine the hscrp levels but not to other inflammatory biomarkers such as il-6 , tnf- and tgf-1 which have also been identified as stronger predictors of clinical events in patients with advanced hf treated with crt . , , furthermore , although debate exists regarding the clinical utility of different hscrp cut - off values for predicting a worse outcome , we used a conventional cut point of 3.0 mg / l for the definition of high versus low hscrp in current study . additionally , the measurement of 6-month follow - up hscrp values and echocardiographic parameters was not available for the whole patients which may potentially influence our conclusions . finally , further studies are needed to elucidate the underlying pathophysiologic mechanisms of the interaction between hscrp levels and lv reverse remodeling as well as clinical prognosis in patients undergoing crt . we have shown that hscrp levels at baseline could predict the risk of death or hf hospitalizations in crt recipients . in addition , a reduction measured at 6-months or maintenance of low hscrp levels was observed in responders to crt , and patients who had persistently high or exhibited an increase in hscrp levels showed inferior lv reverse remodeling and subsequently had an increase in adverse clinical outcomes . taken together , our results suggest that the important prognostic information can be obtained via an early assessment of hscrp levels before and after crt implantation and that this information may be useful for risk stratification and could influence clinical management strategies in crt recipients . firstly , this study is a nonrandomized retrospective analysis , and it involved a relatively small sample size , which may lead to statistical limitations and subsequently affect our findings . moreover , the present study was only designed to examine the hscrp levels but not to other inflammatory biomarkers such as il-6 , tnf- and tgf-1 which have also been identified as stronger predictors of clinical events in patients with advanced hf treated with crt . , , furthermore , although debate exists regarding the clinical utility of different hscrp cut - off values for predicting a worse outcome , we used a conventional cut point of 3.0 mg / l for the definition of high versus low hscrp in current study . additionally , the measurement of 6-month follow - up hscrp values and echocardiographic parameters was not available for the whole patients which may potentially influence our conclusions . finally , further studies are needed to elucidate the underlying pathophysiologic mechanisms of the interaction between hscrp levels and lv reverse remodeling as well as clinical prognosis in patients undergoing crt . we have shown that hscrp levels at baseline could predict the risk of death or hf hospitalizations in crt recipients . in addition , a reduction measured at 6-months or maintenance of low hscrp levels was observed in responders to crt , and patients who had persistently high or exhibited an increase in hscrp levels showed inferior lv reverse remodeling and subsequently had an increase in adverse clinical outcomes . taken together , our results suggest that the important prognostic information can be obtained via an early assessment of hscrp levels before and after crt implantation and that this information may be useful for risk stratification and could influence clinical management strategies in crt recipients .

backgroundthe data on the prognostic values of high sensitivity c - reactive protein ( hscrp ) levels in patients with advanced symptomatic heart failure ( hf ) receiving cardiac resynchronization therapy ( crt ) are scarce . the aim of present study was to investigate the association of serum hscrp levels with left ventricle reverse remodeling after six months of crt as well as long - term outcome.methodsa total of 232 crt patients were included . the assessment of hscrp values , clinical status and echocardiographic data were performed at baseline and after six months of crt . long - term follow - up included all - cause mortality and hospitalizations for hf.resultsduring the mean follow - up periods of 31.3 31.5 months , elevated hscrp ( > 3 mg / l ) prior to crt was associated with a significant 2.39-fold increase ( p = 0.006 ) in the risk of death or hf hospitalizations . at 6-month follow - up , patients who responded to crt showed significant reductions or maintained low in hscrp levels ( 0.5 4.1 mg / l reduction ) compared with non - responders ( 1.7 6.1 mg / l increase , p = 0.018 ) . compared with patients in whom 6-month hscrp levels were reduced or remained low , patients in whom 6-month hscrp levels were increased or maintained high experienced a significantly higher risk of subsequent death or hf hospitalizations ( log - rank p < 0.001 ) . the echocardiographic improvement was also better among patients in whom 6-month hscrp levels were reduced or remained low compared to those in whom 6-month hscrp levels were raised or maintained high.conclusionsour findings demonstrated that measurement of baseline and follow - up hscrp levels may be useful as prognostic markers for timely potential risk stratification and subsequent appropriate treatment strategies in patients with advanced hf undergoing crt .

Introduction Methods Study population Definitions Echocardiographic evaluation CRT implantation procedure Statistics Results Baseline characteristics Baseline hsCRP and clinical outcome Effect of CRT on hsCRP levels HsCRP change and subsequent outcome HsCRP change and LV reverse remodeling Discussion Study limitations Conclusions

it has been firmly shown that cardiac resynchronization therapy ( crt ) is an established treatment option for a subgroup of appropriately selected patients with diverse heart failure ( hf ) severity to improve left ventricular ( lv ) reverse remodeling , reduce clinical symptoms and decrease cardiovascular mortality and morbidity . high sensitivity c - reactive protein ( hscrp ) is synthesized and secreted by hepatocytes in response to proinflammatory cytokines , namely il-6 , and it seems to be the most reliable biomarkers to assess inflammatory processes in clinical practice . however , data regarding the association of hscrp levels with lv reverse remodeling as well as long - term outcome in the subset of patients with hf receiving crt are limited , and the value of hscrp levels in predicting mortality or morbidity in crt recipients currently remains unclear . therefore , aims of the present study were to : ( 1 ) investigate the relation between baseline hscrp levels and long - term outcome after crt ; ( 2 ) evaluate the impact of crt on the concentrations of hscrp during 6-month follow - up ; and ( 3 ) explore the prognostic value of follow - up hscrp assessment following crt . long - term follow - up evaluations after device implantation were performed through the chart review , device interrogation or a telephone interview . we used the recommended conventional cut - off value for hscrp of 3.0 mg / l provided by consensus conference of the centers of disease control ( cdc ) and the american heart association ( aha ) on the use of hscrp in clinical practice , and patients at baseline were dichotomized into high ( > 3.0 mg / l ) and low hscrp groups ( 3.0 mg / l ) according to enrollment hscrp levels . primary endpoint events were defined as all - cause mortality ( due to hf , sudden cardiac death , or non - cardiac cause ) , cardiac transplantation and hospitalizations for decompensated hf . long - term follow - up evaluations after device implantation were performed through the chart review , device interrogation or a telephone interview . we used the recommended conventional cut - off value for hscrp of 3.0 mg / l provided by consensus conference of the centers of disease control ( cdc ) and the american heart association ( aha ) on the use of hscrp in clinical practice , and patients at baseline were dichotomized into high ( > 3.0 mg / l ) and low hscrp groups ( 3.0 mg / l ) according to enrollment hscrp levels . primary endpoint events were defined as all - cause mortality ( due to hf , sudden cardiac death , or non - cardiac cause ) , cardiac transplantation and hospitalizations for decompensated hf . patients were subdivided into high ( > 3 mg / l ) and low ( 3 mg / l ) hscrp groups , with 105 patients in the high hscrp group ( 7.8 4.3 mg / l ) and 127 subjects in the low hscrp group ( 1.3 1.6 mg / l ) . in our study , the mean follow - up duration was 31.3 31.5 months . thirty - two patients ( 13.8% ) experienced all - cause mortality including 24 patients with hf - related death , five patients with sudden death and three patients with non - cardiac death . over the long - term follow - up , patients with hscrp > 3 mg / l had a significantly lower probability of surviving without suffering the combined endpoint of death or hf hospitalizations as compared to those with hscrp 3 mg mg / l ) at baseline were shown to independently predict adverse clinical response ( hr : 2.39 , 95%ci : 1.284.47 , p = 0.006 , table 2 ) . when assessed as a continuous variable , each 1 mg / l increase in hscrp was associated with a corresponding 8.3% increase ( p = 0.005 ) in the risk of the combined endpoint of death or hf hospitalizations . crt : cardiac resynchronization therapy ; hf : heart failure ; hscrp : high sensitivity c - reactive protein . during 6-month follow - up , a total of 170 of 232 ( 73.3% ) patients were classified as responders . baseline hscrp values as well as 6-month hscrp levels were significantly lower in patients who responded to crt than in those who did not ( p = 0.001 and p < 0.001 , respectively ) , with a mean 0.5 4.1 mg / l reduction from baseline for patients who were responders and 1.7 6.1 mg / l increase from baseline for those who were non - responders ( p = 0.018 ) . both baseline and 6-month hscrp values for responders and non - responders to crt are displayed in figure 2 . the survival curve for all - cause mortality or hf hospitalizations among patients with for each of the baseline to 6-month hscrp change group is shown in figure 3 . during long - term follow - up , compared with those with low / high baseline and low 6-month hscrp values , patients with low / high baseline and high 6-month hscrp values displayed higher rate of the combined endpoint of death or hf hospitalizations ( log - rank p < 0.01 ) . after multivariate adjustment , compared with those with 6-month hscrp values which stayed low , patients with 6-month hscrp levels that remained high experienced the highest rate of death or hf hospitalizations ( hr : 7.6 , p < 0.001 ) , and patients whose levels increased from low baseline to high 6-month hscrp values suffered secondary risk ( hr : 2.9 , p = 0.079 ) , and patients whose levels declined from a high baseline to low 6-month hscrp values underwent similar risk ( hr : 1.4 , p = 0.576 , table 3 ) . when assessed as a continuous variable , each 1 mg / l increase in 6-month hscrp levels was related to a corresponding 9.6% increase ( p = 0.001 ) in the risk of death or hf hospitalizations . bl : baseline ; 6 : 6 months ; hf : heart failure ; hscrp : high sensitivity c - reactive protein ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 mg / l ) . similar to the association of clinical outcome with hscrp change , patients with low 6-month hscrp values displayed the largest concurrent mean reductions in lvedd and increases in lvef ( 6.4 7.7 mm reduction in lvedd and 11.0% 8.6 % increase in lvef among those with low baseline hscrp , and 7.1 8.9 mm reduction in lvedd and 11.1% 8.5 % increase in lvef among those with high baseline hscrp ) . in contrast , patients with high 6-month hscrp values showed significantly lower reductions in lvedd and increases in lvef ( 3.9 5.9 mm reduction in lvedd and 6.2% 8.3% increase in lvef among those with low baseline hscrp and 1.5 9.8 mm reduction in lvedd and 5.8% 9.4% increase in lvef among those with high baseline hscrp ; p < 0.01 for the overall difference ) . acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; bl : baseline ; crt : cardiac resynchronization therapy ; hscrp : high sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class ; 6-mo : 6-month . bl : baseline ; hscrp : high sensitivity c - reactive protein ; lvedd : left ventricular end diastolic dimension ; lvef : left ventricular ejection fraction ; 6 : 6 months ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 table 1 shows the baseline demographics and clinical characteristics of 232 patients according to the initial hscrp levels . patients were subdivided into high ( > 3 mg / l ) and low ( 3 mg / l ) hscrp groups , with 105 patients in the high hscrp group ( 7.8 4.3 mg / l ) and 127 subjects in the low hscrp group ( 1.3 1.6 mg / l ) . in our study , the mean follow - up duration was 31.3 31.5 months . over the long - term follow - up , patients with hscrp > 3 mg / l had a significantly lower probability of surviving without suffering the combined endpoint of death or hf hospitalizations as compared to those with hscrp 3 mg / l ( log - rank p < 0.01 , figure 1 ) . after controlling for confounding factors , elevated hscrp levels ( > 3 mg / l ) at baseline were shown to independently predict adverse clinical response ( hr : 2.39 , 95%ci : 1.284.47 , p = 0.006 , table 2 ) . when assessed as a continuous variable , each 1 mg / l increase in hscrp was associated with a corresponding 8.3% increase ( p = 0.005 ) in the risk of the combined endpoint of death or hf hospitalizations . crt : cardiac resynchronization therapy ; hf : heart failure ; hscrp : high sensitivity c - reactive protein . during 6-month follow - up , a total of 170 of 232 ( 73.3% ) patients were classified as responders . baseline hscrp values as well as 6-month hscrp levels were significantly lower in patients who responded to crt than in those who did not ( p = 0.001 and p < 0.001 , respectively ) , with a mean 0.5 4.1 mg / l reduction from baseline for patients who were responders and 1.7 6.1 mg / l increase from baseline for those who were non - responders ( p = 0.018 ) . both baseline and 6-month hscrp values for responders and non - responders to crt are displayed in figure 2 . arb : angiotensin receptor blocker ; bun : blood urea nitrogen ; crt - d : cardiac resynchronization therapy - defibrillator ; hscrp : high - sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; lvmi : left ventricular mass index ; mr : mitral regurgitation ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class . the survival curve for all - cause mortality or hf hospitalizations among patients with for each of the baseline to 6-month hscrp change group is shown in figure 3 . during long - term follow - up , compared with those with low / high baseline and low 6-month hscrp values , patients with low / high baseline and high 6-month hscrp values displayed higher rate of the combined endpoint of death or hf hospitalizations ( log - rank p < 0.01 ) . after multivariate adjustment , compared with those with 6-month hscrp values which stayed low , patients with 6-month hscrp levels that remained high experienced the highest rate of death or hf hospitalizations ( hr : 7.6 , p < 0.001 ) , and patients whose levels increased from low baseline to high 6-month hscrp values suffered secondary risk ( hr : 2.9 , p = 0.079 ) , and patients whose levels declined from a high baseline to low 6-month hscrp values underwent similar risk ( hr : 1.4 , p = 0.576 , table 3 ) . when assessed as a continuous variable , each 1 mg / l increase in 6-month hscrp levels was related to a corresponding 9.6% increase ( p = 0.001 ) in the risk of death or hf hospitalizations . bl : baseline ; 6 : 6 months ; hf : heart failure ; hscrp : high sensitivity c - reactive protein ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 mg / l ) . similar to the association of clinical outcome with hscrp change , patients with low 6-month hscrp values displayed the largest concurrent mean reductions in lvedd and increases in lvef ( 6.4 7.7 mm reduction in lvedd and 11.0% 8.6 % increase in lvef among those with low baseline hscrp , and 7.1 8.9 mm reduction in lvedd and 11.1% 8.5 % increase in lvef among those with high baseline hscrp ) . in contrast , patients with high 6-month hscrp values showed significantly lower reductions in lvedd and increases in lvef ( 3.9 5.9 mm reduction in lvedd and 6.2% 8.3% increase in lvef among those with low baseline hscrp and 1.5 9.8 mm reduction in lvedd and 5.8% 9.4% increase in lvef among those with high baseline hscrp ; p < 0.01 for the overall difference ) . acei : angiotensin - converting enzyme inhibitor ; arb : angiotensin receptor blocker ; bl : baseline ; crt : cardiac resynchronization therapy ; hscrp : high sensitivity c - reactive protein ; lbbb : left bundle branch block ; lvedd : left ventricular end - diastolic diameter ; lvef : left ventricular ejection fraction ; nt - probnp : n - terminal pro - brain natriuretic peptide ; nyha class : new york heart association functional class ; 6-mo : 6-month . bl : baseline ; hscrp : high sensitivity c - reactive protein ; lvedd : left ventricular end diastolic dimension ; lvef : left ventricular ejection fraction ; 6 : 6 months ; ( + ) : high hscrp group ( > 3 mg / l ) ; ( ) : low hscrp group ( 3 mg / l ) . secondly , during 6-month follow - up , plasma levels of hscrp were reduced or remained low in patients who were responders to crt , yet a similar improvement was not observed among patients who were non - responders . finally , the pattern of hscrp changes after six months of crt was significantly associated with the lv reverse parameters and subsequent clinical outcome . found , in a small cohort of 65 patients , that hscrp levels before crt implantation were identified as the strongest predictive factor for cardiac death irrespective of other prognostic factors and that being in the high hscrp group ( > 3 this finding is similar to our results that hscrp levels pre - crt implant independently predict all - cause mortality or hospitalizations for hf following crt after adjusting other established risk factors . the similar effect of hscrp levels on the long - term outcome may also occur in crt recipients , this hypothesis , however , can not be directly confirmed by the current or prior results . reported that crt reduced hscrp status only in patients who showed lv reverse and reduction of hscrp levels were associated with the absence of adverse cardiac events . another small observational study of 27 patients with advanced hf showed similar results to ours , in that hscrp levels at 6-month follow up decreased in responders while such improvements were not observed in non - responders . the findings of these small retrospective studies were mostly congruent with our results that patients who showed significant lv reverse remodeling experienced significant reductions or maintained low 6-month hscrp values , whereas patients who do not exhibit obvious cardiac reverse remodeling experienced a slight increase or remained high with regard to their 6-month hscrp levels after crt . additionally , when patients were further stratified into four subgroups according to hscrp change , we found that the pattern of hscrp change from baseline to 6-month was associated with a lack of favorable lv reverse remodeling parameters with respect to lv dimensions and lvef , and independently predicted subsequent all - cause mortality or hf hospitalizations . further studies are needed to elucidate the mechanisms underlying the changes in hscrp levels and lv reverse remodeling as well as prognosis in patients undergoing crt . moreover , the present study was only designed to examine the hscrp levels but not to other inflammatory biomarkers such as il-6 , tnf- and tgf-1 which have also been identified as stronger predictors of clinical events in patients with advanced hf treated with crt . additionally , the measurement of 6-month follow - up hscrp values and echocardiographic parameters was not available for the whole patients which may potentially influence our conclusions . we have shown that hscrp levels at baseline could predict the risk of death or hf hospitalizations in crt recipients . taken together , our results suggest that the important prognostic information can be obtained via an early assessment of hscrp levels before and after crt implantation and that this information may be useful for risk stratification and could influence clinical management strategies in crt recipients . moreover , the present study was only designed to examine the hscrp levels but not to other inflammatory biomarkers such as il-6 , tnf- and tgf-1 which have also been identified as stronger predictors of clinical events in patients with advanced hf treated with crt . additionally , the measurement of 6-month follow - up hscrp values and echocardiographic parameters was not available for the whole patients which may potentially influence our conclusions . finally , further studies are needed to elucidate the underlying pathophysiologic mechanisms of the interaction between hscrp levels and lv reverse remodeling as well as clinical prognosis in patients undergoing crt . we have shown that hscrp levels at baseline could predict the risk of death or hf hospitalizations in crt recipients . taken together , our results suggest that the important prognostic information can be obtained via an early assessment of hscrp levels before and after crt implantation and that this information may be useful for risk stratification and could influence clinical management strategies in crt recipients .

the l1 family of cell adhesion molecules ( the l1 family ) includes l1 , close homolog of l1 ( chl1 ) , ngcam - related cell adhesion molecule ( nrcam ) and neurofascin . these molecules regulate neuronal development and networking ( burden - gulley et al . , 1997 ; hortsch , 2000 ; katidou et al . , 2008 ) . defects in l1 family members are implicated in various neurological diseases including x - linked mental retardation , multiple sclerosis , low - iq syndrome , developmental delay , and schizophrenia ( kenwrick et al . , 2000 ; knock - out of l1 family members in mice led to guidance errors in corticospinal and retino - collicular axons , hippocampal ca3 mossy fibers , olfactory neurons , and retinal axons ( maness and schachner , 2007 ) . the mice also exhibited abnormal cerebellar development , dendritic misorientation defects , and behavioral deficits . l1 family proteins are type i membrane proteins with heavy glycosylation ; they contain an ectodomain ( 1,100 residues ) with six ig - like domains ( ig1-ig6 ) and five fibronectin - type iii domains ( fn1-fn5 ) , a single pass transmembrane region and a cytoplasmic domain ( 110 residues ) ( hortsch , 1996 ) . homophilic and heterophilic interactions of l1 on the cell surface mediate functions including axonal guidance , neural system development , and cell migration ( maness and schachner , 2007 ) . partners for heterophilic interactions include integrins , neurophilin-1 , tag-1 , and receptor tyrosine phosphatases that potentiate cell mobility and signal transduction ( maness and schachner , 2007 ) . the adhesive function of l1 induces cellular signaling processes involving activation of tyrosine protein kinases ( silletti et al . , 2004 ) . the short cytoplasmic tail binds to cytoskeletons including ankyrin and spectrin , thus mediating intracellular signal transduction ( bennett and baines , 2001 ) . when backcrossed with the c57bl/6 mouse strain , a mouse line lacking l1-l1 homophilic interactions due to the deletion of the sixth ig - like domain of l1 resulted in a severe hydrocephalus and became embryonic lethal after several generations ( itoh et al . , 2004 ) . in inflammatory conditions , homophilic interaction of l1 between dendritic cells ( dcs ) and the vascular endothelium mediates enhanced dc transmigration ( maddaluno et al . , 2009 ) . chl1 plays a pivotal role in neuronal positioning in the visual and somatosensory cortex by coordinating with 1-integrins ( schmid and maness , 2008 ) . homophilic interactions of neurofascin are critical for neurite induction ( pruss et al . , 2004 ) . l1 syndrome diseases include x - linked hydrocephalus as a result of stenosis of the aqueduct of sylvius ( hsas ) , mental retardation , aphasia , shuffling gait , and adducted thumbs ( masa ) syndrome , and x - linked spastic paraplegia ( wong et al . , 1995 ; hortsch , 2000 ) . the studies on the crystal structure of ig - like domain 1 - 4 in neurofascin suggested that many pathological l1 mutations affect conserved amino acid residues within these domains and interfere with homophilic interactions ( liu et al . , 2011 ) . sixteen mutations were found in the n - terminal horseshoe region that mediates homophilic interaction , indicating that disruption of the horseshoe was the likely cause of the diseases . some of these mutations have been investigated by others ( de angelis et al . , 2002 ) and the possible effect on horseshoe conformation discussed . these mutants usually affect adhesion - dependent cell signaling by reducing l1-protein 's adhesive abilities without altering their expression ( nagaraj et al . , 2009 ) . l1 mutations are also associated with fetal alcohol spectrum disorders ( fasd ) ( ramanathan et al . , 1996 ; kenwrick et al . , 2000 ) . a series of structural modeling and mutagenesis studies indicate the presence of an alcohol binding site in the domain interface of the l1 horseshoe region , whose perturbation by alcohol could disrupt homophilic interactions ( arevalo et al . , 2008 ; dou et al . , 2011 cell adhesion molecules such as integrins , cadherins , ig - like cams , and selectins are often aberrantly expressed in human cancers , and contribute to disease progression ( kwak et al . , 2011 ; witzel et al . , 2012 ) . various human tumors with poor prognosis for example , the invasive front of colon cancers expresses l1 ( gavert et al . , 2005 ) , and stable ectopic expression of l1 in fibroblastic cells induces the expression of metastasis - associated genes ( silletti et al . , 2004 ) . due to the wide expression of l1 in cancerous tissues and its cell surface localization , l1 is regarded as a useful target for the diagnosis of advanced cancers and a potential candidate for therapeutic intervention ( knogler et al . the crystal structures of l1 homologs , hemolin ( su et al . , 1998 ) , axonin-1 ( freigang et al . , 2000 ) , tag-1 ( mortl et al . , 2007 ) and dscam ( meijers et al . , 2007 ; sawaya et al . , 2008 ) suggest that the four n - terminal ig - like domains of l1 adopt a horseshoe - shaped conformation in which the first and second ig - like domains ( ig1 and ig2 ) fold back to interact with the fourth and third ig - like domains ( ig4 and ig3 ) . the arg - gly - asp ( rgd ) sequence in ig6 is essential for integrin interactions ( thelen et al . , 2002 ) . other evidence indicates a regulatory role of carbohydrates in l1-mediated homophilic adhesion ( acheson et al . , 1991 ; kleene et al . , 2001 ) . several models for l1 homophilic interactions have been proposed based on l1 homolog structures , including hemolin ( su et al . , 1998 ) , axonin-1 ( freigang et al . , 2000 ) , tag-1 ( mortl et al . , 2007 ) , and dscam ( meijers et al . the structure of neurofascin ( liu et al . , 2011 ) , the first l1 family structure to be defined , showed a novel homophilic interaction mechanism that could be compared with previous models . the cryo - electron tomograms of l1 bound to microsomes also elucidated the patterns of homophilic interaction under physiologically relevant conditions ( he et al . , 2009 ) . in this review , we focus on the recent discoveries in the structural mechanisms of l1 homophilic interactions and compare them to other l1 homologs . understanding the structural mechanism of l1 homophilic interactions may contribute to the development of therapeutic strategies for diseases mediated by defects in l1-related cellular functions . ig - like cell adhesion molecules ( ig - cam ) such as l1 and ncam are the most ancient and diverse family of cell adhesion proteins ( rougon and hobert 2003 ; aricescu and jones , 2007a ) . some of them have homophilic binding specificities , while others have heterophilic specificities , which allow them to interact with other ig - cams or other types of cams . ig - cam ectodomains have a modular structure , with ig - like domains located near the membrane - distal n - terminus and other types of domains such as fibronectin type iii ( fn domain ) connecting them to the membrane - spanning domain ( figure 1 ) ( williams et al . , 1989 ; shapiro et al . , 2007 ) the adhesive function of ig - cams is usually mediated by ig domains ( williams et al . , 1989 ) . in comparison to igs that use a defined structural mode for antigen recognition such as the complementarity - determining loops ( cdrs ) , ig - cams exhibit mechanistic diversity because of the highly diverse arrangements of domain modules . the l1 family of proteins includes mammalian l1 , chl1 , nrcam , and neurofascin ( hortsch , 2000 ; katidou et al . , 2008 ) . other homologs include the insect adhesion protein hemolin ( su et al . , 1998 ) , the glycosyl phosphatidylinositol ( gpi)-anchored neural cell adhesion molecule ( ncam ) axonin-1 ( chicken)/tag-1(mammalian ) ( furley et al . , 1990 ; zuellig et al . , 1992 ) , and the down syndrome protein dscam ( figure 1 ) ( schmucker et al . , 2000 ) . the ectodomain of the l1 family proteins has six ig - like domains and five fn domains , whereas l1 homolog ectodomains exhibit distinct domain structures . the ectodomain of hemolin has four ig - like domains , whereas the ectodomain of axonin-1/tag-1 has six ig - like domains and four fn domains ( figure 1 ) . different dscam isoforms share a common domain structure , with ten ig - like domains , six fn domains , a single transmembrane region , and a c - terminal cytoplasmic tail ( figure 1 ) . other ig - cams that exhibit homophilic interactions include cd2 , cadherin , ncams and others ( figure 1 ) . the t - cell surface protein cd2 ( selvaraj et al . , 1987 ) has two ig - like domains that bind to its homologs cd58 ( selvaraj et al . , 1987 ) and cd48 ( kato et al . , 1992 ) on opposing cells , forming pseudo - homophilic interactions . , 1992 ; bodian et al . , 1994 ) showed a head - to - head interaction between the gfcc'c " sheets of ig - like domains from two interacting molecules . cadherins mediate various cell - cell contacts via homophilic interaction between the same isotypes ( steinberg and mcnutt , 1999 ) . the crystal structure of a c - cadherin ectodomain containing five tandem cadherin domains showed homophilic interaction involving the first cadherin domain in trans ( boggon et al . , 2002 ) . in addition to the trans interaction , the cadherin structure exhibited a cis interaction between the first domain of first cardherin and the interface of the first and second domains of the second cadherin , creating a zipper - like structure ( boggon et al . , 2002 ) . ncam is one of the most extensively studied ig - like domain - containing cams and play vital roles in brain development and function . major types of ncams including ncam-120 , ncam-140 and ncam-180 contain five ig - like domains and two fn domains in their extracellular regions ( maness and schachner , 2007 ) . studies on various ncams suggest the zipper - like models ( walmod et al . , 2004 ) . necl molecules , which play a vital role during synapse assembly , contain three ig - like domains in their extracellular regions . the crystal structure of the n - terminal ig - like domain of necl-1 showed homophilic interactions with a zipper - type arrangement ( dong et al . , 2006 ) . type iib receptor - type protein tyrosine phosphatases ( rptps ) , such as rptp have ectodomains that mediate homophilic ( adhesive ) interactions and intracellular phosphatase domains that dephosphorylate target proteins ( tonks , 2006 ) . type iib rptps share a common architecture , in that their extracellular regions contain one meprin / a5/m ( mam ) domain , one ig - like domain and four fn domains . the structure of a full - length rptp ectodomain ( aricescu et al . , 2007b ) showed that the determinants required for homophilic interaction are the residues of mam , ig1 , fn1 and fn2 domains . homophilic interaction would generate a zipper - like structure via cis - interaction with adjacent molecules ( aricescu and jones , 2007a ) . the crystal structures of ig1 - 4 domains from l1 homologs indicated different modes of homophilic interactions ( su et al . hemolin , an innate immunity - related molecule in insects , contains four ig - like domains ( ig1 - 4 ) that share 38% sequence homology with l1 ig1 - 4 domains ( su et al . , 1998 ) . the crystal structure determination of hemolin ig1 - 4 domains revealed a unique domain arrangement among the ig - like domain - containing cell surface proteins . previously described ig - like domain proteins such as cd4 have a linearly - arranged domain structure ( wu et al . , 1997 ; boggon et al . , cd4 has four ig - like domains in a beads - on - a - string formation ( wu et al . , 1997 ) . in comparison , the ig1 - 4 domains of hemolin form a horseshoe structure where ig1 and ig2 fold back to interact with ig4 and ig3 , respectively ( figure 2 ) ( su et al . , 1998 ) . an extended linker between ig2 and ig3 creates a sharp bend of the ig1 - 2 domains toward the ig3 - 4 domains . the domain interactions between ig1 and ig4 , and between ig2 and ig3 comprise an array of hydrophobic interactions , and residues involved in these interactions are highly conserved in the l1 family . in the crystal of hemolin ( su et al . , 1998 ) , there were no crystal contacts to suggest the mechanism of its homophilic interactions . however , based on the structure and previously described homophilic interactions in ig - like domain - containing molecules such as cd2 and cadherin , su et al . , proposed a model ( figure 2 ) ( su et al . , 1998 ) in which , ig1 - 2 of hemolin move away from ig3 - 4 to form a domain - swapped horseshoe with the ig3'-4 ' of another hemolin molecule from an opposing membrane . the chicken ncam axonin-1 has six ig - like domains and four fn domains ( zuellig et al . , 1992 ; freigang et al . , 2000 the crystal structure of the ig1 - 4 domains of axonin-1 also showed a horseshoe shape , confirming the generality of the ig1 - 4 domain arrangement in hemolin ( figure 2b ) ( freigang et al . , 2000 ) . the inter - molecular interaction continues through the crystal lattice and forms a zipper - like structure . the crystal contact showed a significant burying surface , where the loop ce of ig3 protruded to interact with the central hole in the face of the next molecule , including residues of fg loop of ig2 ' . the c - termini of the dimer point in opposite directions so that facing membranes can be connected by the homophilic interaction . when residues involved in the dimeric interaction ( deletion of residues 187 - 190 and h186a / f189a ) are mutated , the myeloma cells expressing mutant axonin-1 can not aggregate , in comparison to normal axonin-1 expressing cells , which aggregate readily ( freigang et al . later , the ig1 - 4 structure of tag-1 , the human homolog of axonin-1 was determined ( mortl et al . , 2007 ) . although tag-1 has a horseshoe structure similar to that axonin-1 , the zipper - like dimer interaction of axonin-1 was not observed in the tag-1 crystal . instead , a two - fold symmetry - mediated dimer interaction is formed by side - to - side interactions between g strand of ig2 with g strand of ig2 ' , generating an intermolecular -sheet ( figure 2 ) . the buried surface created by dimeric interaction in the tag-1 crystal is 2240 , indicating that the interaction likely has a physiological relevance . in addition , the importance of fg loop in ig2 in myeloma cell aggregation was reported for axonin-1 , the chicken homolog of tag-1 ( freigang et al . , 2000 ) . because the same regions of two tag-1 monomers are used in the dimeric interaction , the interaction can not be extended to form a zipper - like structure as proposed in the case of axonin-1 ( figure 2 ) . the possibility of two different modes of dimeric interaction in these closely related molecules must be resolved by further structural and functional analysis . the drosophila protein dscam , which contains ten ig - like domains and six fn domains , mediates cell - cell interaction in brain cell networking ( schmucker et al . , 2000 ) . among the ten ig - like domains , ig2 , ig3 and ig7 have 12 , 47 and 33 alternative sequences , respectively , giving 18,048 potential derivatives ( schmucker et al . , 2000 ) . homophilic interactions occur only between the same isotypes ; interactions between different isotypes are rare , providing necessary specificity in brain cell networking . the crystal structure of ig1 - 4 domains from two different isotypes of dscam revealed their novel dimeric interactions ( meijers et al . , 2007 ) ( figure 2d ) . in comparison to the dimeric interactions of axonin-1 ( freigang et al . , 2000 ) and tag-1 ( mortl et al . , 2007 ) , where interaction occurred between side regions , the dimeric interaction in dscam involved the main surface of ig2 and ig3 . the ig2 surface interacts with the same surface of ig2 ' and the ig3 surface interacts with the same surface of ig3 ' . a two - fold symmetry axis mediates both interactions , and the c - termini are on opposite sides , such that the opposing membranes can be associated by dscam homophilic interaction . structural studies of dscam ig1 - 8 revealed the same dimeric interaction of the horseshoe region as that occurring in the the ig1 - 4 domain structures ( sawaya et al . , 2008 ) . in addition to the interactions mediated by ig2 and ig3 in the ig1 - 4 region , ig7 of dscam interacts with ig7 ' of the other monomer in a face - to - face interaction with the same two fold symmetry axis as that occurring in the ig2 and ig3 interactions ( sawaya et al . , 2008 ) . tight interactions of the three ig domains in dscam generate a double - s conformation ( figure 3 ) . the double - s conformation of dscam ig1 - 8 indicates the critical role of the domains outside the horseshoe region in the homophilic interactions of l1 homologs . because of the tight intermolecular interaction between two ig1 - 8 molecules , alteration in non - horseshoe domains can affect the orientation of inter - horseshoe regions and direct homophilic interactions mediated by non - horseshoe domains earlier experiments with recombinant l1 produced in escherichia coli indicated that ig2 was sufficient to induce trans - homophilic interaction and neurite elongation ( zhao et al . , 1998 ) . the bacteria - derived fn domain spontaneously formed trimeric and higher - order complexes , suggesting that fn domains were also necessary for l1 homophilic interactions in cis ( de angelis et al . , 1999 ) . later , domain - mapping experiments used eukaryotic cell - derived l1 constructs and found that the homophilic interactions involved more domains . the first four ig domains ( ig1 - 4 ) promoted homophilic cell adhesion and ig1 - 6 were necessary for optimal neurite outgrowth ( haspel et al . an insect cell expression system showed that a protein containing ig1 - 4 domains mediated the homophilic interaction , whereas ig2-fn5 or ig1 - 3 did not , indicating that ig2 was not sufficient for the homophilic interaction ( gouveia et al . co - immunoprecipitation studies of truncated forms of l1 and endogenous full - length l1 showed that the l1 ectodomain ( l1/ecd ) and l1/ig1 - 4 interact homophilically in trans ( gouveia et al . , 2008 ) . kinetic analysis by surface plasmon resonance showed that the kd of the whole ectodomain - whole ectodomain interaction was 116 2 nm , and the kd value of the whole ectodomain - ig1 - 4 interaction was 130 6 nm ( gouveia et al . , 2008 ) . thus , ig1 - 4 was the minimum portion of l1 to exhibit a similar homophilic interaction activity similar to that of full length l1 . consistent with this , insect cells stably expressing l1 adhered only to l1/ecd- and l1/ig1 - 4-coated surfaces or to hek293 cells overexpressing l1 on the cell surface . after the determination of the crystal structure of ig1 - 4 domains of insect hemolin ( su et al . , 1998 ) , su et al . , proposed two mechanisms to explain the homophilic interaction . the first mechanism is that ig1 - 4 domains exist in equilibrium between the extended and folded - back conformations and that the extended conformation mediates the homophilic interaction in trans . in this model , all ig domains interact with ig domains of the partner molecule in an antiparallel orientation ( i.e. ig1-ig4 ' , ig2-ig3 ' , ig3-ig2 ' , and ig4-ig1 ' ) ( figure 2a ) . the second mechanism suggests that the folded - back ( horseshoe ) conformation is the active form . in this model , the compact horseshoe structures of opposing l1 molecules do not open , but rather interact with each other . although there is no clear structural evidence to favor either model , experimental data seem to rule out the extended conformation model . deletion of ig1 or ig4 was sufficient to impair the homophilic interaction ( gouveia et al . , 2008 ) , supporting the latter mechanism because deletion of these domains disrupted the horseshoe structure , but not the antiparallel interaction between the linearized domains . ethanol inhibits cell - cell adhesion mediated by l1 in neuroblastoma - glioma hybrid cells , cerebella granule cells , and human l1-transfected murine fibroblasts ( charness et al . , 1994 ; ramanathan et al . , 1996 ; wilkemeyer and charness , 1998 ) , indicating that ethanol can disrupt homophilic interaction of l1 . indeed , mutations in the l1 gene produce neuropathological abnormalities similar to those found in fasd caused by prenatal ethanol exposure . these abnormalities include growth deficiency , mental retardation and facial malformations ( sampson et al . , 1997 ; hoyme et al . , the alcohol effect on l1 occurs for methanol through 1-butanol , but disappears for alcohols larger than 1-pentanol , indicating the presence of an alcohol binding pocket of a limited size in l1 ( ramanathan et al . , purified l1 ig1 - 4 protein was photo - labeled by photoactivatable analogs of alcohol , 3-azibutanol ( 1-butanol analog ) and 3-azioctanol ( 3-octanol analog ) ( arevalo et al . , 2008 ) . the photoactivatable analogs labeled tyr418 on ig4 and several n - terminal residues including glu33 and glu24-glu27 on ig1 . in a modeled structure of the l1 ig1 - 4 based on the horseshoe structure of axonin-1 ( freigang et al . , 2000 ) , the photo - labeled residues were found at the interface between ig1 and ig4 , indicating that the surface formed by these residues was likely the alcohol binding site ( arevalo et al . , 2008 ) . in the model , glu33 and tyr418 were located closely with the distance of 2.8 between them , indicating that the residues formed a strong hydrogen bond . the n - terminal regions including glu33 and glu24-glu27 could not be modeled because the axonin-1 structure did not contain the region . alcohol binding to the pocket in the domain interface of glu33 and tyr418 likely affected the conformation of the horseshoe structure of l1 , preventing of ideal interaction between the horseshoe regions . the proximity of glu33 and tyr418 and their role in homophilic interaction were tested by cysteine substitution mutations ( the e33c / y418c mutant ) ( dou et al . , 2011 ) . cells transfected with the cysteine mutant showed significantly increased l1 adhesion as compared to those transfected with the wild - type protein or single cysteine mutants . moreover , reducing conditions decreased the effect of the mutation , indicating that reversible disulfide bond formation between cysteines stabilized the horseshoe conformation and the resulting homophilic interaction . the inhibitory effect of ethanol on l1-mediated adhesion decreased in cells expressing the e33c / y418c mutant l1 . although the modeling studies and mutation analyses convincingly present the potential alcohol - binding site in l1 , the shape of the binding pocket and the interactions between alcohol and l1 need to be defined by experimental structure studies . the role of the n - terminal residues including glu33 and glu24-glu27 also has to be resolved . the horseshoe structures found in l1 homologs ( su et al . , 1998 ; freigang et al . , 2000 , 2007 ; sawaya et al . , 2008 ) suggest that l1 has a similar structure . however , the structures proposed different models for the homophilic interaction ; therefore , a physiologically relevant analysis was necessary . cryo - electron tomography of l1 addressed some of the questions surrounding the homophilic interaction mechanism ( he et al . , 2009 ) . tomographic reconstructions of the interfaces between l1-presenting liposomes showed regularly spaced globular densities appearing as dots in a row among the overall diffuse densities ( he et al . , 2009 ) . the approximate size of the dots with tomogram resolution of 4 - 5 nm was consistent with the size of dscam horseshoe pairs of 60 70 80 and 60 60 100 ( pdb files 2v5r and 2v5s ) , indicating that the dots represented the paired dimer of l1 horseshoes . the discrete arrangement of dots in the area of membrane interaction suggested that l1 homophilic interaction did not occur as proposed in the zipper model . the interacting horseshoes in the zipper model with indefinite repeats of horseshoe domains should appear as a continuous and solid line of density in tomograms . moreover , the domain - swapped multimerization model where domains 1 and 2 forms a horseshoe with domains 4 and 3 of the opposing membrane ( figure 2a ) , was not consistent with the l1 tomography observations . with the domain - swapped multimerization model , the tomograms should show a zig - zag pattern of individual horseshoe density . instead of the zipper or domain - swapped multimerization models , the discrete and regularly spaced dots fit the model of a dimer seen in the dscam ( meijers et al . , 2007 ; sawaya et al . , 2008 ) and tag-1 structures ( mortl et al . , 2007 ) . in dscam and tag-1 dimers ( figures 2c and 2d ) , one region of the first horseshoe interacts with the same region of the second horseshoe ( d2-d2 ' and d3-d3 ' in dscam ; d2-d2 ' in tag-1 ) through a two - fold symmetry axis . this dimeric interaction produces an isolated dimer , which is consistent with the discrete dots in l1 tomograms . although the domain - swapped multimerization model does not fit the tomographic observation , one - to - one domain - swapping may result in an isolated dimer ( figure 2a ) , which can not be ruled out as a possible model for the observed dots . l1 has twenty - one glycosylations consisting of four , five and twelve sites in the horseshoe , ig5 - 6 and fn domains , respectively . various studies have shown that l1 glycosylation affects its homophilic interaction ( acheson et al . , 1991 ; kleene et al . , 2001 ) . carbohydrate - protein or carbohydrate - carbohydrate interactions often regulate homophilic interaction of cell surface proteins ( bucior and burger , 2004 ) . furthermore , the l1 ectodomain binds to sialic acid , revealing that l1 functions as a sialic acid - binding lectin ( acheson et al . , 1991 ; kleene et al . in an experiment to analyze the role of carbohydrates in l1 , he et al . . , 2009 ) expressed l1 in mammalian and insect cells with attached complex n - linked and oligomannoside carbohydrates , respectively , on their membrane proteins . when the two types of l1 were reconstructed with liposomes , they produced similar dots between the two interacting liposomes ; however , the dots had different inter - dot distances : 100 for the mammalian cell - derived l1 and 140 for the insect cell - derived l1 ( figure 4 ) . thus , the regular spacing of dots in tomograms likely represents interactions with long chains of carbohydrates attached to l1 ectodomains . when the terminal sialic acids on mammalian cell - derived l1 were removed by sialidase or cell - growth in a medium containing kifunensine , the liposome interaction pattern changed ( he et al . , 2009 ) . these observations indicate that the carbohydrates on l1 play a pivotal role in determining the distances between horseshoe dimers , thus modulating homophilic interaction patterns ( figure 4 ) . the carbohydrate attachment sites of mammalian l1 are completely conserved , and there are no additional sites , further indicating the importance of carbohydrates in l1 function . however , the mechanism by which the flexible carbohydrate chain maintains the precise distance between the dots observed in the tomograms is of interest . it is unlikely that the carbohydrate alone can hold the interacting horseshoe regions in a regular fashion . the remaining domains ( ig5 - 6 and fn1 - 5 domains ) are also likely to mediate the homophilic interaction and regular inter - dot spacing . the role of carbohydrates in homophilic cell adhesion interactions was shown in ncam ( kadmon et al . , 1990 ; acheson et al . , 1991 ) and coxsackievirus and adenovirus receptors ( excoffon et al . , 2007 ) . the terminal sialic acids are also vital for ncam homophilic interactions ( fujimoto et al . , 2001 ; varki , 2007 ) as well as heterophilic interactions between l1 and cd24 ( kleene et al . , 2001 ) recently , the first structure of the l1 family proteins was determined for neurofascin ( liu et al . , 2011 ) . the ig1 - 4 domains of neurofascin were overexpressed and purified from an insect expression system . the purified neurofascin ig1 - 4 shows a 47 kda band on sds - page , as expected from the calculation of its theoretical molecular weight . however , in a gel filtration experiment , the protein produced a peak at 100 kda position , which was twice the monomeric size , indicating that neurofascin ig1 - 4 forms a dimer in solution . the neurofascin ig1 - 4 ( liu et al . , 2011 ) has a horseshoe structure as that of l1 homologs ( su et al . , 1998 ; freigang et al . , 2000 the ig1:ig4 and ig2:ig3 interdomain interactions involve large surfaces with hydrophobic patches , indicating that the horseshoe structure of neurofascin ig1 - 4 is likely stable . the residues involved in the domain interactions of neurofascin ig1 - 4 are conserved in other members of the l1 family ( liu et al . , 2011 ) . consistent with the dimer formation in solution , the two different crystal lattices of neurofascin ig1 - 4 contain a common neurofascin ig1 - 4 dimer with extensive monomer - monomer contacts ( liu et al . , 2011 ) . the neurofascin ig1 - 4 dimer formation is mediated by the side of ig2 domain , resulting in the formation of a supramolecular -sheet with a two - fold symmetry ( figure 5a ) . when mutations were introduced in the residues involved in the dimeric interaction , most mutant proteins showed a shift in gel filtration peak positions towards the monomeric molecular weight . among the mutations , the substitution of thr 222 to ala ( t222a ) showed a clear movement of the peak . thr 222 of one monomer forms a hydrogen bond with gln 224 of the other monomer at the center of the supramolecular -sheet , and the mutation result indicates its importance in the dimeric interaction . suparmolecular -sheet formation of neurofascin ig1 - 4 is similar to the dimeric interaction in tag-1 ( figure 5b ) ( mortl et al . , 2007 ) . the two - fold symmetry - related dimeric interaction between the side regions of neurofascin ig2 produced an isolated dimer , consistent with the discrete inter - membrane dots observed in l1 tomograms ( he et al . although the dscam horseshoe dimeric interaction ( meijers et al . , 2007 ; sawaya et al . , 2008 ) also generates an isolated dimer without further continuation of the interaction , the dscam interaction occurs throughout the domain face ; this differs from the edge interaction observed in neurofascin ig1 - 4 . because both neurofascin ig1 - 4 and tag-1 horseshoes exhibit the same suparmolecular -sheet formation , this mode of interaction may represent homophilic interaction of l1 family molecules and their homologs . the neurofascin ig1 - 4 structure was used to map pathological mutations in neurological disorders such as hsas , masa syndrome , and x - linked spastic paraplegia ( wong et al . , 1995 ; most mutations were found in the domain interfaces ( l120v , g121s , g370r , r184q / w , and y194c ) of the horseshoe formation or in the hydrophobic core of individual domains ( i179s , h210q , p240l , c264y , g268d , p333r , w335r , and l391p ) , confirming the importance of the horseshoe structure in l1 function . in contrast , one mutation ( i219 t ) corresponding to ile213 in neurofascin ig1 - 4 produced a neurite branching defect and was located in the hydrophobic cluster that mediates dimeric interaction in neurofascin ig1 - 4 crystals , supporting the model of homophilic interaction proposed by the crystal structures . although the neurofascin ig1 - 4 structure is useful for explaining pathological mutations in the horseshoe region , it does not provide information on other domains , including ig5 - 6 and fn domains ( fn1 - 5 ) where various disease - inducing mutations occur ( de angelis et al . , 2002 ) . the lack of homophilic interaction of l1 in a mouse line carrying a deletion of the ig5 domain ( itoh et al . , 2004 ) also indicates that involvement of domains other than the horseshoe region can affect the homophilic interaction of l1 family proteins . mutations in non - horseshoe regions may change the structure of the horseshoe region or direct intermolecular interactions . structural analysis and carefully designed mutational and functional studies of molecules containing domains outside the horseshoe region are required to answer this question . homophilic interactions between l1 family proteins are essential for neural system development and brain cell wiring . because homophilic interaction - mediated signal transduction activates cell growth signaling , it is believed that cancer progression also involves the homophilic interaction of l1 . various models for homophilic interaction have been proposed for cell adhesion proteins ( figure 2 ) , including zipper , domain - swapped monomer , domain - swapped multimer , two - fold symmetry related surface interaction and two - fold symmetry related edge interaction models based on the structures of axonin-1 , hemolin , dscam and tag-1/neurofascin , respectively . the two - fold symmetry - related surface or edge interaction and domain - swapped monomer models are consistent with the discrete and equally spaced dots observed in cryo - electron tomograms of l1 . interestingly , dimeric interaction via the edge of ig2 domain was observed in tag-1 and neurofascin , suggesting that the symmetry - related edge model may represent the homophilic interaction mechanism of l1 and its homologs . current understanding of homophilic interaction in l1 family molecules is based mainly on the crystal structures of the first four ig - like domains of a horseshoe structure . however , for a complete understanding of homophilic interactions , structural determination of l1 including other ig - like and fn domains is necessary . such studies may reveal novel supra - structures such as the double - s conformation observed in the dscam ig1 - 8 dimer .

homophilic interaction of the l1 family of cell adhesion molecules plays a pivotal role in regulating neurite outgrowth and neural cell networking in vivo . functional defects in l1 family members are associated with neurological disorders such as x - linked mental retardation , multiple sclerosis , low - iq syndrome , developmental delay , and schizophrenia . various human tumors with poor prognosis also implicate the role of l1 , a representative member of the l1 family of cell adhesion molecules , and ectopic expression of l1 in fibroblastic cells induces metastasis - associated gene expression . previous studies on l1 homologs indicated that four n - terminal immunoglobulin - like domains form a horseshoe - like structure that mediates homophilic interactions . various models including the zipper , domain - swap , and symmetry - related models are proposed to be involved in structural mechanism of homophilic interaction of the l1 family members . recently , cryo - electron tomography of l1 and crystal structure studies of neurofascin , an l1 family protein , have been performed . this review focuses on recent discoveries of different models and describes the possible structural mechanisms of homophilic interactions of l1 family members . understanding structural mechanisms of homophilic interactions in various cell adhesion proteins should aid the development of therapeutic strategies for l1 family cell adhesion molecule - associated diseases .

Introduction Cell adhesion molecules with immunoglobulin-like domains Homophilic interaction of L1 homologs Analysis of L1 homophilic interactions Alcohol binding site in L1 Cryo-electron tomography of L1 Structure of the L1 family protein neurofascin Conclusions

the l1 family of cell adhesion molecules ( the l1 family ) includes l1 , close homolog of l1 ( chl1 ) , ngcam - related cell adhesion molecule ( nrcam ) and neurofascin . defects in l1 family members are implicated in various neurological diseases including x - linked mental retardation , multiple sclerosis , low - iq syndrome , developmental delay , and schizophrenia ( kenwrick et al . , 2000 ; knock - out of l1 family members in mice led to guidance errors in corticospinal and retino - collicular axons , hippocampal ca3 mossy fibers , olfactory neurons , and retinal axons ( maness and schachner , 2007 ) . l1 family proteins are type i membrane proteins with heavy glycosylation ; they contain an ectodomain ( 1,100 residues ) with six ig - like domains ( ig1-ig6 ) and five fibronectin - type iii domains ( fn1-fn5 ) , a single pass transmembrane region and a cytoplasmic domain ( 110 residues ) ( hortsch , 1996 ) . homophilic and heterophilic interactions of l1 on the cell surface mediate functions including axonal guidance , neural system development , and cell migration ( maness and schachner , 2007 ) . when backcrossed with the c57bl/6 mouse strain , a mouse line lacking l1-l1 homophilic interactions due to the deletion of the sixth ig - like domain of l1 resulted in a severe hydrocephalus and became embryonic lethal after several generations ( itoh et al . in inflammatory conditions , homophilic interaction of l1 between dendritic cells ( dcs ) and the vascular endothelium mediates enhanced dc transmigration ( maddaluno et al . chl1 plays a pivotal role in neuronal positioning in the visual and somatosensory cortex by coordinating with 1-integrins ( schmid and maness , 2008 ) . homophilic interactions of neurofascin are critical for neurite induction ( pruss et al . l1 syndrome diseases include x - linked hydrocephalus as a result of stenosis of the aqueduct of sylvius ( hsas ) , mental retardation , aphasia , shuffling gait , and adducted thumbs ( masa ) syndrome , and x - linked spastic paraplegia ( wong et al . the studies on the crystal structure of ig - like domain 1 - 4 in neurofascin suggested that many pathological l1 mutations affect conserved amino acid residues within these domains and interfere with homophilic interactions ( liu et al . sixteen mutations were found in the n - terminal horseshoe region that mediates homophilic interaction , indicating that disruption of the horseshoe was the likely cause of the diseases . a series of structural modeling and mutagenesis studies indicate the presence of an alcohol binding site in the domain interface of the l1 horseshoe region , whose perturbation by alcohol could disrupt homophilic interactions ( arevalo et al . , 2011 cell adhesion molecules such as integrins , cadherins , ig - like cams , and selectins are often aberrantly expressed in human cancers , and contribute to disease progression ( kwak et al . various human tumors with poor prognosis for example , the invasive front of colon cancers expresses l1 ( gavert et al . , 2005 ) , and stable ectopic expression of l1 in fibroblastic cells induces the expression of metastasis - associated genes ( silletti et al . due to the wide expression of l1 in cancerous tissues and its cell surface localization , l1 is regarded as a useful target for the diagnosis of advanced cancers and a potential candidate for therapeutic intervention ( knogler et al . the crystal structures of l1 homologs , hemolin ( su et al . , 2008 ) suggest that the four n - terminal ig - like domains of l1 adopt a horseshoe - shaped conformation in which the first and second ig - like domains ( ig1 and ig2 ) fold back to interact with the fourth and third ig - like domains ( ig4 and ig3 ) . several models for l1 homophilic interactions have been proposed based on l1 homolog structures , including hemolin ( su et al . , 2011 ) , the first l1 family structure to be defined , showed a novel homophilic interaction mechanism that could be compared with previous models . the cryo - electron tomograms of l1 bound to microsomes also elucidated the patterns of homophilic interaction under physiologically relevant conditions ( he et al . in this review , we focus on the recent discoveries in the structural mechanisms of l1 homophilic interactions and compare them to other l1 homologs . understanding the structural mechanism of l1 homophilic interactions may contribute to the development of therapeutic strategies for diseases mediated by defects in l1-related cellular functions . ig - like cell adhesion molecules ( ig - cam ) such as l1 and ncam are the most ancient and diverse family of cell adhesion proteins ( rougon and hobert 2003 ; aricescu and jones , 2007a ) . ig - cam ectodomains have a modular structure , with ig - like domains located near the membrane - distal n - terminus and other types of domains such as fibronectin type iii ( fn domain ) connecting them to the membrane - spanning domain ( figure 1 ) ( williams et al . the l1 family of proteins includes mammalian l1 , chl1 , nrcam , and neurofascin ( hortsch , 2000 ; katidou et al . , 1998 ) , the glycosyl phosphatidylinositol ( gpi)-anchored neural cell adhesion molecule ( ncam ) axonin-1 ( chicken)/tag-1(mammalian ) ( furley et al . the ectodomain of the l1 family proteins has six ig - like domains and five fn domains , whereas l1 homolog ectodomains exhibit distinct domain structures . the ectodomain of hemolin has four ig - like domains , whereas the ectodomain of axonin-1/tag-1 has six ig - like domains and four fn domains ( figure 1 ) . different dscam isoforms share a common domain structure , with ten ig - like domains , six fn domains , a single transmembrane region , and a c - terminal cytoplasmic tail ( figure 1 ) . , 1987 ) has two ig - like domains that bind to its homologs cd58 ( selvaraj et al . , 1994 ) showed a head - to - head interaction between the gfcc'c " sheets of ig - like domains from two interacting molecules . the crystal structure of a c - cadherin ectodomain containing five tandem cadherin domains showed homophilic interaction involving the first cadherin domain in trans ( boggon et al . in addition to the trans interaction , the cadherin structure exhibited a cis interaction between the first domain of first cardherin and the interface of the first and second domains of the second cadherin , creating a zipper - like structure ( boggon et al . ncam is one of the most extensively studied ig - like domain - containing cams and play vital roles in brain development and function . major types of ncams including ncam-120 , ncam-140 and ncam-180 contain five ig - like domains and two fn domains in their extracellular regions ( maness and schachner , 2007 ) . studies on various ncams suggest the zipper - like models ( walmod et al . necl molecules , which play a vital role during synapse assembly , contain three ig - like domains in their extracellular regions . the crystal structure of the n - terminal ig - like domain of necl-1 showed homophilic interactions with a zipper - type arrangement ( dong et al . homophilic interaction would generate a zipper - like structure via cis - interaction with adjacent molecules ( aricescu and jones , 2007a ) . the crystal structures of ig1 - 4 domains from l1 homologs indicated different modes of homophilic interactions ( su et al . hemolin , an innate immunity - related molecule in insects , contains four ig - like domains ( ig1 - 4 ) that share 38% sequence homology with l1 ig1 - 4 domains ( su et al . the crystal structure determination of hemolin ig1 - 4 domains revealed a unique domain arrangement among the ig - like domain - containing cell surface proteins . previously described ig - like domain proteins such as cd4 have a linearly - arranged domain structure ( wu et al . in comparison , the ig1 - 4 domains of hemolin form a horseshoe structure where ig1 and ig2 fold back to interact with ig4 and ig3 , respectively ( figure 2 ) ( su et al . the domain interactions between ig1 and ig4 , and between ig2 and ig3 comprise an array of hydrophobic interactions , and residues involved in these interactions are highly conserved in the l1 family . however , based on the structure and previously described homophilic interactions in ig - like domain - containing molecules such as cd2 and cadherin , su et al . the chicken ncam axonin-1 has six ig - like domains and four fn domains ( zuellig et al . , 2000 the crystal structure of the ig1 - 4 domains of axonin-1 also showed a horseshoe shape , confirming the generality of the ig1 - 4 domain arrangement in hemolin ( figure 2b ) ( freigang et al . the inter - molecular interaction continues through the crystal lattice and forms a zipper - like structure . the c - termini of the dimer point in opposite directions so that facing membranes can be connected by the homophilic interaction . although tag-1 has a horseshoe structure similar to that axonin-1 , the zipper - like dimer interaction of axonin-1 was not observed in the tag-1 crystal . because the same regions of two tag-1 monomers are used in the dimeric interaction , the interaction can not be extended to form a zipper - like structure as proposed in the case of axonin-1 ( figure 2 ) . the drosophila protein dscam , which contains ten ig - like domains and six fn domains , mediates cell - cell interaction in brain cell networking ( schmucker et al . among the ten ig - like domains , ig2 , ig3 and ig7 have 12 , 47 and 33 alternative sequences , respectively , giving 18,048 potential derivatives ( schmucker et al . structural studies of dscam ig1 - 8 revealed the same dimeric interaction of the horseshoe region as that occurring in the the ig1 - 4 domain structures ( sawaya et al . the double - s conformation of dscam ig1 - 8 indicates the critical role of the domains outside the horseshoe region in the homophilic interactions of l1 homologs . because of the tight intermolecular interaction between two ig1 - 8 molecules , alteration in non - horseshoe domains can affect the orientation of inter - horseshoe regions and direct homophilic interactions mediated by non - horseshoe domains earlier experiments with recombinant l1 produced in escherichia coli indicated that ig2 was sufficient to induce trans - homophilic interaction and neurite elongation ( zhao et al . the bacteria - derived fn domain spontaneously formed trimeric and higher - order complexes , suggesting that fn domains were also necessary for l1 homophilic interactions in cis ( de angelis et al . later , domain - mapping experiments used eukaryotic cell - derived l1 constructs and found that the homophilic interactions involved more domains . the first four ig domains ( ig1 - 4 ) promoted homophilic cell adhesion and ig1 - 6 were necessary for optimal neurite outgrowth ( haspel et al . co - immunoprecipitation studies of truncated forms of l1 and endogenous full - length l1 showed that the l1 ectodomain ( l1/ecd ) and l1/ig1 - 4 interact homophilically in trans ( gouveia et al . after the determination of the crystal structure of ig1 - 4 domains of insect hemolin ( su et al . ethanol inhibits cell - cell adhesion mediated by l1 in neuroblastoma - glioma hybrid cells , cerebella granule cells , and human l1-transfected murine fibroblasts ( charness et al . , 1996 ; wilkemeyer and charness , 1998 ) , indicating that ethanol can disrupt homophilic interaction of l1 . in a modeled structure of the l1 ig1 - 4 based on the horseshoe structure of axonin-1 ( freigang et al . the n - terminal regions including glu33 and glu24-glu27 could not be modeled because the axonin-1 structure did not contain the region . alcohol binding to the pocket in the domain interface of glu33 and tyr418 likely affected the conformation of the horseshoe structure of l1 , preventing of ideal interaction between the horseshoe regions . the proximity of glu33 and tyr418 and their role in homophilic interaction were tested by cysteine substitution mutations ( the e33c / y418c mutant ) ( dou et al . although the modeling studies and mutation analyses convincingly present the potential alcohol - binding site in l1 , the shape of the binding pocket and the interactions between alcohol and l1 need to be defined by experimental structure studies . the role of the n - terminal residues including glu33 and glu24-glu27 also has to be resolved . the horseshoe structures found in l1 homologs ( su et al . however , the structures proposed different models for the homophilic interaction ; therefore , a physiologically relevant analysis was necessary . cryo - electron tomography of l1 addressed some of the questions surrounding the homophilic interaction mechanism ( he et al . the approximate size of the dots with tomogram resolution of 4 - 5 nm was consistent with the size of dscam horseshoe pairs of 60 70 80 and 60 60 100 ( pdb files 2v5r and 2v5s ) , indicating that the dots represented the paired dimer of l1 horseshoes . the discrete arrangement of dots in the area of membrane interaction suggested that l1 homophilic interaction did not occur as proposed in the zipper model . moreover , the domain - swapped multimerization model where domains 1 and 2 forms a horseshoe with domains 4 and 3 of the opposing membrane ( figure 2a ) , was not consistent with the l1 tomography observations . instead of the zipper or domain - swapped multimerization models , the discrete and regularly spaced dots fit the model of a dimer seen in the dscam ( meijers et al . carbohydrate - protein or carbohydrate - carbohydrate interactions often regulate homophilic interaction of cell surface proteins ( bucior and burger , 2004 ) . in an experiment to analyze the role of carbohydrates in l1 , he et al . , 2009 ) expressed l1 in mammalian and insect cells with attached complex n - linked and oligomannoside carbohydrates , respectively , on their membrane proteins . when the two types of l1 were reconstructed with liposomes , they produced similar dots between the two interacting liposomes ; however , the dots had different inter - dot distances : 100 for the mammalian cell - derived l1 and 140 for the insect cell - derived l1 ( figure 4 ) . these observations indicate that the carbohydrates on l1 play a pivotal role in determining the distances between horseshoe dimers , thus modulating homophilic interaction patterns ( figure 4 ) . the carbohydrate attachment sites of mammalian l1 are completely conserved , and there are no additional sites , further indicating the importance of carbohydrates in l1 function . the role of carbohydrates in homophilic cell adhesion interactions was shown in ncam ( kadmon et al . , 2001 ) recently , the first structure of the l1 family proteins was determined for neurofascin ( liu et al . , 2011 ) has a horseshoe structure as that of l1 homologs ( su et al . the residues involved in the domain interactions of neurofascin ig1 - 4 are conserved in other members of the l1 family ( liu et al . the two - fold symmetry - related dimeric interaction between the side regions of neurofascin ig2 produced an isolated dimer , consistent with the discrete inter - membrane dots observed in l1 tomograms ( he et al . because both neurofascin ig1 - 4 and tag-1 horseshoes exhibit the same suparmolecular -sheet formation , this mode of interaction may represent homophilic interaction of l1 family molecules and their homologs . the neurofascin ig1 - 4 structure was used to map pathological mutations in neurological disorders such as hsas , masa syndrome , and x - linked spastic paraplegia ( wong et al . , 1995 ; most mutations were found in the domain interfaces ( l120v , g121s , g370r , r184q / w , and y194c ) of the horseshoe formation or in the hydrophobic core of individual domains ( i179s , h210q , p240l , c264y , g268d , p333r , w335r , and l391p ) , confirming the importance of the horseshoe structure in l1 function . in contrast , one mutation ( i219 t ) corresponding to ile213 in neurofascin ig1 - 4 produced a neurite branching defect and was located in the hydrophobic cluster that mediates dimeric interaction in neurofascin ig1 - 4 crystals , supporting the model of homophilic interaction proposed by the crystal structures . the lack of homophilic interaction of l1 in a mouse line carrying a deletion of the ig5 domain ( itoh et al . , 2004 ) also indicates that involvement of domains other than the horseshoe region can affect the homophilic interaction of l1 family proteins . because homophilic interaction - mediated signal transduction activates cell growth signaling , it is believed that cancer progression also involves the homophilic interaction of l1 . various models for homophilic interaction have been proposed for cell adhesion proteins ( figure 2 ) , including zipper , domain - swapped monomer , domain - swapped multimer , two - fold symmetry related surface interaction and two - fold symmetry related edge interaction models based on the structures of axonin-1 , hemolin , dscam and tag-1/neurofascin , respectively . the two - fold symmetry - related surface or edge interaction and domain - swapped monomer models are consistent with the discrete and equally spaced dots observed in cryo - electron tomograms of l1 . interestingly , dimeric interaction via the edge of ig2 domain was observed in tag-1 and neurofascin , suggesting that the symmetry - related edge model may represent the homophilic interaction mechanism of l1 and its homologs . current understanding of homophilic interaction in l1 family molecules is based mainly on the crystal structures of the first four ig - like domains of a horseshoe structure . however , for a complete understanding of homophilic interactions , structural determination of l1 including other ig - like and fn domains is necessary .

the upcoming depletion of fossil carbon resources , like crude oil and natural gas , forces all oil- and gas - consuming industries to scout for suitable substitutes . pyrolysis oil is such resources , produced from organic material [ 1 , 2 ] . generally speaking , in the process of pyrolysis , molecules of higher molecular weight are thermally decomposed into smaller molecules in an oxygen - free atmosphere . during this process , a great variety of chemical reactions , such as elimination , cracking , isomerization , and rearrangements reactions , take place , leading to ultracomplex product mixtures . potential feedstocks are carbon - rich materials , for instance coal [ 513 ] , biomass [ 1417 ] or scrap tyres [ 18 , 19 ] . coal pyrolysates are mainly composed of aliphatic and aromatic compounds , with varying heteroatomic content ( i.e. , s , o , and n ) [ 912 ] . the hyphenation of gas chromatography ( gc ) with high - resolution mass spectrometry ( ms ) represents a method to analyze these complex mixtures in depth . in general , the volatile parts of oil are separated by means of gas chromatography and subsequently identified by mass spectrometry . this hyphenation can be applied , for example , to analyze pyrolysis products with different polarities [ 2123 ] . with this knowledge , much more efficient oil reprocessing and choice of the pyrolysis oil application site are possible . the contents of the pyrolysis oil can be ionized extensively by using a sensitive and selective ion source , like an atmospheric pressure chemical ionization ( apci ) source or an atmospheric pressure laser ionization ( apli ) source , coupled to an ultrahigh - resolving ( uhr ) mass analyzer , such as the fourier transform ion cyclotron resonance mass spectrometer ( ft - icr - ms ) . apci is a special form of chemical ionization ( ci ) , where a corona needle current regulated high voltage direct current ( hv - dc ) gradient is employed to generate a corona plasma . this plasma causes the formation of reactive species , which ionize the analyte molecules in a subsequent secondary ionization process . in comparison , apli is an ionization technique which uses pulsed laser light to produce molecular ions . both ionization methods are much more sensitive and selective than the electron ionization ( ei ) that is commonly used for gc - ms hyphenation [ 2433 ] . apci is mainly adopted to analyze compounds with higher polarity , such as phosphoric acid esters and carbamates . in contrast , apli is a technique to analyze preferably nonpolar compounds , like polycyclic aromatic hydrocarbons ( pahs ) [ 25 , 26 ] . according to the literature , a compound quantification with these soft ionization methods up to 5 fg is possible . in a previous paper , our research group was able to demonstrate the potential of the ft - icr - ms on pyrolysis oil characterization , using an electrospray ionization ( esi ) source . we could identify different single compounds and compound classes for a representative pyrolysis oil from a german brown coal . hence , a fractionation of compounds with the same exact molecular formula was not possible . theoretically , the hyphenation of gc with ft - icr - ms using a soft apci ion source should provide an opportunity to separate and detect molecules with the same exact monoisotopic mass with an ultrahigh mass resolution . the aim of this study is the development of a method for the analysis of a pyrolysis liquid from german brown coal by hyphenation of gc with ft - icr - ms using an apci source . a representative mixture of standard compounds ( rmsc ) was used for determination of optimum parameters for apci ion source and gc . results from apci were analyzed based on type , frequency , and intensity of assigned molecular formulas . the potential of the developed method , that is , soft ionization , isomeric separation , and ultrahigh mass detection , was demonstrated for the actual pyrolysis liquid sample . to the best of our knowledge , the applied gc - apci - ft - icr - ms analyses represent the first application of this analytical method for characterization of a pyrolysis liquid from a german brown coal . a pyrolysis liquid from german brown coal pyrolysis was used for applicability tests and will be denoted as s500 during the discussion . pulverized coal was filled into the tubular reactor and heated by an external oven to the designated temperature of 500c . the first trap was a packed bed and the second a solvent ( tetrahydrofuran ( thf ) ) filled trap . after each experiment , the first trap was flushed with solvent to release the oil . the extract was combined with the solvent from the second trap and the solvent was separated from the oil by distillation . the sample was weighted in an erlenmeyer flask and diluted with benzene to yield a mass concentration of 100 gl . hereinafter , the sample was shaken at 20c and 800 min for 30 min in a vortex shaker and centrifuged for 10 min at 10,000 g. supernatant solution was separated from the undissolved pellet and transferred to amber vials . reference compounds for the rmsc were weighted into amber glass vials and solved in methyl acetate to yield a mass concentration of 1 gl per compound . all reference compounds were purchased from sigma - aldrich either as analytical standard or as highest purity available . the rmsc was composed of aromatic ( e.g. , benzene ) and heterocyclic compounds ( e.g. , thiophene ) , with a varying number of alkyl groups . its detailed composition is shown in the supplementary material ( see section s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/5960916 ) . benzene and methyl acetate were also purchased from sigma - aldrich and both of 99.8% purity . the chromatographic separation of pyrolysis oil constituents was performed using bruker daltonics 450-gc with a varian cp-8400 autosampler . the gas chromatograph was equipped with a supelcowax 10 capillary column ( l = 30 m , d c = 0.25 mm , and d f = 0.25 m ) from supelco , which has a polyethylene glycol stationary phase . the gc temperature program started at a temperature of 100c , which was held for 2 min , then ramped to 250c , at a constant heating rate of 5 kmin , and finally held at this temperature for 30 min , resulting in an overall analysis time of 62 min . gc parameters gas flow , injection volume , and split - radio were optimized during the method development process , as presented hereinafter . a 15 t solarix ft - icr - ms from bruker daltonics was hyphenated , equipped with a bruker daltonics gc - apli / d source , and operated in negative ion mode , with a scan range from 46.06 to 500 da , a transient length of 2 m words , and 300 ms ion accumulation time . these settings resulted in a scan time of 1.003 s per single spectrum and hence a data acquisition rate of 0.997 hz . furthermore , the corona needle current and capillary voltage were also optimized during the method development process . not explicitly optimized parameters of the apci ionization source were chosen as follows : end - plate - offset : 0 v , dry gas flow : 1.5 lmin , dry gas temperature : 250c , nebulizer gas pressure : 3.0 bar , and vaporizer temperature : 350c . external calibration of the gc - apci - ft - icr - ms analyses was performed using a liquid standard that was composed of n - hexane , 250 pmol trifluoroacetic acid ( tfa ) , 50 pmol heptafluorobutyric acid ( hfba ) , and 16 mmol dichloromethane ( dcm ) . the standard solution was introduced using a thermo scientific dionex ultimate 3000 hplc pump at a constant flow rate of 80 lmin . creation and evaluation of the experimental design were performed using statgraphics centurion xvi . however , an experimental design was only necessary for the optimization of the apci parameters that were varied simultaneously . every single optimization experiment was analyzed according to the intensity values of specific compounds of the rmsc that were found in the extracted ion chromatograms ( eics ) . for evaluating the optimization , the following mass traces were examined : ( m / z ) = 93.034588 , ( m / z ) = 107.050238 , ( m / z ) = 109.029503 , ( m / z ) = 121.065888 , ( m / z ) = 123.045153 , ( m / z ) = 151.040068 , ( m / z ) = 151.076453 , ( m / z ) = 163.076453 , and ( m / z ) = 165.092103 . we assumed all investigated compounds to be present as [ m h ] molecular ions . this assumption is primarily based on the high mass accuracy of the ft - icr - ms , which supports the general existence of the [ m h ] molecular ions with a high degree of certainty . in case of the apci optimization , the intensity sum of all investigated peaks in the specific eics was used as validation factor . in comparison to the apci parameter optimization the validation of gc optimization experiments was conducted by comparing peak intensities , peak areas , and observed peak separation of the single compounds in the above described mass traces . the data sets were calibrated and processed with an automated in - house tool , using bruker daltonics software data analysis ( 4.1 sp1 ) . its main task was the calibration and thus ( m / z ) correction of every acquired mass spectrum in each time segment . as described before , calibration was performed using a liquid standard , composed of n - hexane , tfa , hfba , and dcm . these standard compounds themselves formed characteristic molecular ions during the ionization process that were present throughout the analysis and used as reference . this resulted in a processing output , where for each time segment peak and molecular formula lists were created . more precisely , the peak lists contained information about the detected analyte signals in the specific time section with their ( m / z)-ratios , intensities , resolution , and signal - to - noise - ratios ( s / n ) . compared to that , molecular formula lists comprised molecular formula suggestions based on the detected ( m / z)-ratios . application of this data processing method reduced the amount of data from approximately 77.2 gb ( unprocessed raw data ) to 55.3 mb ( entirety of output files ) . in the next processing step , the acquired molecular formula lists were processed with in - house matlab scripts ; that is , the peak and molecular formula lists were loaded into matlab r2015b as .csv - files and different figures for the experiment evaluation were created . by filtering out all calibrant signals that were present throughout the analysis , a flow sheet of the conducted data processing is visualized in figure 1 . for further information on the used molecular ions for data calibration , screen shots of the calibration and processing tool and its outcome files are presented in the supplementary material ( see section s3 ) as well . the optimization of gc and apci source parameters was conducted using a mix of standard compounds , representative for aromatic pyrolysis oil constituents ( rmsc ) . main goal of the gc optimization was to improve three specific parameters : gas flow , injection volume , and split - ratio . their values were varied and optimized in case of the gas flow from 0.6 to 1.2 mlmin in 0.2 mlmin steps , in case of the injection volume from 1.0 to 2.5 l in 0.5 l steps , and in case of the split - ratio from splitless injection to 1 : 20 in five single experiments ( splitless , 1 : 2 , 1 : 5 , 1 : 10 , and 1 : 20 ) . the eics of the rmsc were evaluated in terms of signal intensity to determine the ideal gc parameters . furthermore , the baseline separation of eic peaks of compounds with the same ( m / z)-ratio ( e.g. , o- and m - cresol ( ( m / z ) = 107.050238 ) ) and the areas under the eic peaks were analyzed by integration , using a gaussian filter for peak shape fitting . the mass traces used for experiment evaluation of the gc optimization were chosen as described before in the experimental section . according to the applied analysis , the following gc parameters were determined as ideal : gas flow : 0.8 mlmin , injection volume : 1.0 l , and splitless injection . considering the intensity values , baseline peak separation , and peak areas of the single components in the eics , these parameters yielded the highest intensities and peak areas and thus were applied to the actual sample investigation . the optimization of the apci parameters was performed similar to the gc optimization , using the same mix of standard compounds ( rmsc ) . the main goal of this optimization was to improve two parameters of the apci source simultaneously : corona needle current and capillary voltage . the corona needle current is essential to generate the plasma , which itself is required to induce the ionization process . capillary voltage is an electrical potential that is applied to transport the produced ions to the skimmer and ion funnel . their values were varied in case of the corona needle current from 10 to 15 a in 2.5 a steps and in case of the capillary voltage from + 100 v to + 1100 v in 500 v steps . analogous to a 3-level full factorial design , nine randomized experiments were conducted . according to the optimization , the following ideal values for corona needle current and capillary voltage were determined : corona needle current 15 a and capillary voltage + 100 v. the highest overall intensity sum values as well as the highest single intensity values for most of the eic peaks of the individual components were obtained at these parameters . altogether , the optimization of gc and apci parameters helped to shape an uhr analysis method that was applied hereinafter to an actual pyrolysis liquid sample , namely , s500 . the reproducibility of conducted experiments was investigated by analyzing pyrolysis oil s500 three times successively . in seven different eics , more precisely , following mass traces were evaluated : ( m / z ) = 93.034588 , ( m / z ) = 107.050238 , ( m / z ) = 109.029503 , ( m / z ) = 121.065888 , ( m / z ) = 123.045153 , ( m / z ) = 135.081539 , and ( m / z ) = 151.040068 . peak shape fitted peaks were analyzed based on peak intensity , peak area , and s / n . for each assessment parameter , a mean value of the three sample measurements was computed , separately for each peak and overall for all investigated peaks as well . hereinafter , the absolute and relative standard deviation ( rsd ) of every measurement , according to the deviation from calculated mean values , was determined . thereby , an averaged rsd for all examined peaks of 15.68% in terms of peak intensity ( range of 0.3045.33% ) , 24.47% in terms of peak area ( range of 0.6995.20% ) , and 19.49% in terms of s / in particular , rsd values of 0.30% at ( m / z ) = 135.081539 in terms of signal intensity or 0.69% at ( m / z ) = 151.040068 in terms of peak area demonstrate the good qualitative reproducibility . nonetheless , reproducible quantitative statements are not feasible at this point , due to the high variance in received rsd values , particularly in terms of peak area and s / n . better rsd values and minimized rsd variances could be achieved if experiments with added internal standards are conducted . since ft - icr - ms is a high - resolving analysis technique , it allows very accurate mass measurements and molecular formulas can be derived from the exact masses . the number of potential compounds for one mass trace depends on mass accuracy and the elements ( and isotopes ) taken into account . in conjunction with gc , it was possible to demonstrate a significant advantage of the gc - ms hyphenation in comparison to isolated ms experiments , that is , isomeric separation . two eic examples , exhibiting a successful isomeric separation , are illustrated in figures 2 and 3 . actual sample signals are presented in blue and corresponding gaussian peak shape fitted signals in red . the first example , illustrated in figure 2 , presents the eic at ( m / z ) = 107.050238 . according to our mix of standard compounds , rmsc , this ( m / z)-ratio refers to two different methylphenols ( cresols ) , o- and m - cresol . both peaks were baseline separated . a second example for the successful isomeric separation of liquid oil compounds was observed at ( m / z ) = 135.081539 ( see figure 3 ) . this ( m / z)-ratio can be assigned to propylphenols ( n- and iso-(cumenol ) ) , ethylmethylphenol , and/or trimethylphenols , such as mesitol ( 2,4,6-trimethylphenol ) . altogether , we observed nine different peaks and hence nine compounds in this mass trace . considering the presented eics , the developed gc - apci - ft - icr - ms method finally gives a possibility to separate compounds with the same exact molecular mass and thus the same exact ( m / z)-ratio and detect them with an ultrahigh - resolving mass spectrometric analyzer . therefore , the established gc - apci - ft - icr - ms hyphenation can be considered an uhr analysis method . figure 4 shows the spectrochromatogram of the gc - apci - ms analysis of sample s500 , during the overall analysis time ( in min ) and exhibits ( m / z)-ratios in the range of 90300 da . observed relative intensity values are illustrated as color - coded ones ( white : low intensity , red : medium intensity , and black : high intensity ) . according to figure 4 , we could verify different compounds of the rmsc in this plot , for example , phenol ( ( m / z ) = 93.034588 ) , o- and m - cresol ( ( m / z ) = 107.050238 ) , ethyl- and dimethylphenols ( ( m / z ) = 121.065888 ) , vanillin ( ( m / z ) = 151.040068 ) , and different eugenols ( cis- and trans - isoeugenol , eugenol ) ( ( m / z ) = 163.076453 ) . molecular ions , which were detected throughout the analysis , can be mainly attributed to additionally added reference compounds ( n - hexane , tfa , hfba , and dcm ) . also ubiquitous signals , whose m / z values correspond to analytes of the pyrolysis liquid , were detected over the entire analysis time . for instance , a compound was observed at ( m / z ) = 255.232954 , that was present in a s / n range slightly higher than the corresponding threshold value ( s / n = 5 ) . nonetheless , although an omnipresent background was noticed at these mass traces , clear peak maxima resulted when corresponding compounds eluted . furthermore , we observed deprotonated molecules , that varied in terms of ( m / z)-ratios by 14.015650 da . this refers to an increasing number of ch2 groups and hence longer alkyl chains , resulting in a homologous series of different compounds . we were able to identify six different homologous series of compound classes , according to the observed ( m / z)-ratios : saturated monocarboxylic acids ( alkyl carboxylic acids ) , unsaturated monocarboxylic acids ( alkylene carboxylic acids ) , dicarboxylic acids , alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols . corresponding alkyl dihydroxy benzenes and alkoxy alkyl phenols have same exact molecular masses and thus same exact ( m / z)-ratios , resulting in a detection in the same eics . figure 5 illustrates the ( m / z)-ratios and retention times ( in min ) of all allocated saturated and unsaturated monocarboxylic acids and dicarboxylic acids . as shown in this figure , we could discern 15 alkyl carboxylic acids with a chain length from c4 to c18 , 11 alkylene carboxylic acids with a chain length from c6 to c16 , and six dicarboxylic acids with a chain length from c5 to c10 . table 1 presents an overview of all assigned saturated and unsaturated monocarboxylic acids and dicarboxylic acids , exhibiting assigned compound classes , assigned compounds , assigned number ( see figure 5 ) , retention time t r in min ( represented as confidence interval ) , theoretical calculated ( m / z)-ratios , observed ( m / z)-ratios , ( m / z ) errors in mda , and ( m / z ) errors in ppm . calculated ( m / z ) errors varied in case of the alkyl carboxylic acids from 0.096 to 0.427 ppm ( mean value : 0.210 ppm ) , the alkylene carboxylic acids from 0.021 to 0.367 ppm ( mean value : 0.165 ppm ) , and the dicarboxylic acids from 0.038 to 0.107 ppm ( mean value : 0.070 ppm ) . hence , the applied assignment of observed ( m / z)-ratios to the described compounds is most accurate for the five dicarboxylic acids but also quite accurate for the other two compound classes . figure 6 illustrates the observed ( m / z)-ratios and retention times ( in min ) for the identified formulas of alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols . we could presumably identify 50 different alkyl phenols in nine distinct mass traces and 24 different alkyl dihydroxy benzenes / alkoxy alkyl phenols in five distinct eics . in accordance with the compounds of the rmsc , we were able to identify phenol ( a ) at ( m / z ) = 93.034588 , o - cresol ( b ) and m - cresol ( c ) at ( m / z ) = 107.050238 , and two dimethyl- and ethylphenols , 2,6-dimethylphenol ( d ) , and 2-ethylphenol ( e ) at ( m / z ) = 121.065888 . higher ( m / z ) values , increasing by 14.015650 da ( mass of a ch2 group ) , indicating longer alkyl chains at the phenolic core , for example , trimethylphenol or tetramethylphenol , are plausible . compounds of the rmsc were also verified for alkyl dihydroxy benzenes and alkoxy alkyl phenols as well . for instance , peaks at ( m / z ) = 109.029503 can be identified as hydroquinone ( f ) , ( m / z ) = 123.045153 as guaiacol ( g ) , 4-methylcatechol ( h ) , and 2-methylhydroquinone ( i ) , ( m / z ) = 137.060803 as 4-methylguaiacol ( j ) , ( m / z ) = 151.076453 as 4-ethylguaiacol ( k ) , and ( m / z ) = 165.092103 as 4-propylguaiacol an overview , such as table 1 for the carboxylic acids , is not presented in the main part of this paper for alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols , due to the high number of detected peaks and assignable compounds in the specific mass traces . carboxylic acids were always present in the eics with just one peak and hence could be evaluated more easily . however , the overview for alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols can be found in the supplementary material ( see section s4 ) . compared to pyrolysis liquid sample s500 , retention times of alkyl dihydroxy benzenes , for example , 4-methylcatechol and 2-methylhydroquinone , and alkoxy alkyl phenols , for example , guaiacol and 4-methylguaiacol , were consistent with respect to the retention times of the rmsc . as shown in figure 6 , it was possible to separate and identify alkyl dihydroxy benzenes and alkoxy alkyl benzenes , regardless of their same exact molecular mass and ( m / z)-ratio , by comparing their retention times . additionally , compound clustering of alkyl dihydroxy benzenes and alkoxy alkyl phenols was observed , below and above a retention time of 32 min . alkyl dihydroxy benzenes exhibit stronger interactions with the polar stationary phase than alkoxy alkyl phenols , because of the increased polarity of alkyl dihydroxy benzenes due to their additional second hydroxy group . hence , alkoxy alkyl phenols elute earlier than the corresponding alkyl dihydroxy benzenes . for a better assignment of detected compounds , apart from verified compounds of the rmsc , the concept of double bond equivalent ( dbe ) [ 13 , 33 , 34 , 36 ] was applied . dbe is calculated using the molecular formula , while simplifying the manual search for potential compound structures . the dbe increases by one , if , for example , a double bond , a ring , a carbonyl group , or a carboxylic group is introduced into a structure . figure 7 shows the dbe plotted against the number of carbon atoms ( n c ) for eight different compound classes over the entire analysis time . reference compounds that were ubiquitous , that is , calibrant signals , were filtered out to receive the illustrated figure . in general , predominantly oxygen- and/or sulphur - containing organic species were discovered in liquid sample s500 , and hence only n c - dbe - plots of compound classes containing these elements are illustrated in figure 7 . nitrogen - containing compounds were not observed , due to the applied negative ion mode and thus will not be evaluated further . the presented postulated structures are affiliated to reactions and structural changes of the raw coal during the pyrolysis process , according to existing knowledge about this class of substances . compounds of the rmsc were found in the n c - dbe - plot of compound class cchho1 , such as phenol ( a ) ( n c = 6 , dbe = 4 ) , o- and m - cresol ( b , c ) ( n c = 7 , dbe = 4 ) , and 2,6-dimethylphenol ( d ) and 2-ethylphenol ( e ) ( n c = 8 , dbe = 4 ) ( see also figure 6 ) . higher dbe values might refer to phenolic structures with annulated cycles , for example , cycloalkanes and cycloalkenes . dbe values of 1 in compound class cchho2 match to assigned alkyl carboxylic acids and their corresponding esters as well . besides previously determined monocarboxylic acids with n c 18 ( see table 1 ) , also compounds such as esters of cerotic acid ( c26 ) or montanic acid ( c28 ) were present . these compounds normally appear in montan wax ; that is , they are constituents of the waxy contents of brown coal . dbe values of 2 in compound class cchho2 were assigned to alkylene carboxylic acids and their esters , where compounds with n c values between 5 and 22 were verifiable . dicarboxylic acids and their corresponding esters were assigned to compound class cchho4 at a dbe of 2 with n c values between 5 and 20 . furthermore , alkyl dihydroxy benzenes and alkoxyl alkyl phenols were assigned to compound class cchho2 at a dbe of 4 , for example , n c of 6 matches to hydroquinone ( f ) , n c of 7 to guaiacol ( g ) , 4-methylcatechol ( h ) , and 2-methylhydroquinone ( i ) , n c of 8 to 4-methylguaiacol ( j ) , n c of 9 to 4-ethylguaiacol ( k ) , and n c of 10 to 4-propylguaiacol ( l ) ( see also figure 6 ) . sulphur - containing compounds were assigned to the n c - dbe - plots of compound classes cchhs1 , cchho1s1 , and cchho2s1 . according to the plots of these compound classes , presumed compounds are alkyl derived thiophenes ( cchhs1 , dbe = 3 ) , mono carbonyl derived thiophenes ( cchho1s1 , dbe = 4 ) , and benzothiophenes ( cchho1s1 , dbe = 7 ) , as well as dihydroxy derived benzothiophenes ( cchho2s1 , dbe = 6 ) and mono carboxyl derived thiophenes ( cchho2s1 , dbe = 4 ) and benzothiophenes ( cchho2s1 , dbe = 7 ) . retention time - depending statements are possible by using time separated n c - dbe - plots . the n c - dbe - t - plots of compound classes cchho1 , cchho2 , cchho4 , and cchho2s1 are presented exemplarily in figure 8 . the time - dependent n c - dbe - plots of other evaluated compound classes are presented and interpreted in the supplementary material ( see section s5 ) . the previously described filter procedure for the creation of n c - dbe - plots was also applied to generate the n c - dbe - t - plots . in the illustrated n c - dbe - t - plots of compound class cchho1 , no increase of n c and dbe values with advancing analysis time was observed . as stated before , in this compound class phenol ( a ) and other alkyl phenols , like o- and m - cresol ( b , c ) , can be found at n c = 6 , n c = 7 and dbe = 4 . these assigned compounds were mainly detected in a time range between 20 and 30 min . compared to that , assigned saturated ( dbe = 1 ) and unsaturated monocarboxylic acids ( dbe = 2 ) and their corresponding esters in compound class cchho2 eluted throughout the analysis ( 060 min ) . most likely , this could be attributed to the wider range of detected n c values from 5 to 29 for saturated monocarboxylic acids and 5 to 22 for unsaturated monocarboxylic acids . identified saturated dicarboxylic acids ( dbe = 2 ) in class cchho4 were detected between 0 and 40 min . in general , a slight increase of observed n c values with increasing analysis time was noticed in this class . time - depending n c - dbe - plots of compound class cchho2s1 demonstrated an elution of assumed dihydroxy substituted benzothiophenic molecules ( dbe = 6 ) between 20 and 60 min . in comparison , supposed thiophenes and benzothiophenes , containing a single carboxyl group ( dbe = 4 and dbe = 7 ) , were mainly detected in the second analysis half , that is , from 2050 min and 3060 min . the n c - dbe - t - plots also illustrate the presence of ubiquitous signals at certain ( m / z ) values . for instance , in class cchho2 , a signal leading to an assigned molecular formula with n c = 6 and dbe = 5 was detected over the entire analysis time . analogous to the spectrochromatogram ( see figure 4 ) , varying intensity values were observed . the intensity of the sample compound is almost equal between 0 and 20 min , shows a maximum in the two subsequent time segments , and decreases thereafter until the end of the analysis . hence , an approximate peak maximum identification is also possible from this type of illustration . in this study , the hyphenation of gas chromatography ( gc ) with fourier transform ion cyclotron resonance mass spectrometry ( ft - icr - ms ) , using an atmospheric pressure chemical ionization ( apci ) source , was applied to pyrolysis oil , obtained at a pyrolysis temperature of 500c from a german brown coal . the method development process included optimization of specific gc and apci parameters , for example , by using an 3-level full factorial design . furthermore , the processing of the obtained data was an essential part of the method development . considering all applied method optimization steps , this method allows separating different isomers of a molecule and detecting them with ultrahigh ( m / z ) resolution . this isomer separation was exemplified , as shown in figure 2 , for the separation of two cresol isomers . furthermore , it was possible to demonstrate the high analytical potential of the applied gc - apci - ft - icr - ms hyphenation by verifying different homologous series in the liquid sample analysis . observed compound classes , increasing by the number of ch2 groups , were alkyl carboxylic acids , alkylene carboxylic acids , dicarboxylic acids , alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols . for structural statements , a visualization of double bond equivalent ( dbe ) versus carbon number ( n c ) was applied , illustrated for the entire analysis time ( see figure 7 ) and for six different time segments ( see figure 8) as well . with the help of these plots , the gc retention time - depending appearance of specific compounds could be visualized . mainly saturated and unsaturated monocarboxylic acids , as well as dicarboxylic acids , thiophenes , benzothiophenes , cycloalkanes , cycloalkenes , and pah analogous structures , partially derived by oxygen- and sulphur - containing groups , were assumed and in parts identified . due to the polar supelcowax 10 gc column , a clear separation of molecules of higher polarity was observed , such as saturated and unsaturated monocarboxylic acids , saturated dicarboxylic acids , and alkyl phenols . in particular , the observed separation of different carboxylic acid compounds and compound classes is economically interesting , considering their various potential applications . according to the assigned compounds in the single eics , most molecules were detected as single deprotonated molecules ( [ m h ] ) ; that is , no analyte fragmentation was obvious , which is one of the advantages of the apci ion source . compared to that , an ionization with other commonly used techniques for gc - ms hyphenation , such as ei , normally leads to an intense fragmentation of sample molecules . thus , the developed gc - apci - ft - icr - ms hyphenation makes it easier to assign a molecular formula to the detected molecular ion . in contrast , gc - ei - ms simplifies the clarification of the exact structure of a compound , due to the higher number of detectable fragments with lower ( m / z)-ratios . hence , the combination of results from the developed gc - apci - ft - icr - ms hyphenation with results from gc - ei - ms hyphenation should increase the knowledge about pyrolysis oil to a greater extent . moreover , an improved gc separation , for example , with an ionic liquid gc column or a high - temperature gc column , will also help to separate nonpolar and polar compounds similarly by using a higher gc maximum temperature . furthermore , experiments involving collision - induced dissociation ( cid ) would allow the identification of specific compound structures or typical building units [ 4042 ] , especially of those contained in larger molecules .

pyrolysis oil from the slow pyrolysis of german brown coal from schningen , obtained at a temperature of 500c , was separated and analyzed using hyphenation of gas chromatography with an atmospheric pressure chemical ionization source operated in negative ion mode and fourier transform ion cyclotron resonance mass spectrometry ( gc - apci - ft - icr - ms ) . development of this ultrahigh - resolving analysis method is described , that is , optimization of specific gc and apci parameters and performed data processing . the advantages of gc - apci - ft - icr - ms hyphenation , for example , soft ionization , ultrahigh - resolving detection , and most important isomer separation , were demonstrated for the sample liquid . for instance , it was possible to separate and identify nine different propylphenol , ethylmethylphenol , and trimethylphenol isomers . furthermore , homologous series of different acids , for example , alkyl and alkylene carboxylic acids , were verified , as well as homologous series of alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols .

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

the hyphenation of gas chromatography ( gc ) with high - resolution mass spectrometry ( ms ) represents a method to analyze these complex mixtures in depth . in general , the volatile parts of oil are separated by means of gas chromatography and subsequently identified by mass spectrometry . this hyphenation can be applied , for example , to analyze pyrolysis products with different polarities [ 2123 ] . the contents of the pyrolysis oil can be ionized extensively by using a sensitive and selective ion source , like an atmospheric pressure chemical ionization ( apci ) source or an atmospheric pressure laser ionization ( apli ) source , coupled to an ultrahigh - resolving ( uhr ) mass analyzer , such as the fourier transform ion cyclotron resonance mass spectrometer ( ft - icr - ms ) . both ionization methods are much more sensitive and selective than the electron ionization ( ei ) that is commonly used for gc - ms hyphenation [ 2433 ] . in a previous paper , our research group was able to demonstrate the potential of the ft - icr - ms on pyrolysis oil characterization , using an electrospray ionization ( esi ) source . we could identify different single compounds and compound classes for a representative pyrolysis oil from a german brown coal . theoretically , the hyphenation of gc with ft - icr - ms using a soft apci ion source should provide an opportunity to separate and detect molecules with the same exact monoisotopic mass with an ultrahigh mass resolution . the aim of this study is the development of a method for the analysis of a pyrolysis liquid from german brown coal by hyphenation of gc with ft - icr - ms using an apci source . the potential of the developed method , that is , soft ionization , isomeric separation , and ultrahigh mass detection , was demonstrated for the actual pyrolysis liquid sample . to the best of our knowledge , the applied gc - apci - ft - icr - ms analyses represent the first application of this analytical method for characterization of a pyrolysis liquid from a german brown coal . a pyrolysis liquid from german brown coal pyrolysis was used for applicability tests and will be denoted as s500 during the discussion . pulverized coal was filled into the tubular reactor and heated by an external oven to the designated temperature of 500c . hereinafter , the sample was shaken at 20c and 800 min for 30 min in a vortex shaker and centrifuged for 10 min at 10,000 g. supernatant solution was separated from the undissolved pellet and transferred to amber vials . the gc temperature program started at a temperature of 100c , which was held for 2 min , then ramped to 250c , at a constant heating rate of 5 kmin , and finally held at this temperature for 30 min , resulting in an overall analysis time of 62 min . a 15 t solarix ft - icr - ms from bruker daltonics was hyphenated , equipped with a bruker daltonics gc - apli / d source , and operated in negative ion mode , with a scan range from 46.06 to 500 da , a transient length of 2 m words , and 300 ms ion accumulation time . not explicitly optimized parameters of the apci ionization source were chosen as follows : end - plate - offset : 0 v , dry gas flow : 1.5 lmin , dry gas temperature : 250c , nebulizer gas pressure : 3.0 bar , and vaporizer temperature : 350c . external calibration of the gc - apci - ft - icr - ms analyses was performed using a liquid standard that was composed of n - hexane , 250 pmol trifluoroacetic acid ( tfa ) , 50 pmol heptafluorobutyric acid ( hfba ) , and 16 mmol dichloromethane ( dcm ) . however , an experimental design was only necessary for the optimization of the apci parameters that were varied simultaneously . this assumption is primarily based on the high mass accuracy of the ft - icr - ms , which supports the general existence of the [ m h ] molecular ions with a high degree of certainty . in comparison to the apci parameter optimization the validation of gc optimization experiments was conducted by comparing peak intensities , peak areas , and observed peak separation of the single compounds in the above described mass traces . in the next processing step , the acquired molecular formula lists were processed with in - house matlab scripts ; that is , the peak and molecular formula lists were loaded into matlab r2015b as .csv - files and different figures for the experiment evaluation were created . the optimization of gc and apci source parameters was conducted using a mix of standard compounds , representative for aromatic pyrolysis oil constituents ( rmsc ) . considering the intensity values , baseline peak separation , and peak areas of the single components in the eics , these parameters yielded the highest intensities and peak areas and thus were applied to the actual sample investigation . the optimization of the apci parameters was performed similar to the gc optimization , using the same mix of standard compounds ( rmsc ) . according to the optimization , the following ideal values for corona needle current and capillary voltage were determined : corona needle current 15 a and capillary voltage + 100 v. the highest overall intensity sum values as well as the highest single intensity values for most of the eic peaks of the individual components were obtained at these parameters . altogether , the optimization of gc and apci parameters helped to shape an uhr analysis method that was applied hereinafter to an actual pyrolysis liquid sample , namely , s500 . since ft - icr - ms is a high - resolving analysis technique , it allows very accurate mass measurements and molecular formulas can be derived from the exact masses . in conjunction with gc , it was possible to demonstrate a significant advantage of the gc - ms hyphenation in comparison to isolated ms experiments , that is , isomeric separation . this ( m / z)-ratio can be assigned to propylphenols ( n- and iso-(cumenol ) ) , ethylmethylphenol , and/or trimethylphenols , such as mesitol ( 2,4,6-trimethylphenol ) . considering the presented eics , the developed gc - apci - ft - icr - ms method finally gives a possibility to separate compounds with the same exact molecular mass and thus the same exact ( m / z)-ratio and detect them with an ultrahigh - resolving mass spectrometric analyzer . therefore , the established gc - apci - ft - icr - ms hyphenation can be considered an uhr analysis method . figure 4 shows the spectrochromatogram of the gc - apci - ms analysis of sample s500 , during the overall analysis time ( in min ) and exhibits ( m / z)-ratios in the range of 90300 da . according to figure 4 , we could verify different compounds of the rmsc in this plot , for example , phenol ( ( m / z ) = 93.034588 ) , o- and m - cresol ( ( m / z ) = 107.050238 ) , ethyl- and dimethylphenols ( ( m / z ) = 121.065888 ) , vanillin ( ( m / z ) = 151.040068 ) , and different eugenols ( cis- and trans - isoeugenol , eugenol ) ( ( m / z ) = 163.076453 ) . for instance , a compound was observed at ( m / z ) = 255.232954 , that was present in a s / n range slightly higher than the corresponding threshold value ( s / n = 5 ) . this refers to an increasing number of ch2 groups and hence longer alkyl chains , resulting in a homologous series of different compounds . we were able to identify six different homologous series of compound classes , according to the observed ( m / z)-ratios : saturated monocarboxylic acids ( alkyl carboxylic acids ) , unsaturated monocarboxylic acids ( alkylene carboxylic acids ) , dicarboxylic acids , alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols . corresponding alkyl dihydroxy benzenes and alkoxy alkyl phenols have same exact molecular masses and thus same exact ( m / z)-ratios , resulting in a detection in the same eics . as shown in this figure , we could discern 15 alkyl carboxylic acids with a chain length from c4 to c18 , 11 alkylene carboxylic acids with a chain length from c6 to c16 , and six dicarboxylic acids with a chain length from c5 to c10 . table 1 presents an overview of all assigned saturated and unsaturated monocarboxylic acids and dicarboxylic acids , exhibiting assigned compound classes , assigned compounds , assigned number ( see figure 5 ) , retention time t r in min ( represented as confidence interval ) , theoretical calculated ( m / z)-ratios , observed ( m / z)-ratios , ( m / z ) errors in mda , and ( m / z ) errors in ppm . calculated ( m / z ) errors varied in case of the alkyl carboxylic acids from 0.096 to 0.427 ppm ( mean value : 0.210 ppm ) , the alkylene carboxylic acids from 0.021 to 0.367 ppm ( mean value : 0.165 ppm ) , and the dicarboxylic acids from 0.038 to 0.107 ppm ( mean value : 0.070 ppm ) . figure 6 illustrates the observed ( m / z)-ratios and retention times ( in min ) for the identified formulas of alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols . we could presumably identify 50 different alkyl phenols in nine distinct mass traces and 24 different alkyl dihydroxy benzenes / alkoxy alkyl phenols in five distinct eics . higher ( m / z ) values , increasing by 14.015650 da ( mass of a ch2 group ) , indicating longer alkyl chains at the phenolic core , for example , trimethylphenol or tetramethylphenol , are plausible . compounds of the rmsc were also verified for alkyl dihydroxy benzenes and alkoxy alkyl phenols as well . for instance , peaks at ( m / z ) = 109.029503 can be identified as hydroquinone ( f ) , ( m / z ) = 123.045153 as guaiacol ( g ) , 4-methylcatechol ( h ) , and 2-methylhydroquinone ( i ) , ( m / z ) = 137.060803 as 4-methylguaiacol ( j ) , ( m / z ) = 151.076453 as 4-ethylguaiacol ( k ) , and ( m / z ) = 165.092103 as 4-propylguaiacol an overview , such as table 1 for the carboxylic acids , is not presented in the main part of this paper for alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols , due to the high number of detected peaks and assignable compounds in the specific mass traces . however , the overview for alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols can be found in the supplementary material ( see section s4 ) . compared to pyrolysis liquid sample s500 , retention times of alkyl dihydroxy benzenes , for example , 4-methylcatechol and 2-methylhydroquinone , and alkoxy alkyl phenols , for example , guaiacol and 4-methylguaiacol , were consistent with respect to the retention times of the rmsc . as shown in figure 6 , it was possible to separate and identify alkyl dihydroxy benzenes and alkoxy alkyl benzenes , regardless of their same exact molecular mass and ( m / z)-ratio , by comparing their retention times . additionally , compound clustering of alkyl dihydroxy benzenes and alkoxy alkyl phenols was observed , below and above a retention time of 32 min . alkyl dihydroxy benzenes exhibit stronger interactions with the polar stationary phase than alkoxy alkyl phenols , because of the increased polarity of alkyl dihydroxy benzenes due to their additional second hydroxy group . hence , alkoxy alkyl phenols elute earlier than the corresponding alkyl dihydroxy benzenes . the dbe increases by one , if , for example , a double bond , a ring , a carbonyl group , or a carboxylic group is introduced into a structure . reference compounds that were ubiquitous , that is , calibrant signals , were filtered out to receive the illustrated figure . nitrogen - containing compounds were not observed , due to the applied negative ion mode and thus will not be evaluated further . higher dbe values might refer to phenolic structures with annulated cycles , for example , cycloalkanes and cycloalkenes . dbe values of 1 in compound class cchho2 match to assigned alkyl carboxylic acids and their corresponding esters as well . these compounds normally appear in montan wax ; that is , they are constituents of the waxy contents of brown coal . dbe values of 2 in compound class cchho2 were assigned to alkylene carboxylic acids and their esters , where compounds with n c values between 5 and 22 were verifiable . furthermore , alkyl dihydroxy benzenes and alkoxyl alkyl phenols were assigned to compound class cchho2 at a dbe of 4 , for example , n c of 6 matches to hydroquinone ( f ) , n c of 7 to guaiacol ( g ) , 4-methylcatechol ( h ) , and 2-methylhydroquinone ( i ) , n c of 8 to 4-methylguaiacol ( j ) , n c of 9 to 4-ethylguaiacol ( k ) , and n c of 10 to 4-propylguaiacol ( l ) ( see also figure 6 ) . according to the plots of these compound classes , presumed compounds are alkyl derived thiophenes ( cchhs1 , dbe = 3 ) , mono carbonyl derived thiophenes ( cchho1s1 , dbe = 4 ) , and benzothiophenes ( cchho1s1 , dbe = 7 ) , as well as dihydroxy derived benzothiophenes ( cchho2s1 , dbe = 6 ) and mono carboxyl derived thiophenes ( cchho2s1 , dbe = 4 ) and benzothiophenes ( cchho2s1 , dbe = 7 ) . in comparison , supposed thiophenes and benzothiophenes , containing a single carboxyl group ( dbe = 4 and dbe = 7 ) , were mainly detected in the second analysis half , that is , from 2050 min and 3060 min . for instance , in class cchho2 , a signal leading to an assigned molecular formula with n c = 6 and dbe = 5 was detected over the entire analysis time . the intensity of the sample compound is almost equal between 0 and 20 min , shows a maximum in the two subsequent time segments , and decreases thereafter until the end of the analysis . in this study , the hyphenation of gas chromatography ( gc ) with fourier transform ion cyclotron resonance mass spectrometry ( ft - icr - ms ) , using an atmospheric pressure chemical ionization ( apci ) source , was applied to pyrolysis oil , obtained at a pyrolysis temperature of 500c from a german brown coal . the method development process included optimization of specific gc and apci parameters , for example , by using an 3-level full factorial design . this isomer separation was exemplified , as shown in figure 2 , for the separation of two cresol isomers . furthermore , it was possible to demonstrate the high analytical potential of the applied gc - apci - ft - icr - ms hyphenation by verifying different homologous series in the liquid sample analysis . observed compound classes , increasing by the number of ch2 groups , were alkyl carboxylic acids , alkylene carboxylic acids , dicarboxylic acids , alkyl phenols , alkyl dihydroxy benzenes , and alkoxy alkyl phenols . for structural statements , a visualization of double bond equivalent ( dbe ) versus carbon number ( n c ) was applied , illustrated for the entire analysis time ( see figure 7 ) and for six different time segments ( see figure 8) as well . mainly saturated and unsaturated monocarboxylic acids , as well as dicarboxylic acids , thiophenes , benzothiophenes , cycloalkanes , cycloalkenes , and pah analogous structures , partially derived by oxygen- and sulphur - containing groups , were assumed and in parts identified . due to the polar supelcowax 10 gc column , a clear separation of molecules of higher polarity was observed , such as saturated and unsaturated monocarboxylic acids , saturated dicarboxylic acids , and alkyl phenols . according to the assigned compounds in the single eics , most molecules were detected as single deprotonated molecules ( [ m h ] ) ; that is , no analyte fragmentation was obvious , which is one of the advantages of the apci ion source . compared to that , an ionization with other commonly used techniques for gc - ms hyphenation , such as ei , normally leads to an intense fragmentation of sample molecules . thus , the developed gc - apci - ft - icr - ms hyphenation makes it easier to assign a molecular formula to the detected molecular ion . in contrast , gc - ei - ms simplifies the clarification of the exact structure of a compound , due to the higher number of detectable fragments with lower ( m / z)-ratios . hence , the combination of results from the developed gc - apci - ft - icr - ms hyphenation with results from gc - ei - ms hyphenation should increase the knowledge about pyrolysis oil to a greater extent . moreover , an improved gc separation , for example , with an ionic liquid gc column or a high - temperature gc column , will also help to separate nonpolar and polar compounds similarly by using a higher gc maximum temperature .

lipids constitute a large and diverse group of compounds , which all are united by their hydrophobic nature and consequently their solubility in nonpolar organic solvents . lipids and related lipophilic compounds are found , e.g. in animal and plant fats and oils , as waxes , phospholipids , eicosonoids , terpenes , and lipidsoluble steroids and vitamins . the main sources for the industrial use of lipids are animal fats and plant oils , with the latter ones being forecasted for 20082009 to be produced at a scale of 418 million tonnes worldwide ( http://www.abareconomics.com/interactive/08ac_sept/htm/oilseeds.htm ) . the chemical industry has developed a number of processes for the chemical conversion of lipids into various valuable industrial end products . an overview on the plethora of chemical conversion processes that is being worked upon can be obtained from the 2008 euro fed lipid congress abstracts book ( oils , fats , and lipids in the third millennium , 710 september , 2008 , book of abstracts ) . for example , lipids can serve as raw materials for the production of functional polymers such as polyurethanes , polyamides and polyesters by subjecting unsaturated vegetable oils ( e.g. soy or canola ) to ozonolysis followed by a hydrogenation step ( narine , 2008 ) . or , lipidderived fatty acid monomers are being modified by adding specific functional groups prior to their incorporation into specific polymers , such as polyesters and polyamides ( behr and rothstock , 2008 ) . hydrocarbons for use as hydrocarbonbased biofuels can be synthesized from lipids by thermal and catalytic decarboxylation ( stadlbauer et al . , 2008 ) . rare functionalized unsaturated fatty acids can be produced from calendula and tung oils ( biermann et al . , 2007 ) , while modified fatty acids with reduced transfat content can be obtained from vegetable oils via metalbased catalysis ( golden et al . , 2008 ) . these and other innovative strategies for the production of addedvalue lipids typically still depend on costly and energyconsuming chemical reactions . biotechnology on the other hand provides a wide range of industrial applications that can not easily be achieved by conventional chemistry . although the production of biofuels from oils via lipase catalysis currently represent a major focus in the application of biotechnology to lipid transformation ( liu et al . , 2008 ; watanabe and shimada , 2008 ) , new types of bioconversions are emerging , which complement pure enzymatic catalysis now by whole cellbased biocatalytic ( wcb ) conversion strategies . the former group of enzymatic bioconversions include , apart from lipase catalysed conversions , e.g. the biosynthesis of verylongchain pufas employing desaturases and elongases ( hoppe et al . , 2008 ) or the rational application of acylcoa acyltransferases to modify the relative composition of sunflower oils , aiming at lower transfats content during hydrogenation process while enhancing the proportion of stearic fat ( martinezforce et al . , 2008 ) . wcb on the other hand are employed for the production of conjugated pufas using bifidobacterium species ( gorissen et al . , 2008 ) , for the production of biosurfactants from vegetable oils , often refinery wastes , using different bacteria and yeast species ( bernarski et al . , 2004 ; raza et al . , 2007 ; sarubbo et al . , 2007 ; reviewed in van bogaert et al . , 2007 ) , but also for the targeted production of fine chemicals , such as vanillin from rice bran oil by using aspergillus niger and pycnoporus cinnabarinus ( zheng et al . , 2007 ) . some prominent yeast species , the socalled oleaginous yeasts that in fact were originally isolated from lipid substrates , have evolved sophisticated metabolic pathways that allow them to thrive on a variety of lipid substrates ( reviewed in thevenieau et al . , 2009;2010 ) . yet , only few examples have been reported this far on the use of these yeasts in lipid bioconversion : yarrowia lipolytica as well as some species of the genus candida were employed for the lipidderived production of biosurfactants and of gammadecalactone , a highvalue aroma substance ( endrizzi and belin , 1995 ; wachet al . , 2001 ) . the notable underutilization of oleaginous yeast for lipid bioconversion may correspond to the prominent research focus on conventional yeasts such as saccharomyces cerevisiae , which are thus much better studied in general terms . yet , since recently the sequence of the oleaginous yeast y. lipolytica has been published ( dujon et al . , 2004 ) , the foundations were laid to make this yeast a prime model organism for studying lipid metabolism in oleaginous yeasts . in this review we describe targeted efforts in utilizing y. lipolytica for the bioconversion of lipids into diverse highvalue products , by combining the yeast 's lipidmetabolizing potential with biosynthetic capacities derived from oil and lipiddegrading bacteria . nonconventional yeast primarily due to its ability to grow on a variety of substrates that are rich in lipids such as certain dairy and meat products ( reviewed in wolf , 2003 ; beopoulos et al . , variant strains have also been isolated from soil , sewage and oilpolluted environments ( reviewed in beopoulos et al . , 2010 ) , where in the latter case its ability to grow on a variety of aliphatic hydrocarbons has been exploited for the bioremediation of oily and greasy waste waters ( oswal et al . yarrowia is considered a gras ( generally recognized as safe ) organism that has already been used for some biotechnological applications like , e.g. citric acid production and heterologous protein expression ; however , none of these production processes has exploited lipids as substrates ( madzak et al . , 2004 ; forster et al . , 2007 ) . on the other hand , this yeast has been used to produce neutral lipids consisting of triacylglycerols ( tags ) and sterol esters from oleic acid as substrate ( mlickova et al . , 2004 ) , which accumulate as intracellular lipid bodies making up for about 80% of the cell dry weight ( reviewed in fickers et al . , 2005 ; beopoulos et al . , 2009 ) . yarrowia 's ability to accumulate lipids is now being explored , e.g. to produce in a targeted way either oils rich in saturated fatty acids ( papanikolaou et al . , 2003 ) , or mono and polyunsaturated oils , by corresponding genetic engineering of the yeast ( beopoulos et al . , yarrowia 's versatile lipid metabolism allows it , in response to the prevailing metabolic conditions , to either degrade , store or modify exogenous lipid substrates via distinct compartmentalized metabolic routes ( reviewed in fickers et al . , 2005 ) . yarrowia lipolytica shows remarkable adaptations to lipid substrates in its ability to actually communicate with lipids by producing extracellular protrusions that serve to gather lipid droplets at the cell 's surface , as well as to store significant amounts of lipids intracellularly ( mlickova et al . , 2004 ) . recent research efforts directed at elucidating its lipid metabolism ( reviewed in fickers et al . , 2005 ) together with available genomic data ( dujon et al . , 2004 ) figure 1 demonstrates the scope of y. lipolytica 's storage capacity when grown on lipids ( triolein ) or lipid precursors ( oleic acid ) as compared with a rich nonlipid carbon source ( glucose ) , and for different genetic backgrounds , i.e. the wild type ( wt ) , a mutant deficient in lipid production ( jmy 1877 ) , and an obese mutant ( jmy 1367 ) , where metabolic routes competing with lipid production are blocked . scanning electron and fluorescence microscope visualization of lipid accumulation and lipid body formation depending on genotype of y. lipolytica cells grown on glucose ( ypd ) , triglyceride ( ynbtriolein ) and fatty acid ( ynboleic acid ) . strains used are the wild type ( wt ) , a quadruple lipid bodydeficient mutant deleted for all acyl transferases ( dga1 , lro1 , are1and are2 ) ( jmy1877 ) ( a. beopoulos , r. haddouche , p. kabran , t. chardot and j.m . nicaud , in preparation ) and an obese lipidoverproducing mutant strain deleted for gut2 and the pox genes ( jmy1367 ) ( pox16 andgut2 ) ( beopoulos et al . , upper panel ( no staining ) and lower panel ( lipid are stained with lipidtoxgreen neutral strain ) . yarrowia lipolytica may gradually develop into a new yeast model organism for industrial use with gradually enhanced understanding of its genetics and physiology . a wide range of tools have by now been developed that allow genetic manipulation of yarrowiato be done with the same ease as for s. cerevisiae(fickers et al . 2004 ) . a large set of additional mutations to more specifically alter this yeast 's lipid metabolism are currently being engineered by dr nicaud and colleagues at inra ( r. haddouche , in preparation ) . these strains then serve as tool box for the rational redesign of yarrowia 's lipid flux with modified oxidation , tag biosynthesis and oxidation thus allowing for the production of lipid precursors or for their conversion into other addedvalue derivatives . additional engineering efforts can further optimize and tailor yarrowia 's lipid metabolism to create new branch points for interception , and to increase metabolic flux along these engineered routes . yarrowia lipolytica is considered a nonconventional yeast primarily due to its ability to grow on a variety of substrates that are rich in lipids such as certain dairy and meat products ( reviewed in wolf , 2003 ; beopoulos et al . , 2010 ) . variant strains have also been isolated from soil , sewage and oilpolluted environments ( reviewed in beopoulos et al . , 2010 ) , where in the latter case its ability to grow on a variety of aliphatic hydrocarbons has been exploited for the bioremediation of oily and greasy waste waters ( oswal et al . , 2002 ; yarrowia is considered a gras ( generally recognized as safe ) organism that has already been used for some biotechnological applications like , e.g. citric acid production and heterologous protein expression ; however , none of these production processes has exploited lipids as substrates ( madzak et al . , 2004 ; forster et al . , 2007 ) . on the other hand , this yeast has been used to produce neutral lipids consisting of triacylglycerols ( tags ) and sterol esters from oleic acid as substrate ( mlickova et al . , 2004 ) , which accumulate as intracellular lipid bodies making up for about 80% of the cell dry weight ( reviewed in fickers et al . , 2005 ; beopoulos et al . , 2009 ) . yarrowia 's ability to accumulate lipids is now being explored , e.g. to produce in a targeted way either oils rich in saturated fatty acids ( papanikolaou et al . , 2003 ) , or mono and polyunsaturated oils , by corresponding genetic engineering of the yeast ( beopoulos et al . , 2008 ) . yarrowia 's versatile lipid metabolism allows it , in response to the prevailing metabolic conditions , to either degrade , store or modify exogenous lipid substrates via distinct compartmentalized metabolic routes ( reviewed in fickers et al . , 2005 ) . yarrowia lipolytica shows remarkable adaptations to lipid substrates in its ability to actually communicate with lipids by producing extracellular protrusions that serve to gather lipid droplets at the cell 's surface , as well as to store significant amounts of lipids intracellularly ( mlickova et al . , 2004 ) . recent research efforts directed at elucidating its lipid metabolism ( reviewed in fickers et al . , 2005 ) together with available genomic data ( dujon et al . , 2004 ) make it now possible to intercept its branched lipid metabolism for biotechnological purposes . figure 1 demonstrates the scope of y. lipolytica 's storage capacity when grown on lipids ( triolein ) or lipid precursors ( oleic acid ) as compared with a rich nonlipid carbon source ( glucose ) , and for different genetic backgrounds , i.e. the wild type ( wt ) , a mutant deficient in lipid production ( jmy 1877 ) , and an obese mutant ( jmy 1367 ) , where metabolic routes competing with lipid production are blocked . scanning electron and fluorescence microscope visualization of lipid accumulation and lipid body formation depending on genotype of y. lipolytica cells grown on glucose ( ypd ) , triglyceride ( ynbtriolein ) and fatty acid ( ynboleic acid ) . strains used are the wild type ( wt ) , a quadruple lipid bodydeficient mutant deleted for all acyl transferases ( dga1 , lro1 , are1and are2 ) ( jmy1877 ) ( a. beopoulos , r. haddouche , p. kabran , t. chardot and j.m . nicaud , in preparation ) and an obese lipidoverproducing mutant strain deleted for gut2 and the pox genes ( jmy1367 ) ( pox16 andgut2 ) ( beopoulos et al . , 2008 ) . upper panel ( no staining ) and lower panel ( lipid are stained with lipidtoxgreen neutral strain ) . yarrowia lipolytica may gradually develop into a new yeast model organism for industrial use with gradually enhanced understanding of its genetics and physiology . a wide range of tools have by now been developed that allow genetic manipulation of yarrowiato be done with the same ease as for s. cerevisiae(fickers et al . , 2003 ; madzak et al . , 2004 ) . a large set of additional mutations to more specifically alter this yeast 's lipid metabolism are currently being engineered by dr nicaud and colleagues at inra ( r. haddouche , in preparation ) . these strains then serve as tool box for the rational redesign of yarrowia 's lipid flux with modified oxidation , tag biosynthesis and oxidation thus allowing for the production of lipid precursors or for their conversion into other addedvalue derivatives . additional engineering efforts can further optimize and tailor yarrowia 's lipid metabolism to create new branch points for interception , and to increase metabolic flux along these engineered routes . the term hydrocarbonoclastic bacteria refers to group of marine bacteria that are capable of utilizing aliphatic hydrocarbons as their sole carbon and energy source . these bacteria are present in low abundance in marine environments but bloom after an oil spill ( golyshin et al . , 2003 ) . one species , alcanivorax borkumensis , appears to dominate microbial communities associated with oilpolluted waters and is assumed to be mostly responsible for the removal of marine oil contaminations ( golyshin et al . , 2003 ) . this bacterium is particularly efficient at degrading a wide range of aliphatic hydrocarbons , which initiated research aimed at finding enzymes that could be used for bioremediation , biocatalysis and biosynthesis . earlier completed genome sequence of a. borkumensis ( schneiker et al . , 2006 ) and its first genome and proteome analyses revealed multiple alkane degradation pathways as well as metabolic routes for the accumulation of carbon storage compounds from scavenged alkanes ( sabirova et al . , 2006 ) . more recently , genome sequences of other hydrocarbonoclastic marine bacteria [ marinobacter aquaeolei , idiomarina loihiensis , oleospira antarctica ( marinobacter and oleospira are currently being sequenced ) ] have become available , which provides additional sources for the identification of novel enzyme functions for the biocatalytic conversion of lipophilic compounds into valuable products , such as putative hydrolases and oxygenases . marine hydrocarbonoclastic bacteria have not only evolved efficient mechanisms for the degradation of aliphatic hydrocarbons , but also for the formation of storage compounds enabling them to survive periods of poor nutrient conditions ( reviewed in steinbchel et al . , alcanivorax has been shown to accumulate significant amounts of wax esters ( wes ) and tags ( kalscheuer et al . , 2007 ) , while marinobacter hydrocarbonoclasticus is known to make wes from recalcitrant isoprenoid alcohols that are released from accumulated ( bacterio)chlorophylls in marine sediments ( rontani et al . , 1999 ) . other yet undiscovered hydrocarbonoclastic bacteria may have evolved other scavenging mechanisms that allow them to utilize other types of hydrocarbons and make new types of storage compounds , and hence it is anticipated that targeted screening and metagenomics efforts focussed on this group of marine bacteria will uncover new metabolic functions that can be exploited for the conversion of hydrocarbons and lipids into highvalue products . research efforts under the project described here all aim at producing highvalue fine chemicals , carotenoids and biopolymers from bulk materials like cheap animal fats or vegetable oils . . 2 ) and are used as highperformance lubricants for engines , transmission and hydraulic systems ( lea , 2002 ) . currently , these compounds are produced primarily from mineral oils , although the production of biodegradable lubricants from renewable sources increasingly attracts interest ( goodstein , 2004 ) . however , as the most accessible natural sources of wes , whale sperm oil and jojoba oil , are too expensive for wide range use , researchers have explored microbial sources for the synthesis of biowes and thus some gramnegative bacteria such as , e.g. acinetobacter , alcanivorax and marinobacter species have been reported to synthesize different types of wes as carbon storage compounds ( kalscheuer , 2010 ) . fatty acidfatty alcohol ( top ) and isoprenoid we ( bottom ) . polyhydroxyalkanoates ( phas ) are biodegradable biopolymers synthesized by bacteria and used as bioplastics , e.g. for a variety of packaging and medical applications . chemically being composed of 3hydroxy fatty acid monomers , these polymers can be classified as shortchainlength ( scl ) , mediumchainlength ( mcl ) or longchain length ( lcl ) phas . sclphas contain monomers with a chain length of three to five carbons , while mclphas contain 3hydroxy acid monomers with 616 carbon chains , and longchainlength phas contain monomers longer than with 16 carbon atoms ( fig . 3 ) . currently , the bestcharacterized pha is the sclpha polyhydroxybutyrate phb ( fourcarbonchain length ) which can be produced in an industrial scale . however , phb is a rather brittle and inflexible biopolymer which compromised in its range of applications . to increase the utility of these biopolymers it is necessary to produce phas with different monomer compositions , e.g. longer chain length for improved mechanical properties . short and mediumchainlength phas . mclpha 's : poly(3hydroxyhexanoate ) ( c6 ) , poly(3hydroxyoctanoate ) , poly(3hydroxydecanoate ) ( c10 ) , poly(3hydroxydodecanoate ) ( c12 ) , poly(3hydroxytetradecanoate ) ( c14 ) , and poly(3hydroxyhexadecanoate ) ( c16 ) . yeasts with their more active metabolism yielding pha precursors appears to be a better choice , as compared with bacterial hosts , for largescale pha production , because of their ability to grow on a wider range of lowcost feedstocks and because their sizes allow easier recovery during downstream processing ( terentiev et al . recently attempts have therefore been aimed to produce phas in s. cerevisiae , pichia pastoris and arxula adeninivorans by overexpressing either pseudomonas aeruginosaphac synthase or the entire phbabc cluster of ralstonia eutropha ( poirier et al . , 2001 ; 2002 ; however , pha production rates so far obtained in yeasts were low compared with those obtained with bacteria . yarrowia lipolytica specialized in efficient mobilization , storage , and conversion of lipids into pha , is expected to be more appropriate yeast as heterologous host for pha production . hydroxylated fatty acids ( hfas ) have been used as chiral building blocks for the synthesis of a number of fine chemicals ( seebach et al . , 1986 ; lee et al . , moreover , some of hfas have high potential for antimicrobial agents or biomedical applications ( hou , 2008 ) . enantiomerically pure hfas can be derived from phas by chemical hydrolysis , which is a not very costeffective production route . alternatively , hfas can be produced from phas by enzymatic depolymerization using pha depolymerases that in phaproducing bacteria catalyse the mobilization of storage compounds for growth ( ren et al . , 2005 ) . the most desirable process for hfas production , however , would be conversion of 3hydroxy acylcoa intermediates from microbial oxidation of fatty acids into r3hydroxy acyl acids . by employing genetic engineering of yarrowia , we will target 3hydroxy intermediates of oxidation into these valuable compounds . carotenoids represent a large group of structurally diverse pigments synthesized by microorganisms and plants ( fig . 5 ) . carotenoids are used commercially as food colorants , feed supplements , nutraceuticals and for cosmetic and pharmaceutical purposes . of the more than 600 different carotenoids identified in nature , only a small number can be synthesized in useful quantities by chemical synthesis , extraction from their natural sources or microbial fermentation ( reviewed in lee and schmidtdannert , 2002 ) . genes encoding the enzymes of the carotenoid biosynthetic pathways leading to carotenoids with c30 , c40 or c50 carbon backbones have been cloned from microorganisms and plants . biosynthesis of these pigments in noncarotenogenic hosts such as escherichia colior yarrowia requires first the extension of its isoprenoid pathway with a gene encoding geranylgeranyl diphosphate synthase ( crte ) , which catalyses headtotail condensation of an additional c5 isoprenoid unit to the general isoprenoid precursor farnesyl diphosphate ( c15 ) to yield geranylgeranyl diphosphate ( c20 , ggdp ) . an additional recombinant enzyme , phytoene synthase ( crtb ) , is then required for the headtohead condensation of two ggdp molecules to the colourless c40 carotenoid phytoene . to obtain coloured carotenoids in e. coli , a phytoene desaturase ( crti ) extension of this core pathway with known enzymes that catalyse cyclization , glucosylation and diverse oxygenation reactions allows the production of an array of acyclic and cyclic carotenoids and xanthophylls in e. coli.production of c30 carotenoids occurs from fpp via a headtohead condensation reaction catalysed by crtm and desaturation of the resulting dehydrosqualene by crn to diaponeurosporene or diapolycopene . expression of these enzymes in the host e. colifacilitated their characterization and allowed the recombinant biosynthesis of diverse cyclic and acyclic carotenoids in e. coli(schmidtdannert et al . , 2000 ) . dr schmidtdannert 's group at the university of minnesota has extensively explored combinatorial , evolutionary and genomic strategies for the production of diverse carotenoid structures and carotenoid cleavage compounds in engineered microbial hosts ( schmidtdannert , 2001 ; mijts et al . , 2004 ; however , despite successes in creating these diverse recombinant carotenoid pathways , there is a pressing need to find ways of producing carotenoid structures ( most can not be chemically synthesized ) in sufficient quantities for commercial applications . 6 ) also referred to as electronic plastics have been developed for applications in an emerging class of optoelectronic devices such as lasers , ultra fast image processors , thinfilm transistors , highly sensitive plastic photodiodes and photodiode arrays and allpolymer integrated circuits . these new polymers , like other commonly used plastics , are chemically synthesized from petroleumderived building blocks . biological routes to functional materials independent petroleumbased economy could instead be described as a biorefinery process in which biomass components are converted into highvalue compounds . nature produces an array of stable conducting organic molecules designed and optimized to carry out electron transport and lightharvesting functions , the latter via photoinduced electron transfer . the largest class of molecules with delocalized electron systems the conjugated polyisoprenoids resemble the first chemically synthesized electronic polymer polyacetylene , but contain additional methylgroups along the chain and are in an alltrans conformation , which exert a stabilizing effect on the molecule and increases electrical conductivity . dr schmidtdannert 's group has developed a unique biosynthetic platform for the production of endgroup functionalized polyenic building blocks in metabolically engineered e. colicells ( mijts et al . , 2005 ) . microbial genome mining for novel biosynthetic enzymes together with combinatorial biosynthesis and in vitro evolution of biosynthetic enzymes was used to engineer e. colistrains that produce fully conjugated isoprenoid chains containing carboxylic acid , aldehyde or alcohol functions at both terminal ends . the produced conjugated and oxygenated isoprenoid chains ( c30 and c40 carotenoids ) are extremely stable ( in fact , the compounds appear selfassemble and their unusual stability may be due to chain stacking ) and represent ideal building blocks for the synthesis of conducting polymers . their lightabsorbing properties make them ideally suited for photoinduced electron transfer , a muchdesired property of electronic plastics . the lipoyeasts project combines biosynthetic potential of hydrocarbonoclastic and other hydrocarbon and lipiddegrading bacteria of various lipidcontaminated environments with the lipidmobilizing potential of the oleaginous yeast y. lipolytica to produce valuable lipidderived byproducts , such as poly and wax esters , hfas , carotenoids and carotenoid esters . for this purpose , the lipid metabolism of yarrowiais to be adjusted in such a way , that it creates a high pool of lipidderived compounds , precursors of highvalue products . more specifically , we will concentrate on the construction of strains with reduced capacity in the storage of fatty acid into lipid bodies and with reduced capacity to degrade fatty acids by either or oxidation , as shown in fig . lipids such as triacylglycerols ( tags ) , fatty acids and nalkanes are used as carbon sources . they get hydrolysed ( tags ) extracellularly and get transported into the cell , where fatty acids get either stored as tags in lipids bodies or get further oxidized in or oxidation cycles . oxidation cycle provides precursors for hydroxylated fatty acids ( hfas ) and polyhydroxyalkanoates ( phas ) . modifications of pox16 genes catalysing the first step of oxidation will be explored for modulating monomer composition of the produced hfas and phas . finally , the oxidation will be also blocked to avoid the production of undesired dicarboxylic fatty acids . the resulted pool of fatty acids and fatty acid alcohols will be used for the production of wes , carotenoids or carotenoid esters by expressing the corresponding bacterial biosynthetic genes . to develop y. lipolytica into a fermentation platform for the conversion of lipid feed stocks into commercially interesting products , we propose the following complementary focus areas ( wps ) of research and development : ( i ) engineering of metabolic precursor pools in y. lipolytica for the production of value added products from lipids , ( ii ) conversion of metabolic precursor pools in yarrowiato valueadded products by overexpressing heterologous biosynthetic enzymes , ( iii ) discovery and characterization of novel aliphatic enzyme activities by metagenomic screening of marine hydrocarbonoclastic microbial communities , ( iv ) pilot process development and identification of commercial applications , and ( v ) project coordination and ipr management . already available for this project are expression vectors and several deletion strains of yarrowia as well as a collection of genes encoding enzymes for the production of wes , 3hfas , phas and carotenoids . we can therefore immediately begin with wp1 to improve lipid precursor pools in yarrowia and implementation of the proposed heterologous biosynthetic capabilities into yarrowia ( wp2 ) . parallel to these efforts we will add new enzyme functions identified in metagenomic libraries to our toolbox of lipidmodifying enzymes ( wp3 ) for expression in yarrowia . once the first recombinant yeast producer strains have been generated , pilot processes for midscale production will be developed and applications for produced compounds identified . current imbalance between chemical technologies and biotechnologies to make use of bulk fats and oils is addressed by this collaborative project . it aims to create new possibilities to convert lipids into valuable products by using the oleaginous yeast y. lipolytica as a lipidmobilizing platform . this research will also hopefully bridge the gap between enzyme discovery studies based on the enormous amount of genomic information generated via genome sequencing projects on one hand and availability of easily genetically modifiable and wellstudied lipidmobilizing yeast on the other hand . by combining all these valuable and rather separated experiences together , we plan to make the practical use of this unique combination of available information and strains , by constructing yeast cell factories for the production of different highvalue industrial compounds . thus we hope to expand the knowledge : ( i ) in yeast metabolism by studying fluxes of fatty acids intermediates , ( ii ) of recently discovered but not fully characterized enzymes derived from hydrocarbonoclastic bacteria and to understand their function by their heterologous expression in yarrowia , ( iii ) in enzyme mining and in establishment and improvement of screening protocols applied to metagenomes , ( iv ) in new enzymes and their unique properties , and ( v ) in novel applicational aspects of the produced compounds . the consortium collectively has all tools and expertise at their disposal to meet this goal via close interaction between the partners . the progress of the work will be posted on the consortium webpage ( http://www.lipoyeasts.ugent.be/ ) inviting a wide range of feedback from the scientific community .

summarythe oleochemical industry is currently still dominated by conventional chemistry , with biotechnology only starting to play a more prominent role , primarily with respect to the biosurfactants or lipases , e.g. as detergents , or for biofuel production . a major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates , which are then to be transformed into highvalue biotechnological , nutritional or pharmacological products . under the eusponsored lipoyeasts project we are developing the oleaginous yeast yarrowia lipolytica into a versatile and highthroughput microbial factory that , by use of specific enzymatic pathways from hydrocarbonoclastic bacteria , efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipidderived compounds like wax esters ( we ) , isoprenoidderived compounds ( carotenoids , polyenic carotenoid ester ) , polyhydroxyalkanoates ( phas ) and free hydroxylated fatty acids ( hfas ) . different lipid stocks ( petroleum , alkane , vegetable oil , fatty acid ) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of y. lipolytica , in order to modulate the composition and yields of the produced addedvalue products .

Lipids and biotechnology None Hydrocarbonoclastic bacteria as sources for novel enzymes Highvalue target compounds LipoYeasts project Conclusion

lipids and related lipophilic compounds are found , e.g. the main sources for the industrial use of lipids are animal fats and plant oils , with the latter ones being forecasted for 20082009 to be produced at a scale of 418 million tonnes worldwide ( http://www.abareconomics.com/interactive/08ac_sept/htm/oilseeds.htm ) . for example , lipids can serve as raw materials for the production of functional polymers such as polyurethanes , polyamides and polyesters by subjecting unsaturated vegetable oils ( e.g. or , lipidderived fatty acid monomers are being modified by adding specific functional groups prior to their incorporation into specific polymers , such as polyesters and polyamides ( behr and rothstock , 2008 ) . , 2007 ) , while modified fatty acids with reduced transfat content can be obtained from vegetable oils via metalbased catalysis ( golden et al . these and other innovative strategies for the production of addedvalue lipids typically still depend on costly and energyconsuming chemical reactions . biotechnology on the other hand provides a wide range of industrial applications that can not easily be achieved by conventional chemistry . although the production of biofuels from oils via lipase catalysis currently represent a major focus in the application of biotechnology to lipid transformation ( liu et al . , 2008 ; watanabe and shimada , 2008 ) , new types of bioconversions are emerging , which complement pure enzymatic catalysis now by whole cellbased biocatalytic ( wcb ) conversion strategies . the former group of enzymatic bioconversions include , apart from lipase catalysed conversions , e.g. wcb on the other hand are employed for the production of conjugated pufas using bifidobacterium species ( gorissen et al . , 2008 ) , for the production of biosurfactants from vegetable oils , often refinery wastes , using different bacteria and yeast species ( bernarski et al . , 2007 ) , but also for the targeted production of fine chemicals , such as vanillin from rice bran oil by using aspergillus niger and pycnoporus cinnabarinus ( zheng et al . yet , only few examples have been reported this far on the use of these yeasts in lipid bioconversion : yarrowia lipolytica as well as some species of the genus candida were employed for the lipidderived production of biosurfactants and of gammadecalactone , a highvalue aroma substance ( endrizzi and belin , 1995 ; wachet al . the notable underutilization of oleaginous yeast for lipid bioconversion may correspond to the prominent research focus on conventional yeasts such as saccharomyces cerevisiae , which are thus much better studied in general terms . yet , since recently the sequence of the oleaginous yeast y. lipolytica has been published ( dujon et al . , 2004 ) , the foundations were laid to make this yeast a prime model organism for studying lipid metabolism in oleaginous yeasts . in this review we describe targeted efforts in utilizing y. lipolytica for the bioconversion of lipids into diverse highvalue products , by combining the yeast 's lipidmetabolizing potential with biosynthetic capacities derived from oil and lipiddegrading bacteria . yarrowia is considered a gras ( generally recognized as safe ) organism that has already been used for some biotechnological applications like , e.g. on the other hand , this yeast has been used to produce neutral lipids consisting of triacylglycerols ( tags ) and sterol esters from oleic acid as substrate ( mlickova et al . , 2004 ) , which accumulate as intracellular lipid bodies making up for about 80% of the cell dry weight ( reviewed in fickers et al . yarrowia 's ability to accumulate lipids is now being explored , e.g. to produce in a targeted way either oils rich in saturated fatty acids ( papanikolaou et al . , 2003 ) , or mono and polyunsaturated oils , by corresponding genetic engineering of the yeast ( beopoulos et al . , yarrowia 's versatile lipid metabolism allows it , in response to the prevailing metabolic conditions , to either degrade , store or modify exogenous lipid substrates via distinct compartmentalized metabolic routes ( reviewed in fickers et al . yarrowia lipolytica shows remarkable adaptations to lipid substrates in its ability to actually communicate with lipids by producing extracellular protrusions that serve to gather lipid droplets at the cell 's surface , as well as to store significant amounts of lipids intracellularly ( mlickova et al . recent research efforts directed at elucidating its lipid metabolism ( reviewed in fickers et al . , 2004 ) figure 1 demonstrates the scope of y. lipolytica 's storage capacity when grown on lipids ( triolein ) or lipid precursors ( oleic acid ) as compared with a rich nonlipid carbon source ( glucose ) , and for different genetic backgrounds , i.e. scanning electron and fluorescence microscope visualization of lipid accumulation and lipid body formation depending on genotype of y. lipolytica cells grown on glucose ( ypd ) , triglyceride ( ynbtriolein ) and fatty acid ( ynboleic acid ) . strains used are the wild type ( wt ) , a quadruple lipid bodydeficient mutant deleted for all acyl transferases ( dga1 , lro1 , are1and are2 ) ( jmy1877 ) ( a. beopoulos , r. haddouche , p. kabran , t. chardot and j.m . nicaud , in preparation ) and an obese lipidoverproducing mutant strain deleted for gut2 and the pox genes ( jmy1367 ) ( pox16 andgut2 ) ( beopoulos et al . yarrowia lipolytica may gradually develop into a new yeast model organism for industrial use with gradually enhanced understanding of its genetics and physiology . a large set of additional mutations to more specifically alter this yeast 's lipid metabolism are currently being engineered by dr nicaud and colleagues at inra ( r. haddouche , in preparation ) . these strains then serve as tool box for the rational redesign of yarrowia 's lipid flux with modified oxidation , tag biosynthesis and oxidation thus allowing for the production of lipid precursors or for their conversion into other addedvalue derivatives . , 2002 ; yarrowia is considered a gras ( generally recognized as safe ) organism that has already been used for some biotechnological applications like , e.g. on the other hand , this yeast has been used to produce neutral lipids consisting of triacylglycerols ( tags ) and sterol esters from oleic acid as substrate ( mlickova et al . , 2004 ) , which accumulate as intracellular lipid bodies making up for about 80% of the cell dry weight ( reviewed in fickers et al . yarrowia 's ability to accumulate lipids is now being explored , e.g. to produce in a targeted way either oils rich in saturated fatty acids ( papanikolaou et al . , 2003 ) , or mono and polyunsaturated oils , by corresponding genetic engineering of the yeast ( beopoulos et al . yarrowia 's versatile lipid metabolism allows it , in response to the prevailing metabolic conditions , to either degrade , store or modify exogenous lipid substrates via distinct compartmentalized metabolic routes ( reviewed in fickers et al . yarrowia lipolytica shows remarkable adaptations to lipid substrates in its ability to actually communicate with lipids by producing extracellular protrusions that serve to gather lipid droplets at the cell 's surface , as well as to store significant amounts of lipids intracellularly ( mlickova et al . figure 1 demonstrates the scope of y. lipolytica 's storage capacity when grown on lipids ( triolein ) or lipid precursors ( oleic acid ) as compared with a rich nonlipid carbon source ( glucose ) , and for different genetic backgrounds , i.e. the wild type ( wt ) , a mutant deficient in lipid production ( jmy 1877 ) , and an obese mutant ( jmy 1367 ) , where metabolic routes competing with lipid production are blocked . scanning electron and fluorescence microscope visualization of lipid accumulation and lipid body formation depending on genotype of y. lipolytica cells grown on glucose ( ypd ) , triglyceride ( ynbtriolein ) and fatty acid ( ynboleic acid ) . strains used are the wild type ( wt ) , a quadruple lipid bodydeficient mutant deleted for all acyl transferases ( dga1 , lro1 , are1and are2 ) ( jmy1877 ) ( a. beopoulos , r. haddouche , p. kabran , t. chardot and j.m . nicaud , in preparation ) and an obese lipidoverproducing mutant strain deleted for gut2 and the pox genes ( jmy1367 ) ( pox16 andgut2 ) ( beopoulos et al . yarrowia lipolytica may gradually develop into a new yeast model organism for industrial use with gradually enhanced understanding of its genetics and physiology . a large set of additional mutations to more specifically alter this yeast 's lipid metabolism are currently being engineered by dr nicaud and colleagues at inra ( r. haddouche , in preparation ) . these strains then serve as tool box for the rational redesign of yarrowia 's lipid flux with modified oxidation , tag biosynthesis and oxidation thus allowing for the production of lipid precursors or for their conversion into other addedvalue derivatives . , 2006 ) and its first genome and proteome analyses revealed multiple alkane degradation pathways as well as metabolic routes for the accumulation of carbon storage compounds from scavenged alkanes ( sabirova et al . more recently , genome sequences of other hydrocarbonoclastic marine bacteria [ marinobacter aquaeolei , idiomarina loihiensis , oleospira antarctica ( marinobacter and oleospira are currently being sequenced ) ] have become available , which provides additional sources for the identification of novel enzyme functions for the biocatalytic conversion of lipophilic compounds into valuable products , such as putative hydrolases and oxygenases . , alcanivorax has been shown to accumulate significant amounts of wax esters ( wes ) and tags ( kalscheuer et al . other yet undiscovered hydrocarbonoclastic bacteria may have evolved other scavenging mechanisms that allow them to utilize other types of hydrocarbons and make new types of storage compounds , and hence it is anticipated that targeted screening and metagenomics efforts focussed on this group of marine bacteria will uncover new metabolic functions that can be exploited for the conversion of hydrocarbons and lipids into highvalue products . research efforts under the project described here all aim at producing highvalue fine chemicals , carotenoids and biopolymers from bulk materials like cheap animal fats or vegetable oils . currently , these compounds are produced primarily from mineral oils , although the production of biodegradable lubricants from renewable sources increasingly attracts interest ( goodstein , 2004 ) . however , as the most accessible natural sources of wes , whale sperm oil and jojoba oil , are too expensive for wide range use , researchers have explored microbial sources for the synthesis of biowes and thus some gramnegative bacteria such as , e.g. polyhydroxyalkanoates ( phas ) are biodegradable biopolymers synthesized by bacteria and used as bioplastics , e.g. chemically being composed of 3hydroxy fatty acid monomers , these polymers can be classified as shortchainlength ( scl ) , mediumchainlength ( mcl ) or longchain length ( lcl ) phas . to increase the utility of these biopolymers it is necessary to produce phas with different monomer compositions , e.g. yarrowia lipolytica specialized in efficient mobilization , storage , and conversion of lipids into pha , is expected to be more appropriate yeast as heterologous host for pha production . hydroxylated fatty acids ( hfas ) have been used as chiral building blocks for the synthesis of a number of fine chemicals ( seebach et al . enantiomerically pure hfas can be derived from phas by chemical hydrolysis , which is a not very costeffective production route . alternatively , hfas can be produced from phas by enzymatic depolymerization using pha depolymerases that in phaproducing bacteria catalyse the mobilization of storage compounds for growth ( ren et al . the most desirable process for hfas production , however , would be conversion of 3hydroxy acylcoa intermediates from microbial oxidation of fatty acids into r3hydroxy acyl acids . of the more than 600 different carotenoids identified in nature , only a small number can be synthesized in useful quantities by chemical synthesis , extraction from their natural sources or microbial fermentation ( reviewed in lee and schmidtdannert , 2002 ) . genes encoding the enzymes of the carotenoid biosynthetic pathways leading to carotenoids with c30 , c40 or c50 carbon backbones have been cloned from microorganisms and plants . biosynthesis of these pigments in noncarotenogenic hosts such as escherichia colior yarrowia requires first the extension of its isoprenoid pathway with a gene encoding geranylgeranyl diphosphate synthase ( crte ) , which catalyses headtotail condensation of an additional c5 isoprenoid unit to the general isoprenoid precursor farnesyl diphosphate ( c15 ) to yield geranylgeranyl diphosphate ( c20 , ggdp ) . an additional recombinant enzyme , phytoene synthase ( crtb ) , is then required for the headtohead condensation of two ggdp molecules to the colourless c40 carotenoid phytoene . to obtain coloured carotenoids in e. coli , a phytoene desaturase ( crti ) extension of this core pathway with known enzymes that catalyse cyclization , glucosylation and diverse oxygenation reactions allows the production of an array of acyclic and cyclic carotenoids and xanthophylls in e. coli.production of c30 carotenoids occurs from fpp via a headtohead condensation reaction catalysed by crtm and desaturation of the resulting dehydrosqualene by crn to diaponeurosporene or diapolycopene . dr schmidtdannert 's group at the university of minnesota has extensively explored combinatorial , evolutionary and genomic strategies for the production of diverse carotenoid structures and carotenoid cleavage compounds in engineered microbial hosts ( schmidtdannert , 2001 ; mijts et al . dr schmidtdannert 's group has developed a unique biosynthetic platform for the production of endgroup functionalized polyenic building blocks in metabolically engineered e. colicells ( mijts et al . the produced conjugated and oxygenated isoprenoid chains ( c30 and c40 carotenoids ) are extremely stable ( in fact , the compounds appear selfassemble and their unusual stability may be due to chain stacking ) and represent ideal building blocks for the synthesis of conducting polymers . the lipoyeasts project combines biosynthetic potential of hydrocarbonoclastic and other hydrocarbon and lipiddegrading bacteria of various lipidcontaminated environments with the lipidmobilizing potential of the oleaginous yeast y. lipolytica to produce valuable lipidderived byproducts , such as poly and wax esters , hfas , carotenoids and carotenoid esters . for this purpose , the lipid metabolism of yarrowiais to be adjusted in such a way , that it creates a high pool of lipidderived compounds , precursors of highvalue products . more specifically , we will concentrate on the construction of strains with reduced capacity in the storage of fatty acid into lipid bodies and with reduced capacity to degrade fatty acids by either or oxidation , as shown in fig . lipids such as triacylglycerols ( tags ) , fatty acids and nalkanes are used as carbon sources . oxidation cycle provides precursors for hydroxylated fatty acids ( hfas ) and polyhydroxyalkanoates ( phas ) . modifications of pox16 genes catalysing the first step of oxidation will be explored for modulating monomer composition of the produced hfas and phas . finally , the oxidation will be also blocked to avoid the production of undesired dicarboxylic fatty acids . the resulted pool of fatty acids and fatty acid alcohols will be used for the production of wes , carotenoids or carotenoid esters by expressing the corresponding bacterial biosynthetic genes . to develop y. lipolytica into a fermentation platform for the conversion of lipid feed stocks into commercially interesting products , we propose the following complementary focus areas ( wps ) of research and development : ( i ) engineering of metabolic precursor pools in y. lipolytica for the production of value added products from lipids , ( ii ) conversion of metabolic precursor pools in yarrowiato valueadded products by overexpressing heterologous biosynthetic enzymes , ( iii ) discovery and characterization of novel aliphatic enzyme activities by metagenomic screening of marine hydrocarbonoclastic microbial communities , ( iv ) pilot process development and identification of commercial applications , and ( v ) project coordination and ipr management . already available for this project are expression vectors and several deletion strains of yarrowia as well as a collection of genes encoding enzymes for the production of wes , 3hfas , phas and carotenoids . we can therefore immediately begin with wp1 to improve lipid precursor pools in yarrowia and implementation of the proposed heterologous biosynthetic capabilities into yarrowia ( wp2 ) . current imbalance between chemical technologies and biotechnologies to make use of bulk fats and oils is addressed by this collaborative project . it aims to create new possibilities to convert lipids into valuable products by using the oleaginous yeast y. lipolytica as a lipidmobilizing platform . by combining all these valuable and rather separated experiences together , we plan to make the practical use of this unique combination of available information and strains , by constructing yeast cell factories for the production of different highvalue industrial compounds . thus we hope to expand the knowledge : ( i ) in yeast metabolism by studying fluxes of fatty acids intermediates , ( ii ) of recently discovered but not fully characterized enzymes derived from hydrocarbonoclastic bacteria and to understand their function by their heterologous expression in yarrowia , ( iii ) in enzyme mining and in establishment and improvement of screening protocols applied to metagenomes , ( iv ) in new enzymes and their unique properties , and ( v ) in novel applicational aspects of the produced compounds .

autism typically develops in childhood , and it is considered as a systemic spectrum disorder with multiple development trajectories with an incidence four times higher in males than in females ( grossi and terruzzi 2014 ) . in addition to behavioural traits , gi abnormalities such as diarrhoea , constipation , bloating and abdominal pain are common in autism and they seem to contribute to , and exacerbate , overall behaviour of children ( irritability , sleeplessness , posturing ) ( van de sande , van buul and brouns 2014 ) . a crosstalk exists between the gut microbiota and central nervous system ( cns ) mediated via a range of different chemical , immunological and signalling interactions that form part of the gut brain axis . several studies have demonstrated the role of the gut microbiota in neurodevelopment and mental health ( foster and mcvey neufeld 2013 ) , and there is increasing evidence associating gut microbial dysbiosis with gi problems that might affect autistic children . ( 2002 ) found nine unique species of clostridia in autistic children compared with controls . song , liu and finegold ( 2004 ) , using qpcr analysis , found higher levels of clostridium bolteae and clostridium clusters i and xi . furthermore , parracho et al . ( 2005 ) , using fish analysis , found greater number of species derived from the c. histolyticum group ( clostridium clusters i and ii ) . desulfovibrio group was found to be 10 times higher in the gut microbiota of autistic children compared with controls ( finegold et al . high - throughput sequencing has been used in more recent studies to determine bacterial composition of faecal samples from autistic children . the genera prevotella , coprococcus and unclassified veillonellaceae have been found in lower abundance in autistic individuals ( kang et al . in addition , bifidobacterium species decreased in asd , comparing with the non - autistic control ( de angelis et al . metabolic associations have also been identified with asd and may be attributed to gut dysbiosis in autistic individuals . abnormalities have been reported in tryptophan metabolism where higher amount of indole derivatives in the blood and higher levels of iag ( indolyl - acryloyl - glycine ) in the urine of autistic children have been identified . may contribute to these metabolic alterations as these organisms can metabolise tryptophan ( bingham 2003 ) . 2010 ) and amino acid deficiencies in autism with restricted diets , modified gut microbial population and gi symptoms being suggested as potential contributors ( ming et al . modulation of gut microbiota is an interesting potential strategy to reduce presence of harmful microorganisms and their metabolites that might be involved in negative stimulation of cns and affect behaviour ( shaw , kassen and chaves 1995 ; sandler et al . 2000 ) . treating gi disorders in asd with antibiotics or pro / prebiotics has been postulated to regulate microbiota and improve gut symptoms , but the evidence is scarce , especially for prebiotics . the bifidogenic properties of b - gos ( bimuno , clasado biosciences ltd , buckinghamshire , uk ) have been investigated in vitro and in human intervention studies involving healthy volunteers , and conditions that have a purported microbial input such as ibs , travellers diarrhoea and obesity ( tzortzis et al . recently , b - gos was also shown to reduce cortisol secretion and anxiety in healthy volunteers ( schmidt et al . b - gos supplementation lowered cortisol reactivity and modulated attention to emotional stimuli compared with a placebo group , supporting the hypothesis that gut microbiota might have a role in behavioural traits ( schmidt et al . our study aimed to assess the effects of b - gos ( 65% gos content ) on gut microbial ecology and metabolic end products of microbial fermentation . we used in vitro , three - stage , continuous gut model systems , inoculated with faecal samples of autistic and non - autistic children , which simulated different physicochemical characteristics of the proximal , transverse and distal colons . the mixture was in syrup format consisting of 65% ( w / v ) gos , 10.1% ( w / v ) lactose , 22% ( w / v ) glucose and 1.8% ( w / v ) galactose . faecal samples were obtained from three non - autistic children and three autistic child donors ( male , aged 510 years old ) who were free of any metabolic and gastrointestinal diseases , were not taking probiotic or prebiotic supplements and had not taken antibiotics 6 months before faecal sample donation . all parents were then provided written informed consent for use of their children 's faeces in the study . this study was approved by the university of reading research ethics committee ( urec 15/20 ) . faecal samples were placed in an anaerobic jar ( anaerojartm 2.5 l , oxoid ltd ) including a gas - generating kit ( anaerogentm , oxoid ) . an aliquot of 20 g of samples was diluted in 100 ml anaerobic pbs ( 0.1 mol / l phosphate buffer solution , ph 7.4 , w / w ) and homogenised ( stomacher 400 , seward , west sussex , uk ) for 2 min at 240 paddle beats per minute . physicochemical conditions in the colon were replicated in a continuous culture system , comprised of a cascade of three glass fermenters of increasing working volume connected in series . a small - scale version of the validated system described by macfarlane , macfarlane and gibson ( 1998 ) was used in this study , with vessels ( v ) representing the proximal ( v1 , 80 ml , ph = 5.5 ) , transverse ( v2 , 100 ml , ph = 6.2 ) and distal colon ( v3 , 120 ml , ph = 6.8 ) . the systems were inoculated with 20% ( wt : v ) faecal homogenate from either non - autistic and autistic children volunteers in a growth medium ( macfarlane , macfarlane and gibson 1998 ) . following inoculation , the colonic model was run as a batch culture for 24 h in order to stabilise bacterial populations prior to the initiation of medium flow . after 24 h ( t0 ) , the medium flow was initiated and the system ran for at least 8 full volume turnovers to allow for steady state to be achieved ( ss1 ) . short - chain fatty acid ( scfa ) profiles ( 5% ) were assessed before starting b - gos administration . taking into account the operating volume ( 300 ml ) and the retention time ( 48 h , flow rate 6.25 ml / h ) of the colonic model system , a syrup containing gos ( 2 g / daily , equivalent to 1 g of gos ) was added daily into v1 . the syrup was added to the system for at least a further 8 volume turnovers upon which steady state 2 ( ss2 ) was achieved . the production of scfas in the fermentations was determined by hplc ( merck , nj ) as previously described by rodriguez - colinas et al . peaks were integrated using atlas lab managing software ( thermo lab systems , mainz , germany ) . quantification of the samples was obtained through calibration curves of lactic , acetic , propionic , butyric and formic acids in concentrations 12.5 , 25 , 50 , 75 and 100 mm , respectively . bacterial composition in the gut models was analysed for using fluorescence in situ hybridization combined with flow cytometry ( fish - fcm ) . seven hundred and fifty microlitres of samples were centrifuged at 1136 g for 5 min . pellets were resuspended in 375 l of filtered pbs ( using a 0.22-m pvdf membrane ) and fixed in 1125 l of 4% ( v / v ) paraformaldehyde . after 4 h of incubation at 4c , samples were washed twice using 1 ml of pbs , resuspended in 600 l pbs - ethanol ( 1:1 , v / v ) and stored at 20c . permeabilisation steps were performed using 30 l of the fixed samples added to 500 l pbs and centrifuged at 1136 g for 3 min . pellets were resuspended using 100 l of filtered te - fish ( tris / hcl 1 m ph 8 , edta 0.5 m ph 8 , distilled h2o , 0.22 m pvdf membrane ) containing lysozyme ( 1 mg / ml of 50 000 u / mg protein ) and incubated for 10 min at room temperature . solutions containing the samples were then vortexed and centrifuged at 1136 g for 3 min . pellets were washed with 500 l pbs and centrifuged ( 1136 g , 3 min ) . hybridisations were performed by resuspending the pellets in 150 l of hybridisation buffer ( 5 m nacl , 1 m tris / hcl ph 8 , 30% formamide , ddh2o , 10% sds ) , vortexed and centrifuged ( 1136 g , 3 min ) . pellets were then resuspended in 1 ml of hybridisation buffer and 50 l aliquoted into eppendorf tubes . the probes used ( sigma aldrich ltd , poole , dorset , uk ) are reported in table 1 ( devereux et al . 1992 ; wallner , amann and beisker 1993 ; langendijk et al . 1995 ; poulsen et al . 1995 ; manz et al . 1996 1999 , 2000 , 2002 ; hold et al.2003 ; lay et al . 2005 ; walker et al . 2005 ; kong et al . non eub338 and eub338 i - ii - iii linked at their 5 end either to alexa488 or alexa647 . four microlitres of each probe and 4 l of eub338 i - ii - iii ( linked to alexa488 ) were added to the working solution and incubated overnight at 35c in a heating block . after 12 h of incubation , an aliquot of 150 l hybridisation buffer was added to the working solution , vortexed and centrifuged ( 1136 g , 3 min ) . one hundred and fifty microlitres of supernatant were removed from each sample and the remaining volume was centrifuged ( 1136 g , 3 min ) . the pellets were washed with 200 l of washing buffer ( 5 m nacl , 1 m tris / hcl ph 8 , 0.5 m edta ph 8 , ddh2o , 10% sds ) , homogenised by vortexing and incubated for 20 min at 37c in a heating block . afterwards the samples were centrifuged ( 1136 g , 3 min ) and supernatants were removed . negative control samples ( no probes added ) were screened by fcm to detect background before the probe samples were resuspended in an appropriate amount of pbs . numbers of specific and total bacteria were determined taking into account dilution factor , calculated from different volumes used in samples preparation steps , and events/l obtained from non eub338 and eub338 i - ii - iii probes analysed by fcm . three consecutive days of the three biological replicates for each group ( autistic and non - autistic ) of all time points ( before and after treatment ) were analysed by h - nmr ( n = 27 , each group ) . fermentation supernatants were defrosted , vortexed and centrifuged at 599 g for 5 min . the supernatants were filtered using 0.22 m low protein binding durapore polyvianylidene fluoride ( pvdf ) membranes ( millex ; emd millipore , billerica , ma , usa ) and 400 l transferred into fresh eppendorf tubes . filtered samples were combined with 200 l of phosphate buffer ( 0.2 m ( ph 7.4 ) in d2o plus 0.001% tsp ) , mixed by vortexing , centrifuged at 1136 g for 10 min and then 550 l was transferred into 5 mm nmr tubes for analysis . all nmr spectra were acquired on a bruker avance drx 500 mhz nmr spectrometer ( bruker biopsin , rheinstetten , germany ) operating at 500 mhz . they were acquired using a standard 1d pulse sequence [ recycle delay ( rd)-90-t1 - 90-tm-90-acquire free induction decay ( fid ) ] with water suppression applied during rd of 2 s , a mixing time tm of 100 ms and a 90 pulse set at 7.70 s . for each spectrum , a total of 128 scans were accumulated into 64 k data points with a spectral width of 12 001 ppm . all spectra were manually phased , baseline corrected and calibrated to the chemical shift of tsp ( 3-(trimethylsilyl)-[2,2,3,3,h4]-propionic acid , 0.00 ) . spectra were digitised using an in - house matlab ( version r2014a , the mathworks , inc . ; the spectral region containing the water resonance was removed to minimise distortions in the baseline arising from imperfect water saturation . median fold normalization was performed for both groups : non - autistic and autistic children . before and after administration of b - gos , principal components analysis ( pca ) using mean - centred data was applied . orthogonal projection to latent structure discriminant analysis ( opls - da ) models was constructed using unit variance scaling for pairwise comparisons of the different experimental groups and time points . correlation coefficients plots were generated from the model outputs by back scaling transformation to display the contribution of each variable ( metabolites ) to sample classification ( e.g. before and after treatment ) . colour represents the significance of correlation ( r ) for each metabolite to class membership . predictive strength ( qy ) of the models was obtained using a 7-fold cross - validation method and these were validated using permutation testing ( number of permutations = 10 000 ) . data from hplc and fmc - fish analyses were analysed using paired t - test in order to assess significance of results , comparing the two time points ss1 and ss2 , before and after treatment , respectively . analyses were performed using graphpad prism 5.0 ( graphpad software , la jolla , ca , usa ) . changes in bacterial compositions in gut model systems are reported in fig . 1 . the data showed lower numbers of bifidobacteria in asd models compared with non - autistic ones . , following the addition of b - gos to models containing both autistic and non - autistic samples , were seen . in autistic models , a significant increase of bifidobacteria occurred from 5.32 to 7.27 log10 cells / ml ( p < 0.01 ) , from 4.81 to 6.79 log10 cfu / ml ( p < 0.001 ) and from 5.57 to 6.83 log log10 cells / ml ( p < 0.05 ) , in v1 , v2 and v3 , respectively . a slight but significant increase in clostridium cluster xi in v2 for autistic children was also found , as well as significant decrease in v2 in veillonellaceae group from 6.06 to 5 log10 cfu / ml ( p < 0.05 ) . in non - autistic models , there was a significant increase in numbers of bifidobacteria in v1 , from 5.83 to 7.16 log log10 cells / ml ( p < 0.01 ) , and in v3 , from 4.97 to 6.73 log10 cells / ml ( p < 0.001 ) and in lactic acid bacteria ( lab158 ) in v3 from 5.13 to 6.01 log10 cells / ml ( p < 0.05 ) . additionally , b - gos slightly increased roseburia spp . in v1 and v3 ( p < 0.05 ) and / ml and faecalibacterium prausnitzii from 6.78 to 5.27 log10 cells / ml ( p < 0.05 for both ) in the second vessel , while increasing atopobium spp . from 5 to 5.92 log10 cells / ml ( p < 0.05 ) in the third vessel of non - autistic models . in these models , numbers of clostridium coccoides eubacterium rectale were also increased from 6.76 to 7.08 log10 cells / ml ( p < 0.01 ) in v1 and sutterella spp . significantly decreased in v1 from 7.05 to 6.49 ( p < 0.01 ) and v2 from 7.02 to 6.37 log10 cfu / ml ( p < 0.05 ) after b - gos administration . there was a general trend to increase all other bacterial groups analysed in all vessels but this was not significant . exceptions were seen for bacteroides ( v1 ) , clostridial cluster ix ( v1 ) , f. prausnitzii ( v1 ) , escherichia coli ( v3 ) , ruminococcus spp . , clostridium leptum ( v2 ) , sutterella spp . and veillonellaceae ( all vessels ) in autistic models , and for clostridium coccoides eubacterium rectale ( v2 ) , atopobium spp . ( v1 ) , clostridial cluster ix ( v2 ) , clostridium cluster xi ( v1 , v2 ) , e. coli ( v2 ) , sutterella spp . and veillonellaceae ( all vessels ) in non - autistic models that slightly decreased . bacterial groups detected by fish - fcm ( log10 cfu / ml ) in culture broth recovered from each vessel ( v1 , v2 and v3 ) of a colonic model before ( ss1 ) and after ( ss2 ) the daily administration of b - gos ( 2 g / d , equivalent to 1 g gos ) . significant difference after the treatment : * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . ( lab158 ) , most bacteroidaceae and prevotellaceae ( bac303 ) , clostridium coccoides eubacterium rectale group ( erec482 ) , roseburia subcluster ( rrec584 ) , f. prausnitzii ( fprau655 ) , clostridium cluster xi ( clit135 ) , sutterella spp . our data show a lower concentration of butyrate and propionate in autistic models , compared with non - autistic models , but no differences in acetate before adding b - gos into the system . after the administration of b - gos , acetate and butyrate were the main end products of microbial fermentation . supplementation of b - gos to gut models inoculated with faecal samples from autistic children led to a significant increase of acetate and butyrate in v1 and v2 , simulating the proximal and transverse colons ( p < 0.05 ) , respectively , while concentration of propionate was decreased ( p < 0.05 ) in v3 mimicking distal colon . in models simulating the colon of non - autistic children , the fermentation of b - gos mediated significant production of acetate ( p < 0.05 ) and butyrate ( p < 0.001 ) in v2 and v3 , simulating the transverse and distal colon , respectively . there was no effect on propionate . acetate , propionate and butyrate concentrations in culture broths recovered from vessels ( v1 , v2 and v3 ) of in vitro gut model systems before ( ss1 ) and after ( ss2 ) administration of b - gos ( 1 g / daily gos ) . results are reported as means ( mm ) of the data ( n = 3 ) : ( a ) autistic children and ( b ) non - autistic children . significant difference after the treatment : * p < 0.05 ; * * * p < 0.001 . pca was performed on mean - centred data to summarise variance with the dataset . the scores plot ( pc1 versus pc2 ) shown in fig . 3a showed separation between autistic and non - autistic models after treatment , indicating that b - gos supplementation contributed to the largest source of variance in the metabolic data . comparison of the spectra profiles from gut models before and after treatment identified that a number of metabolites changed following b - gos supplementation to characterise the metabolic variation associated with asd , b - gos supplementation and differences in microbial response to b - gos between the asd and non - asd microbiota . a significant opls - da model was obtained comparing the metabolic profiles of the autistic and non - autistic models at baseline ( qy = 0.07 ; p < 0.05 ; fig . supernatants from the autistic models contained greater amounts of ethanol , glycine , tyrosine , tyramine , 5-aminopentoanate , acetate , 4-aminobutyrate and betaine , and lower amounts of butyrate compared with the non - autistic models . b - gos supplementation was found to modulate the metabolic profile of the autistic models ( qy = 0.08 ; p < 0.05 ) increasing ethanol , lactate , acetate and butyrate and decreasing propionate and trimethylamine ( fig . sb - i , supporting information ) . increased butyrate and acetate production was also observed in the non - autistic models following the addition of bgos ( qy = 0.12 ; p < 0.01 ; fig . sb - ii , supporting information ) . comparing the metabolic profiles of the autistic and non - autistic models after b - gos feeding ( qy = 0.17 ; p < 0.01 ) revealed that some of the metabolic variation was reduced ( fig . there was no longer variation in 4-aminobutyrate between the models ; however , the difference in ethanol and acetate between autistic and non - autistic models was increased being higher in the autistic models . h - nmr data analysis . ( a ) pca score plot shows a separation between models inoculated with stool samples of non - asd and asd children after administration of b - gos . dark and light blue dots represent replicates of samples from gut models inoculated with faecal samples of autistic children , before ( ss1 ) and after ( ss2 ) treatment , respectively . yellow and red dots represent replicates of samples from gut models inoculated with faecal samples of non - autistic children , before ( ss1 ) and after ( ss2 ) treatment , respectively . ( b ) correlation coefficients indicating the associations of identified metabolites with autism and their alteration upon b - gos administration . recent studies have focused on the effect of pre / probiotics on the gut brain axis ( liu , cao and zhang 2015 ) . this study investigated the influence of b - gos on a small scale , in vitro , gut model system inoculated with faeces from autistic and non - autistic children . the results showed a positive modulation of bacterial populations , using an automated fish method combined with fcm . lower concentrations of scfas have previously been found in children with asd by adams et al . suggesting a reduced fermentation capacity by the asd microbiota . it was hypothesised that this was due to a compromised microbiota characterised by a lower number of bifidobacteria , consistent with microbial signatures observed here ( adams et al . concomitant with these population changes , functional alterations were also observed in both autistic and non - autistic models with acetate and butyrate , the main end products of microbial fermentation , being increased . these fermentation products can cross the blood brain barrier and might influence early brain development . the synthesis of neuroactive compounds such as dopamine and serotonin can be modulated by scfa and they are able to produce reversible psychological and physiological changes in rats similar to those found in asds ( wang et al . experimental evidence using intraventricular infusion in rats indicates that propionic acid can produce brain and behavioural changes similar to asd ( macfabe et al . recent asd studies have shown increase in numbers of sutterella spp . and decrease in veillonellaceae group . in this study , the results did not show any significant differences between asd and non - asd group . however , a general decrease in those bacterial groups after treatment was highlighted , suggesting that b - gos administration might have an impact on the growth of these asd - associated bacteria . following b - gos feeding , the microbiota of autistic children produced greater amount of ethanol and lactate while the amount of amino acids and the scfa propionate , present in the model , was reduced . these metabolic alterations were not observed when the faecal microbiota of non - autistic individuals were fed b - gos . in a healthy colon , lactate production is generally low due to its conversion to other organic acids by many bacteria and because lactate can be used as a substrate for dissimilation of sulphate by some bacteria ( e.g. desulfovibrio spp . ) ( fite et al . 2004 ; marquet et al . 2009 ; flint et al . the presence of lactate is interesting because its accumulation has been associated with neurological problems , in particular studies show the effect of lactate infusions on anxiety and panic disorders ( cowley et al . cowley and colleagues in their findings showed that lactate infusion in patients suffering from panic disorder provokes higher panic symptoms reaction compared with controls ( dillon et al . dillon et al . have showed similar results in in vivo , where panic and anxiety reaction has been measured using acute panic inventory scores . after lactate infusions , the scores were much higher in patients with panic and anxiety disorders compared with normal controls ( cowley et al . the lysine degradation product , 5-aminopentanoic acid , was also higher in the autistic compared with the non - autistic models . it is believed to act as a methylene homologue of -aminobutyric acid ( gaba ) and functions as a weak gaba agonist ( callery and geelhaar 1985 ) . interestingly , gaba was also higher in the autistic models compared with the non - autistic models pre - treatment but these differences were not present following b - gos treatment . certain bacteria , such as lactobacilli , are able to produce molecules that act as neurotransmitters and directly affect the brain ( wall et al . ethanol was found in higher amount in asd children comparing with non - asd children . the vast majority of bacteria form ethanol from acetyl - coa and the glycolytic pathway ( macfarlane and macfarlane 2003 ) . microorganisms are able to oxidase ethanol and the impact of bacterial overgrowth on ethanol production has previously been studied ( baraona et al . metabolism of ethanol can lead to the production of toxic end products such as acetaldehyde , which may affect the gastrointestinal mucosa . the role of acetaldehyde in asd has been recently evaluated in particular for its role in oxidative stress and dna damage . under healthy conditions , ethanol is converted into acetic acid in the liver by a two - step process involving alcohol dehydrogenase and aldehyde dehydrogenase ( aldh ) . mutation of the aldh gene has been shown to increase the accumulation of acetaldehyde and result in cancers within different regions of the gastrointestinal tract and alzheimer 's disease ( jurnak 2015 ) . the potential role of this toxic compound in neurological disorders , including autism , warrants further exploration . this in vitro study showed promising and positive results in that supplementing the microbiota of children with asd with 65%b - gos may manipulate the gut bacterial population and alter metabolic activity towards a configuration that might represent a health benefit to the host . however , further work will be required to assess such changes in an in vivo human intervention study . grg and ac were involved in designing and coordination of the study and revising the manuscript critically for important intellectual content . jv and gt are employed by clasado biosciences ltd , who provided the b - gos product , marketed as bimuno(r ) , used within this research . this does not alter the authors adherence to all the fems policies on sharing data and materials . the funders had no role in study design , data collection and interpretation , or the decision to submit the work for publication .

abstractchildren with autism spectrum disorders ( asd ) often suffer gastrointestinal problems consistent with imbalances in the gut microbial population . treatment with antibiotics or pro / prebiotics has been postulated to regulate microbiota and improve gut symptoms , but there is a lack of evidence for such approaches , especially for prebiotics . this study assessed the influence of a prebiotic galactooligosaccharide ( b - gos ) on gut microbial ecology and metabolic function using faecal samples from autistic and non - autistic children in an in vitro gut model system . bacteriology was analysed using flow cytometry combined with fluorescence in situ hybridization and metabolic activity by hplc and 1h - nmr . consistent with previous studies , the microbiota of children with asd contained a higher number of clostridium spp . and a lower number of bifidobacteria compared with non - autistic children . b - gos administration significantly increased bifidobacterial populations in each compartment of the models , both with autistic and non - autistic - derived samples , and lactobacilli in the final vessel of non - autistic models . in addition , changes in other bacterial population have been seen in particular for clostridium , rosburia , bacteroides , atopobium , faecalibacterium prausnitzii , sutterella spp . and veillonellaceae . furthermore , the addition of b - gos to the models significantly altered short - chain fatty acid production in both groups , and increased ethanol and lactate in autistic children .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS SUPPLEMENTARY DATA AUTHORS' CONTRIBUTIONS

in addition to behavioural traits , gi abnormalities such as diarrhoea , constipation , bloating and abdominal pain are common in autism and they seem to contribute to , and exacerbate , overall behaviour of children ( irritability , sleeplessness , posturing ) ( van de sande , van buul and brouns 2014 ) . a crosstalk exists between the gut microbiota and central nervous system ( cns ) mediated via a range of different chemical , immunological and signalling interactions that form part of the gut brain axis . several studies have demonstrated the role of the gut microbiota in neurodevelopment and mental health ( foster and mcvey neufeld 2013 ) , and there is increasing evidence associating gut microbial dysbiosis with gi problems that might affect autistic children . ( 2002 ) found nine unique species of clostridia in autistic children compared with controls . furthermore , parracho et al . desulfovibrio group was found to be 10 times higher in the gut microbiota of autistic children compared with controls ( finegold et al . high - throughput sequencing has been used in more recent studies to determine bacterial composition of faecal samples from autistic children . the genera prevotella , coprococcus and unclassified veillonellaceae have been found in lower abundance in autistic individuals ( kang et al . in addition , bifidobacterium species decreased in asd , comparing with the non - autistic control ( de angelis et al . metabolic associations have also been identified with asd and may be attributed to gut dysbiosis in autistic individuals . abnormalities have been reported in tryptophan metabolism where higher amount of indole derivatives in the blood and higher levels of iag ( indolyl - acryloyl - glycine ) in the urine of autistic children have been identified . 2010 ) and amino acid deficiencies in autism with restricted diets , modified gut microbial population and gi symptoms being suggested as potential contributors ( ming et al . treating gi disorders in asd with antibiotics or pro / prebiotics has been postulated to regulate microbiota and improve gut symptoms , but the evidence is scarce , especially for prebiotics . the bifidogenic properties of b - gos ( bimuno , clasado biosciences ltd , buckinghamshire , uk ) have been investigated in vitro and in human intervention studies involving healthy volunteers , and conditions that have a purported microbial input such as ibs , travellers diarrhoea and obesity ( tzortzis et al . recently , b - gos was also shown to reduce cortisol secretion and anxiety in healthy volunteers ( schmidt et al . b - gos supplementation lowered cortisol reactivity and modulated attention to emotional stimuli compared with a placebo group , supporting the hypothesis that gut microbiota might have a role in behavioural traits ( schmidt et al . our study aimed to assess the effects of b - gos ( 65% gos content ) on gut microbial ecology and metabolic end products of microbial fermentation . we used in vitro , three - stage , continuous gut model systems , inoculated with faecal samples of autistic and non - autistic children , which simulated different physicochemical characteristics of the proximal , transverse and distal colons . faecal samples were obtained from three non - autistic children and three autistic child donors ( male , aged 510 years old ) who were free of any metabolic and gastrointestinal diseases , were not taking probiotic or prebiotic supplements and had not taken antibiotics 6 months before faecal sample donation . this study was approved by the university of reading research ethics committee ( urec 15/20 ) . faecal samples were placed in an anaerobic jar ( anaerojartm 2.5 l , oxoid ltd ) including a gas - generating kit ( anaerogentm , oxoid ) . physicochemical conditions in the colon were replicated in a continuous culture system , comprised of a cascade of three glass fermenters of increasing working volume connected in series . a small - scale version of the validated system described by macfarlane , macfarlane and gibson ( 1998 ) was used in this study , with vessels ( v ) representing the proximal ( v1 , 80 ml , ph = 5.5 ) , transverse ( v2 , 100 ml , ph = 6.2 ) and distal colon ( v3 , 120 ml , ph = 6.8 ) . the systems were inoculated with 20% ( wt : v ) faecal homogenate from either non - autistic and autistic children volunteers in a growth medium ( macfarlane , macfarlane and gibson 1998 ) . following inoculation , the colonic model was run as a batch culture for 24 h in order to stabilise bacterial populations prior to the initiation of medium flow . short - chain fatty acid ( scfa ) profiles ( 5% ) were assessed before starting b - gos administration . taking into account the operating volume ( 300 ml ) and the retention time ( 48 h , flow rate 6.25 ml / h ) of the colonic model system , a syrup containing gos ( 2 g / daily , equivalent to 1 g of gos ) was added daily into v1 . the production of scfas in the fermentations was determined by hplc ( merck , nj ) as previously described by rodriguez - colinas et al . quantification of the samples was obtained through calibration curves of lactic , acetic , propionic , butyric and formic acids in concentrations 12.5 , 25 , 50 , 75 and 100 mm , respectively . bacterial composition in the gut models was analysed for using fluorescence in situ hybridization combined with flow cytometry ( fish - fcm ) . permeabilisation steps were performed using 30 l of the fixed samples added to 500 l pbs and centrifuged at 1136 g for 3 min . four microlitres of each probe and 4 l of eub338 i - ii - iii ( linked to alexa488 ) were added to the working solution and incubated overnight at 35c in a heating block . after 12 h of incubation , an aliquot of 150 l hybridisation buffer was added to the working solution , vortexed and centrifuged ( 1136 g , 3 min ) . negative control samples ( no probes added ) were screened by fcm to detect background before the probe samples were resuspended in an appropriate amount of pbs . three consecutive days of the three biological replicates for each group ( autistic and non - autistic ) of all time points ( before and after treatment ) were analysed by h - nmr ( n = 27 , each group ) . filtered samples were combined with 200 l of phosphate buffer ( 0.2 m ( ph 7.4 ) in d2o plus 0.001% tsp ) , mixed by vortexing , centrifuged at 1136 g for 10 min and then 550 l was transferred into 5 mm nmr tubes for analysis . all spectra were manually phased , baseline corrected and calibrated to the chemical shift of tsp ( 3-(trimethylsilyl)-[2,2,3,3,h4]-propionic acid , 0.00 ) . spectra were digitised using an in - house matlab ( version r2014a , the mathworks , inc . ; the spectral region containing the water resonance was removed to minimise distortions in the baseline arising from imperfect water saturation . median fold normalization was performed for both groups : non - autistic and autistic children . before and after administration of b - gos , principal components analysis ( pca ) using mean - centred data was applied . orthogonal projection to latent structure discriminant analysis ( opls - da ) models was constructed using unit variance scaling for pairwise comparisons of the different experimental groups and time points . predictive strength ( qy ) of the models was obtained using a 7-fold cross - validation method and these were validated using permutation testing ( number of permutations = 10 000 ) . data from hplc and fmc - fish analyses were analysed using paired t - test in order to assess significance of results , comparing the two time points ss1 and ss2 , before and after treatment , respectively . changes in bacterial compositions in gut model systems are reported in fig . the data showed lower numbers of bifidobacteria in asd models compared with non - autistic ones . , following the addition of b - gos to models containing both autistic and non - autistic samples , were seen . in autistic models , a significant increase of bifidobacteria occurred from 5.32 to 7.27 log10 cells / ml ( p < 0.01 ) , from 4.81 to 6.79 log10 cfu / ml ( p < 0.001 ) and from 5.57 to 6.83 log log10 cells / ml ( p < 0.05 ) , in v1 , v2 and v3 , respectively . in non - autistic models , there was a significant increase in numbers of bifidobacteria in v1 , from 5.83 to 7.16 log log10 cells / ml ( p < 0.01 ) , and in v3 , from 4.97 to 6.73 log10 cells / ml ( p < 0.001 ) and in lactic acid bacteria ( lab158 ) in v3 from 5.13 to 6.01 log10 cells / ml ( p < 0.05 ) . additionally , b - gos slightly increased roseburia spp . in v1 and v3 ( p < 0.05 ) and / ml and faecalibacterium prausnitzii from 6.78 to 5.27 log10 cells / ml ( p < 0.05 for both ) in the second vessel , while increasing atopobium spp . from 5 to 5.92 log10 cells / ml ( p < 0.05 ) in the third vessel of non - autistic models . in these models , numbers of clostridium coccoides eubacterium rectale were also increased from 6.76 to 7.08 log10 cells / ml ( p < 0.01 ) in v1 and sutterella spp . significantly decreased in v1 from 7.05 to 6.49 ( p < 0.01 ) and v2 from 7.02 to 6.37 log10 cfu / ml ( p < 0.05 ) after b - gos administration . there was a general trend to increase all other bacterial groups analysed in all vessels but this was not significant . , clostridium leptum ( v2 ) , sutterella spp . and veillonellaceae ( all vessels ) in autistic models , and for clostridium coccoides eubacterium rectale ( v2 ) , atopobium spp . ( v1 ) , clostridial cluster ix ( v2 ) , clostridium cluster xi ( v1 , v2 ) , e. coli ( v2 ) , sutterella spp . and veillonellaceae ( all vessels ) in non - autistic models that slightly decreased . bacterial groups detected by fish - fcm ( log10 cfu / ml ) in culture broth recovered from each vessel ( v1 , v2 and v3 ) of a colonic model before ( ss1 ) and after ( ss2 ) the daily administration of b - gos ( 2 g / d , equivalent to 1 g gos ) . ( lab158 ) , most bacteroidaceae and prevotellaceae ( bac303 ) , clostridium coccoides eubacterium rectale group ( erec482 ) , roseburia subcluster ( rrec584 ) , f. prausnitzii ( fprau655 ) , clostridium cluster xi ( clit135 ) , sutterella spp . our data show a lower concentration of butyrate and propionate in autistic models , compared with non - autistic models , but no differences in acetate before adding b - gos into the system . after the administration of b - gos , acetate and butyrate were the main end products of microbial fermentation . supplementation of b - gos to gut models inoculated with faecal samples from autistic children led to a significant increase of acetate and butyrate in v1 and v2 , simulating the proximal and transverse colons ( p < 0.05 ) , respectively , while concentration of propionate was decreased ( p < 0.05 ) in v3 mimicking distal colon . in models simulating the colon of non - autistic children , the fermentation of b - gos mediated significant production of acetate ( p < 0.05 ) and butyrate ( p < 0.001 ) in v2 and v3 , simulating the transverse and distal colon , respectively . acetate , propionate and butyrate concentrations in culture broths recovered from vessels ( v1 , v2 and v3 ) of in vitro gut model systems before ( ss1 ) and after ( ss2 ) administration of b - gos ( 1 g / daily gos ) . results are reported as means ( mm ) of the data ( n = 3 ) : ( a ) autistic children and ( b ) non - autistic children . 3a showed separation between autistic and non - autistic models after treatment , indicating that b - gos supplementation contributed to the largest source of variance in the metabolic data . comparison of the spectra profiles from gut models before and after treatment identified that a number of metabolites changed following b - gos supplementation to characterise the metabolic variation associated with asd , b - gos supplementation and differences in microbial response to b - gos between the asd and non - asd microbiota . a significant opls - da model was obtained comparing the metabolic profiles of the autistic and non - autistic models at baseline ( qy = 0.07 ; p < 0.05 ; fig . supernatants from the autistic models contained greater amounts of ethanol , glycine , tyrosine , tyramine , 5-aminopentoanate , acetate , 4-aminobutyrate and betaine , and lower amounts of butyrate compared with the non - autistic models . b - gos supplementation was found to modulate the metabolic profile of the autistic models ( qy = 0.08 ; p < 0.05 ) increasing ethanol , lactate , acetate and butyrate and decreasing propionate and trimethylamine ( fig . increased butyrate and acetate production was also observed in the non - autistic models following the addition of bgos ( qy = 0.12 ; p < 0.01 ; fig . comparing the metabolic profiles of the autistic and non - autistic models after b - gos feeding ( qy = 0.17 ; p < 0.01 ) revealed that some of the metabolic variation was reduced ( fig . there was no longer variation in 4-aminobutyrate between the models ; however , the difference in ethanol and acetate between autistic and non - autistic models was increased being higher in the autistic models . ( a ) pca score plot shows a separation between models inoculated with stool samples of non - asd and asd children after administration of b - gos . dark and light blue dots represent replicates of samples from gut models inoculated with faecal samples of autistic children , before ( ss1 ) and after ( ss2 ) treatment , respectively . yellow and red dots represent replicates of samples from gut models inoculated with faecal samples of non - autistic children , before ( ss1 ) and after ( ss2 ) treatment , respectively . ( b ) correlation coefficients indicating the associations of identified metabolites with autism and their alteration upon b - gos administration . this study investigated the influence of b - gos on a small scale , in vitro , gut model system inoculated with faeces from autistic and non - autistic children . the results showed a positive modulation of bacterial populations , using an automated fish method combined with fcm . lower concentrations of scfas have previously been found in children with asd by adams et al . it was hypothesised that this was due to a compromised microbiota characterised by a lower number of bifidobacteria , consistent with microbial signatures observed here ( adams et al . concomitant with these population changes , functional alterations were also observed in both autistic and non - autistic models with acetate and butyrate , the main end products of microbial fermentation , being increased . the synthesis of neuroactive compounds such as dopamine and serotonin can be modulated by scfa and they are able to produce reversible psychological and physiological changes in rats similar to those found in asds ( wang et al . in this study , the results did not show any significant differences between asd and non - asd group . however , a general decrease in those bacterial groups after treatment was highlighted , suggesting that b - gos administration might have an impact on the growth of these asd - associated bacteria . following b - gos feeding , the microbiota of autistic children produced greater amount of ethanol and lactate while the amount of amino acids and the scfa propionate , present in the model , was reduced . these metabolic alterations were not observed when the faecal microbiota of non - autistic individuals were fed b - gos . the presence of lactate is interesting because its accumulation has been associated with neurological problems , in particular studies show the effect of lactate infusions on anxiety and panic disorders ( cowley et al . cowley and colleagues in their findings showed that lactate infusion in patients suffering from panic disorder provokes higher panic symptoms reaction compared with controls ( dillon et al . after lactate infusions , the scores were much higher in patients with panic and anxiety disorders compared with normal controls ( cowley et al . the lysine degradation product , 5-aminopentanoic acid , was also higher in the autistic compared with the non - autistic models . interestingly , gaba was also higher in the autistic models compared with the non - autistic models pre - treatment but these differences were not present following b - gos treatment . ethanol was found in higher amount in asd children comparing with non - asd children . microorganisms are able to oxidase ethanol and the impact of bacterial overgrowth on ethanol production has previously been studied ( baraona et al . the role of acetaldehyde in asd has been recently evaluated in particular for its role in oxidative stress and dna damage . under healthy conditions , ethanol is converted into acetic acid in the liver by a two - step process involving alcohol dehydrogenase and aldehyde dehydrogenase ( aldh ) . mutation of the aldh gene has been shown to increase the accumulation of acetaldehyde and result in cancers within different regions of the gastrointestinal tract and alzheimer 's disease ( jurnak 2015 ) . this in vitro study showed promising and positive results in that supplementing the microbiota of children with asd with 65%b - gos may manipulate the gut bacterial population and alter metabolic activity towards a configuration that might represent a health benefit to the host . however , further work will be required to assess such changes in an in vivo human intervention study . grg and ac were involved in designing and coordination of the study and revising the manuscript critically for important intellectual content . jv and gt are employed by clasado biosciences ltd , who provided the b - gos product , marketed as bimuno(r ) , used within this research .

the actin - bundling protein fascin has been linked to tumor progression , invasion , and metastasis , a fatal development of the disease . fascin has recently emerged as a novel therapeutic target for treatment of cancer metastasis . a cell - based fascin bioassay identified compounds with potential antimetastasis functions , with diverse chemical structural features . however , no follow - up studies of biological activities were reported to confirm that the compounds indeed target fascin to inhibit migration , invasion , and metastasis . from shape - based molecular modeling and subsequent rational design and synthesis , we have recently identified a thiazole lead compound 1 ( figure 1 ) that effectively blocked cell migration and invasion via interactions with the protein fascin . with potent activities ( ic50 in the 100 nm range ) in inhibiting migration and invasion of several metastatic human breast cancer cell lines such as mda - mb-231 , hela , and a549 , this compound exhibited no cytotoxicity at concentrations exceeding 100 m . the finding of thiazole compounds as antimigration and antiinvasion agents opened up new possibilities of fascin - targeting molecules that can be further optimized for greater potency and bioavailability while maintaining minimal cytotoxicity . to further explore and optimize the structure activity relationships , we have designed and synthesized 63 additional thiazole derivatives where we sought to ( 1 ) homologize the two lead structures by varying the substitution on the thiazole nitrogen , ( 2 ) change the linker structure between thiazole and phenyl groups , and ( 3 ) modify other substitution groups on both the thiazole ring and the phenyl rings ( figure 1 ) . these thiazole analogues were then biologically evaluated to determine their cytotoxicity , antimigration , and antiinvasion activities . further , molecular modeling was performed to assist in the elucidation of observed structure activity - relationships . for the most potent thiazole derivatives , an in vivo assay utilizing chick embryo chorioallantoic membrane ( cam ) finally we investigated their mode of action by overexpressing the protein fascin in cancer cell lines to determine if the activities of the compounds can abrogate the enhanced migration and invasion of the transfected cell lines . as shown in scheme 1 , compounds 5 and 7 and their isomers 6 and 8 were respectively obtained from the n - alkylation of the amides 3 and 4 which were prepared following the literature procedure . 4-(2,4-dimethylphenyl)thiazol-2-amine ( 9 ) was treated with benzenesulfonic chloride to give 10 which was transformed to the analogue 11 and its isomer 12 by the n - methylation reaction in thf . the acylation of the 2-aminothiazoles 13 by acyl chloride provided the amides 14 at room temperature in dichloromethane , and further methylation of 14 led to the desired analogues 15 and their corresponding isomers 16 . a transwell migration assay was used to determine the effects of the synthesized thiazole derivatives on the migratory capacity of mda - mb-231 , an invasive and metastatic breast cancer cell line . the cancer cells were seeded at a density of 2.5 10 per well in the upper chamber in serum - free media . in the presence of varying concentrations of the thiazole derivatives , the cells ability to migrate to the lower chamber with media containing 5% fbs was measured by counting the total number of cells in the lower chamber after 24 h. the concentration of individual thiazole compounds at which inhibition of migration is observed at 50% is defined as the ic50 value listed in table 1 . to evaluate possible contributions of cell viability loss to reduced migration , the thiazole derivatives were also subjected to cell survival assays to determine their cytotoxicities . these were conducted by treating the mda - mb-231 cells with individual thiazole compounds for 5 days and counting the surviving cells . the 63 newly synthesized thiazole derivatives displayed a wide range of antimigration activities as reflected in the ic50 values going from a low of 24 nm to greater than 50 m or no apparent activity . such variations in activity appears to be dependent on minor structural modifications , revealing some interesting trends that may help our understanding of the structure activity - relationships for further optimization of pharmacological index of thiazole derivatives . compounds 5l r are homologues from the lead structure 1 that were designed and prepared to investigate the effect of thiazole - n substitution on gain or loss of the antimigration activity . for comparison , 1 , the most potent thiazole derivative from our previous study , was also included in the assay . when the thiazole n - methyl is changed to an ethyl group ( 5l ) , there was a marked decrease of activity from 0.218 to 1.945 m . interestingly , an n - propyl substitution at the same position ( 5 m ) confers similar antimigration activity with an ic50 of 0.292 m . this trend continues as the alkyl chain length increased to n - butyl ( 5n , ic50 = 0.196 m ) , n - hexyl ( 5o , ic50 = 0.045 m ) , and n - dodecyl ( 5p , ic50 = 0.024 m ) . in addition , allyl substitution at the thiazole nitrogen ( 5q ) yielded activity comparable to n - propyl ( 5 m ) , but changing the substitution ( 5r ) to propynyl leads to a significant loss of the activity , resulting in an ic50 value of 3.89 m . on another note , putting the propynyl group on the amide nitrogen ( 6 ) appears to restore the activity . if the amide linker is replaced by a sulfamide structure , antimigration activity was largely lost in 12 but not in 11 . thiazole n - methyl substitution confers , whereas the amide n - methyl substitution deprives , antimigration activity . the lead compound 2 was a potent thiazole derivative discovered in a previous study , which also displayed significant cytotoxicity in several cancer cell lines at 10 m . in the current study we further investigated if modifications on n - alkyl substitutions could improve the activity and toxicity profile . thus , when the alkyl substitution at the thiazole nitrogen varied from methyl ( 5j ) , ethyl ( 7a ) , propyl ( 7b ) to allyl ( 7c ) and propynyl ( 7d ) , the cytotoxicity of the compounds remained significant , with relative survival ratio after 5-day treatment at 10 m ranging from 20.4% to 70.9% . however , the cytotoxicity is largely abrogated when the methyl and ethyl substitution is on the amide nitrogen ( 4e , 8a , 8b ) while strong antimigration activity was retained . it was also noted that these compounds ( 7a d ) exhibited moderate to strong inhibition of cell growth , suggesting that , in addition to blocking fascin - mediated migration , these compounds may have other molecular targets that regulate cell survival and growth . next we examine how modifications on the thiazole ring affect antimigration activity . a methyl substitution on the 5-position of the thiazole ring ( 14h ) results in a near complete loss of activity . adding a fluorine atom at the para - position of the 1-thiazole phenyl brings back cytotoxicity without a significant gain of antimigration activity ( 14i ) . an attempt to move the thiazole phenyl ring to the 5-position results in a more linear analogue that has stronger growth inhibitory effect but also more antimigratory ( 14j ) . interestingly , adding a thiazole n - methyl group to the above three analogues gives a more potent 15h ( compared to 14h ) and 15i ( compared to 15i ) but an inactive 15j ( compared to 14j ) . in comparison of 15j ( thiazole n - methyl ) to 16f ( amide n - methyl ) , it was noted that the analogue with amide n - methyl substitution displayed significantly greater toxicity and antimigration activity than that with a thiazole n - methyl group . modifications on the thiazole phenyl ring by halogen and trifluoromethyl substitutions afforded six analogues 14b , 14c , 14d , 14e , 14f , 14 g , of which only 14e and 14f showed strong antimigration activities with moderate inhibition of cell growth at the same time . it was noted that chlorine substitution at meta position ( 14e ) is far superior to ortho position ( 14b ) . adding a methyl group on the thiazole nitrogen to some of these analogues appears to endow more potent cytotoxicity while increasing the overall inhibitory effect on cell motility . 15c , 15d , 15e , 15f , and 15 g all displayed potent antimigration activities with ic50 values ranging from 0.096 m ( 15f ) to 1.07 m ( 15 g ) . attempts to modify both thiazole phenyl and amide phenyl ring yielded eight analogues ( 14k through 14r ) . these compounds showed almost no antimigration activities except 14o with an ic50 value of 1.03 m . however , if these analogues are modified further by adding a thiazole n - methyl substitution ( 15k through 15 m ) , some modest increases in the activity are obtained . for example , 15l exhibited an ic50 value of 0.758 m . on the other hand , if the methyl substitution is on the amide nitrogen ( 16 g through 16 m ) , the results are mixed . for instance , compared to 14k , ic50 value of 16 g improved from > 25 to 0.312 m , an increase of potency by nearly 100-fold . also , from 15o to 16j , the position change of methyl substitution from thiazole nitrogen to amide nitrogen resulted in an increase of antimigration activity by 4 times . to determine the antiinvasion activities of the thiazole compounds , the most potent antimigration analogues were selected for a matrigel invasion assay . as shown in figure 2 , at 10 m , all tested compounds demonstrate 50% or greater inhibition of cell invasion through the matrigel . notably , compound 5o exhibited the greatest potency in blocking the cancer cell invasion . it is also noted that the antiinvasion activities of the selected thiazole derivatives do not always correlate with their antimigration activities . given the association of cell migration with angiogenesis and the possible involvement of fascin in angiogenesis , we next investigated if the thiazole compounds might have any antiangiogenic properties using the chick chorioallantoic membrane ( cam ) assay . in this test the vascular system of a fertilized chicken embryo is used as a model . figure 3 demonstrates the effects of 5o and 5p on the development of embryonal blood vessels compared to a negative control ( pbs ) and a positive control ( 10 ng / plug basic fibroblast growth factor ( bfgf ) and 30 ng / plug vascular endothelial growth factor ( vegf ) ) without the thiazole compounds . the antiangiogenesis activity of the three thiazole compounds was determined by the suppression of angiogenic action of bv ( bfgf + vegf ) when each compound was added to a collagen containing bv and placed on the chorioallantoic membrane of 10-day old chick embryos for 3 days . as shown in figure 3 , the analogues 5o and 5p were seen to potently block the angiogenic action of bv . effect of 5o and 5p on angiogenesis in chick embryo ex ovo chorioallantoic membrane ( cam ) assay . angiogenesis was scored by two scorers in a blinded fashion on a scale from 0 to 3 where 0 contained no new vessels and 3 exhibited extreme angiogenesis determined by the presence of new vessels radiating out from the plug in addition to the regular repeating pattern of the normal cam vasculature . dashed lines in representative images ( below ) indicate the top edges of the collagen and drug - containing plugs from which angiogenesis was determined . in these experiments , the thiazole analogues 5o and 5p significantly inhibited the induction of angiogenesis by bfgf and vegf ( bv ) after 3 days . our previous study found that the thiazole derivatives blocked migration and invasion of cancer cells by impairing the cytoskeleton dynamics accompanied by reduced colocalization of the actin - bundling protein fascin . to provide further evidence that fascin might be involved in the antimigratory action of the newly synthesized thiazole analogues , we overexpressed fascin in mda - mb-231 cells to investigate the resulting cell motility and the effect of the thiazole compounds on the cells overexpressing fascin . figure 4 shows the higher level of fascin expression in the transfected cells than the native level of fascin in the control cells ( figure 4a ) . consistent with previous findings , the fascin+ cells exhibited significantly higher ( 2.4-fold increase ) migration than the control cells ( vector ) . treatment of the fascin+ cells with our most potent thiazole analogues , 5o and 5p , nearly completely abrogated the enhanced migration as illustrated in figure 5 . at 10 m 5o and 5p , the control mda - mb-231 cells lost about 80% migratory capacity . the thiazole compounds also inhibited the fascin+ mda - mb-231 cells by about the same percentage ( figure 5 ) . these results provided additional evidence that the thiazole derivatives likely acted through interaction with fascin . enhanced cell migration induced by fascin - overexpression is abrogated by thiazole analogues 5l , 5o , and 5p . to investigate if the thiazole analogues inhibit cell migration and invasion by impairing the f - actin network , actin filaments of hela cells treated with selected thiazole analogues were stained with phalloidin tagged with fluorescent agent ( acti - stain 488 fluorescent phalloidin ) . as shown in figure 6 , the control cells showed a distinct presence of f - actin filaments along the cells and had more evenly distributed f - actin structures in the cytoplasm . in contrast , after treatment with the three thiazole analogues 5o , 5p , and 8b each at 10 m , the cells exhibited various degrees of disorganization of actin filaments and a decrease of f - actin staining intensity near the cell membranes . these results suggest that the antimigration and antiinvasion activities of the thiazole analogues may be mediated by interfering with the actin filament network . effects of thiazole analogues 5o , 5p , and 8b on the f - actin structure of hela cells . cells were plated on glass coverslips and incubated with vehicle ( dmso ) , 5o , 5p , or 8b for 24 h and were fixed and stained with acti - stain 488 phalloidin . cells were observed under a fluorescent microscope , a digital ccd camera , and 40 objective . in summary , the 63 new thiazole derivatives designed and prepared by further structural modifications of the previously discovered lead compounds 1 and 2 produced more potent fascin inhibitors . among the active compounds , the 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs . the most potent analogue , 5p , inhibited 50% of cell migration at a concentration of 24 nm . as expected , all thiazole derivatives that were selected for matrigel invasion assays were found to have potent antiinvasion activities . in addition to antimigration and antiinvasion properties , two representative thiazole derivatives , 5o and 5p , also showed strong antiangiogenesis activity , blocking new blood vessel formation in a chicken embryo membrane assay . a functional study in which enhanced cell motility due to fascin overexpression was nearly completely abrogated by the most potent analogues 5o and 5p provided additional evidence that the action of the thiazole derivatives involved targeting the f - actin bundling protein fascin . this proposed mechanism of action is further supported by the observation that cellular f - actin structures were significantly impaired by the treatment of the thiazole analogues . all reagents and solvents were purchased from ak scientific , sigma - aldrich chemical co. , fisher scientific , acros , and pharmco - aaper and were used as received . all organic solvents ( pharmco - aaper ) used were of reagent grade quality and were used without further purification . crude synthetic products were purified by the following methods : chromatography on silica gel ( 60100 mesh , fisher scientific ) column . analytical thin layer chromatography ( tlc ) was performed on 250 m fluorescent plates ( agela technologies , de , usa ) and visualized by using uv light . for all products , the purity was ascertained to be greater than 95% by the hplc method using a shimadzu ( columbia , md ) 2010 hplc uv / ms system with a c-18 reverse phase column and by gc ms analyses using an agilent technologies 5975c inert msd mass spectrometer . to a cooled mixture of nah ( 0.26 g , 60% in oil , 6.5 mmol ) in thf ( 20 ml ) was added a solution of compound 3 or 4 ( 5 mmol ) in thf ( 10 ml ) dropwise . after that , the mixture was cooled to 0 c again and mei or rbr ( 6.5 mmol ) was added dropwise . the mixture was then warmed to room temperature and stirred for 2 h. water ( 5 ml ) was added to quench the reaction , and the mixture was further diluted with water ( 50 ml ) . after removal of all the solvent , the residue was purified by silica gel chromatography ( hexane / etoac = 4:1 ) to afford products 5 ( more polar ) and 6 or 7 ( more polar ) and 8 as solids . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 1.8 and 8.1 hz , 2h ) , 7.497.42 ( m , 3h ) , 7.187.10 ( m , 3h ) , 6.45 ( s , 1h ) , 4.35 ( m , 1h ) , 3.82 ( m , 1h ) , 1.24 ( t , j = 6.9 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 167.8 , 140.1 , 137.82 , 137.75 , 137.2 , 131.3 , 131.2 , 130.6 , 129.2 , 128.0 , 127.2 , 126.8 , 106.8 , 42.1 , 21.3 , 19.6 , 13.8 . hrms ( esi(+ ) ) calcd for c20h21n2os ( m + h ) : 337.1375 . found : 337.1365 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( m , 2h ) , 7.527.42 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.45 ( s , 1h ) , 4.28 ( m , 1h ) , 3.71 ( m , 1h ) , 2.41 ( s , 3h ) , 2.13 ( s , 3h ) , 1.70 ( m , 2h ) , 0.81 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 168.1 , 140.0 , 138.2 , 137.7 , 137.2 , 131.3 , 131.2 , 130.7 , 129.2 , 128.0 , 127.3 , 126.8 , 106.8 , 48.5 , 21.8 , 21.3 , 19.6 , 11.2 . gc ms : 350 ( m ) . hrms ( esi(+ ) ) calcd for c21h23n2os ( m + h ) : 351.1531 . found : 351.1525 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( m , 2h ) , 7.527.42 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.45 ( s , 1h ) , 4.33 ( m , 1h ) , 3.73 ( m , 1h ) , 2.40 ( s , 3h ) , 2.14 ( s , 3h ) , 1.64 ( m , 2h ) , 1.22 ( m , 2h ) , 0.81 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.1 , 140.1 , 138.2 , 137.7 , 137.2 , 131.3 , 131.2 , 130.7 , 129.2 , 128.0 , 127.3 , 126.8 , 106.9 , 46.7 , 30.4 , 21.3 , 19.9 , 19.6 , 13.6 . gc ms : 364 ( m ) . hrms ( esi(+ ) ) calcd for c22h25n2os ( m + h ) : 365.1688 . found : 365.1682 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 1.5 and 7.8 hz , 2h ) , 7.527.42 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.45 ( s , 1h ) , 4.33 ( m , 1h ) , 3.72 ( m , 1h ) , 2.41 ( s , 3h ) , 2.14 ( s , 3h ) , 1.61 ( m , 2h ) , 1.231.17 ( m , 6h ) , 0.81 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.0 , 140.1 , 138.1 , 137.7 , 137.2 , 131.3 , 131.2 , 130.7 , 129.2 , 128.0 , 127.3 , 126.8 , 106.9 , 46.9 , 31.1 , 28.2 , 26.2 , 22.4 , 21.3 , 19.6 , 13.9 . hrms ( esi(+ ) ) calcd for c24h29n2os ( m + h ) : 393.2001 . found : 393.1986 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( m , 2h ) , 7.497.26 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.44 ( s , 1h ) , 4.32 ( m , 1h ) , 3.73 ( m , 1h ) , 2.40 ( s , 3h ) , 2.13 ( s , 3h ) , 1.591.50 ( m , 4h ) , 1.281.17 ( m , 16h ) , 0.88 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.1 , 140.0 , 138.1 , 137.7 , 137.3 , 131.3 , 131.2 , 130.7 , 129.2 , 128.0 , 127.3 , 126.8 , 106.9 , 46.9 , 32.8 , 31.9 , 29.7 , 29.6 , 29.5 , 29.4 , 29.3 , 28.9 , 28.2 , 26.5 , 25.8 , 22.7 , 21.3 , 19.6 , 14.1 . ms ( esi ) : 477 ( m + h ) . hrms ( esi(+ ) ) calcd for c30h41n2os ( m + h ) : 477.2940 . found : 477.2945 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( m , 2h ) , 7.517.41 ( m , 3h ) , 7.147.07 ( m , 3h ) , 6.46 ( s , 1h ) , 5.87 ( m , 1h ) , 5.10 ( dd , j = 1.2 and 10.2 hz , 1h ) , 4.98 ( dd , j = 1.2 and 17.1 hz , 2h ) , 4.42 ( m , 1h ) , 2.40 ( s , 3h ) , 2.13 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.2 , 168.1 , 140.1 , 137.8 , 137.1 , 131.3 , 131.1 , 130.8 , 129.2 , 128.0 , 127.0 , 126.6 , 118.4 , 106.8 , 49.1 , 21.3 , 19.7 . hrms ( esi(+ ) ) calcd for c21h21n2os ( m + h ) : 349.1375 . found : 349.1370 . h nmr ( 300 mhz , cdcl3 ) : 8.40 ( m , 2h ) , 7.527.42 ( m , 3h ) , 7.237.11 ( m , 3h ) , 6.47 ( s , 1h ) , 4.80 ( m , 2h ) , 2.40 ( s , 3h ) , 2.21 ( m , 1h ) , 2.19 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.1 , 140.4 , 138.1 , 137.1 , 136.9 , 131.6 , 131.4 , 130.8 , 129.4 , 128.1 , 126.9 , 126.4 , 106.9 , 77.3 , 72.4 , 36.0 , 21.4 , 19.9 . ms ( esi ) : 347 ( m + h ) . hrms ( esi(+ ) ) calcd for c21h19n2os ( m + h ) : 347.1218 . found : 347.1216 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 1.5 and 8.1 hz , 2h ) , 7.527.43 ( m , 4h ) , 7.20 ( s , 1h ) , 7.18 ( s , 2h ) , 4.75 ( m , 2h ) , 2.42 ( s , 3h ) , 2.22(m , 1h ) , 2.21 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.7 , 167.3 , 141.0 , 138.5 , 136.2 , 135.9 , 131.9 , 131.6 , 130.5 , 129.5 , 128.1 , 127.3 , 124.8 , 98.0 , 76.97 , 72.7 , 37.1 , 21.5 , 19.6 . ms ( esi ) : 347 ( m + h ) . hrms ( esi(+ ) ) calcd for c21h19n2os ( m + h ) : 347.1218 . found : 347.1210 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 1.5 and 7.5 hz , 2h ) , 7.517.40 ( m , 8h ) , 6.52 ( s , 1h ) , 4.26 ( q , j = 6.9 hz , 2h ) , 1.34 ( t , j = 6.9 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.2 , 168.1 , 139.1 , 137.1 , 130.9 , 129.6 , 129.4 , 129.2 , 128.9 , 128.0 , 107.2 , 42.5 , 14.0 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 1.2 and 7.5 hz , 2h ) , 7.507.39 ( m , 8h ) , 6.52 ( s , 1h ) , 4.19 ( q , j = 7.5 hz , 2h ) , 1.76 ( m , 2h ) , 0.83 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.5 , 139.3 , 137.2 , 131.3 , 130.9 , 129.6 , 129.5 , 129.2 , 128.8 , 128.7 , 128.0 , 126.0 , 107.2 , 48.8 , 21.9 , 11.1 . hrms ( esi(+ ) ) calcd for c19h19n2os ( m + h ) : 323.1218 . found : 323.1210 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( dd , j = 1.8 and 6.3 hz , 2h ) , 7.497.43 ( m , 8h ) , 6.55 ( s , 1h ) , 5.98 ( m , 1h ) , 5.19 ( d , j = 10.2 hz , 1h ) , 4.98 ( d , j = 17.4 hz , 1h ) , 4.82 ( d , j = 5.1 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 174.3 , 168.5 , 139.3 , 137.0 , 131.9 , 131.4 , 130.6 , 129.7 , 129.6 , 129.2 , 128.7 , 128.0 , 118.0 , 107.1 , 49.6 . hrms ( esi(+ ) ) calcd for c19h17n2os ( m + h ) : 321.1062 . found : 321.1054 . h nmr ( 300 mhz , cdcl3 ) : 8.40 ( d , j = 6.6 hz , 2h ) , 7.577.44 ( m , 8h ) , 6.56 ( s , 1h ) , 4.92 ( d , j = 2.1 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.5 , 138.6 , 136.8 , 131.6 , 130.1 , 129.9 , 129.44 , 129.37 , 129.0 , 128.1 , 77.8 , 72.8 , 36.9 . . hrms ( esi(+ ) ) calcd for c19h15n2os ( m + h ) : 319.0905 . found : 319.0897 . h nmr ( 300 mhz , cdcl3 ) : 7.92 ( m , 2h ) , 7.557.48 ( m , 5h ) , 7.42 ( m , 2h ) , 7.34 ( m , 1h ) , 7.25 ( s , 1h ) , 4.27 ( q , j = 7.5 hz , 2h ) , 1.38 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.6 , 159.0 , 149.5 , 135.2 , 134.8 , 130.4 , 128.68 , 128.65 , 127.8 , 126.7 , 126.0 , 109.2 , 45.2 , 14.1 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . found : 309.1053 . h nmr ( 300 mhz , cdcl3 ) : 7.91 ( dd , j = 1.5 and 6.9 hz , 2h ) , 7.537.32 ( m , 8h ) , 7.24 ( s , 1h ) , 4.18 ( q , j = 7.5 hz , 2h ) , 1.83 ( m , 2h ) , 0.83 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.8 , 159.3 , 149.5 , 135.2 , 134.8 , 130.4 , 128.68 , 128.66 , 128.5 , 127.9 , 127.0 , 126.0 , 109.2 , 51.6 , 21.9 , 11.2 . hrms ( esi(+ ) ) calcd for c19h19n2os ( m + h ) : 323.1218 . found : 323.1211 . to a solution of 9 ( 0.61 g , 3.0 mmol ) and pyridine ( 0.96 ml , 12 mmol ) in anhydrous dichloromethane was added benzenesulfonic chloride ( 0.76 ml , 6 mmol ) in dichloromethane dropwise at 0 c . after being stirred at room temperature for 2 h under nitrogen atmosphere , the reaction mixture was concentrated in vacuo . the saturated na2co3 solution was added to quench the reaction , and the solution was extracted with ethyl acetate , dried over mgso4 , and concentrated in vacuo . the residue was purified by flash chromatography to give product 10 ( 0.57 g , 55% yield ) as a solid . h nmr ( 300 mhz , cdcl3 ) : 9.85 ( bs , 1h ) , 7.93 ( d , j = 7.8 hz , 2h ) , 7.537.43 ( m , 3h ) , 7.16 ( d , j = 7.8 hz , 1h ) , 7.087.04 ( m , 3h ) , 6.26 ( s , 1h ) , 2.34 and 2.32 ( ds , 6h ) . c nmr ( 75 mhz , cdcl3 ) : 168.7 , 141.9 , 140.1 , 136.3 , 136.1 , 132.2 , 132.0 , 128.9 , 128.7 , 127.2 , 126.5 , 125.7 , 103.6 , 21.2 , 20.4 . hrms ( esi(+ ) ) calcd for c17h17n2o2s2 ( m + h ) : 345.0731 . found : 345.0727 . to a cooled mixture of nah ( 0.07 g , 60% in oil , 1.6 mmol ) in thf ( 5 ml ) was added a solution of compound 10 ( 0.34 g , 1 mmol ) in thf ( 5 ml ) dropwise . after that , the mixture was cooled to 0 c again and mei ( 0.25 ml , 4.0 mmol ) was added dropwise . the mixture was then warmed to room temperature and stirred for 2 h. water ( 3 ml ) was added to quench the reaction , and the mixture was further diluted with water ( 10 ml ) . after removal of all the solvent , the residue was purified by silica gel chromatography ( hexane / etoac = 4:1 ) to afford products 11 ( more polar ) ( 0.27 g , 75% yield ) and 12 ( 0.034 g , 9% yield ) as solids . 11 : h nmr ( 300 mhz , cdcl3 ) : 8.058.02 ( m , 2h ) , 7.587.43 ( m , 3h ) , 7.127.01 ( m , 3h ) , 6.27 ( s , 1h ) , 3.19 ( s , 3h ) , 2.36 ( s , 3h ) , 2.11 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 166.8 , 142.3 , 140.6 , 139.1 , 137.6 , 131.9 , 131.4 , 130.5 , 128.6 , 127.1 , 126.6 , 126.4 , 103.5 , 33.6 , 21.3 , 19.5 . hrms ( esi(+ ) ) calcd for c18h19n2o2s2 ( m + h ) : 359.0888 . found : 359.0879 . 12 : h nmr ( 300 mhz , cdcl3 ) : 7.83 ( d , j = 7.2 hz , 2h ) , 7.587.38 ( m , 4h ) , 7.026.98 ( m , 2h ) , 6.88 ( s , 1h ) , 3.45(s , 3h ) , 2.32 and 2.30 ( ds , 6h ) . c nmr ( 75 mhz , cdcl3 ) : 160.0 , 151.2 , 137.9 , 136.8 , 135.9 , 133.8 , 131.8 , 131.4 , 129.4 , 129.3 , 127.4 , 126.6 , 112.0 , 36.7 , 21.2 , 21.1 . hrms ( esi(+ ) ) calcd for c18h19n2o2s2 ( m + h ) : 359.0888 . found : 359.0880 . to a solution of 13 ( 0.01 mol ) and dmap ( 1.24 g , 0.01 mol ) in anhydrous dichloromethane was added the acyl chloride in dichloromethane dropwise at 0 c . after being stirred at room temperature for 2 h under nitrogen atmosphere , the saturated na2co3 solution was added to quench the reaction , and the solution was extracted with ethyl acetate , dried over mgso4 , and concentrated in vacuo . the residue was purified by flash chromatography to give product 14 as a solid . h nmr ( 300 mhz , cdcl3 ) : 7.99 ( dd , j = 1.5 and 8.4 hz , 2h ) , 7.527.47 ( m , 3h ) , 6.55 ( d , j = 0.9 hz , 1h ) , 2.11 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 172.0 , 165.3 , 159.3 , 146.8 , 133.1 , 132.8 , 132.3 , 130.0 , 128.9 , 128.3 , 127.8 , 108.4 , 16.4 . hrms ( esi(+ ) ) calcd for c11h11n2os ( m + h ) : 219.0592 . found : 219.0589 . h nmr ( 300 mhz , cdcl3 ) : 11.5 ( bs , 1h ) , 7.75 ( dd , j = 1.2 and 8.4 hz , 2h ) , 7.63 ( dd , j = 2.1 and 7.2 hz , 1h ) , 7.517.45 ( m , 2h ) , 7.387.28 ( m , 3h ) , 7.197.08 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 165.3 , 158.5 , 146.3 , 132.8 , 132.7 , 132.0 , 131.7 , 130.8 , 130.4 , 128.9 , 128.7 , 127.4 , 126.8 , 113.2 . . hrms ( esi(+ ) ) calcd for c16h12cln2os ( m + h ) : 315.0359 . found : 315.0351 . h nmr ( 300 mhz , cdcl3 ) : 11.68 ( bs , 1h ) , 8.177.15 ( m , 10h ) . c nmr ( 75 mhz , cdcl3 ) : 171.3 , 165.3 , 160.1 , 148.3 , 135.9 , 133.5 , 133.1 , 133.0 , 131.7 , 131.1 , 130.3 , 130.2 , 130.1 , 129.5 , 129.3 , 128.9 , 128.4 , 127.8 , 124.9 , 122.9 , 109.3 . hrms ( esi(+ ) ) calcd for c16h12brn2os ( m + h ) : 358.9854 . found : 358.9847 . h nmr ( 300 mhz , cdcl3 ) : 10.34 ( bs , 1h ) , 7.90 ( m , 2h ) , 7.677.41 ( m , 7h ) , 7.20 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 ) : 164.9 , 158.7 , 149.1 , 133.1 , 133.0 , 131.83 , 131.79 , 129.0 , 127.6 , 127.4 , 122.1 , 108.5 . gc ms : 358 , 360 ( m ) . hrms ( esi(+ ) ) calcd for c16h12brn2os ( m + h ) : 358.9854 . found : 358.9848 . h nmr ( 300 mhz , cdcl3 ) : 10.19 ( bs , 1h ) , 7.90 ( m , 2h ) , 7.77 ( t , j = 1.8 hz , 1h ) , 7.667.56 ( m , 2h ) , 7.497.44 ( m , 1h ) , 7.327.26 ( m , 2h ) , 7.20 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 158.5 , 148.7 , 135.9 , 134.7 , 133.0 , 131.8 , 130.0 , 129.0 , 128.0 , 127.4 , 126.2 , 124.1 , 109.1 . hrms ( esi(+ ) ) calcd for c16h12cln2os ( m + h ) : 315.0359 . found : 315.0353 . h nmr ( 300 mhz , cdcl3 ) : 10.08 ( bs , 1h ) , 8.24 ( s , 2h ) , 7.93(m , 2h ) , 7.77 ( s , 1h ) , 7.60 ( m , 1h ) , 7.517.45 ( m , 2h ) , 7.39 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 ) : 165.0 , 159.1 , 147.3 , 136.3 , 133.4 , 132.5 , 132.0 , 131.7 , 129.3 , 127.6 , 126.2 , 125.3 , 121.7 , 110.9 . hrms ( esi(+ ) ) calcd for c18h11f6n2os ( m + h ) : 417.0496 . found : 417.0486 . h nmr ( 300 hz , cdcl3 ) : 10.10 ( 1h , bs ) , 7.93 ( 2h , j = 7.2 hz , d ) , 7.59 ( 1h , m ) , 7.527.47 ( 2h , m ) , 7.13 ( 1h , s ) , 7.04 ( 2h , s ) , 3.92 ( 6h , s ) , 3.87 ( 3h , s ) . c nmr ( 75 hz , cdcl3 ) : 164.6 , 158.1 , 153.4 , 150.0 , 138.1 , 133.0 , 131.8 , 130.0 , 129.0 , 127.3 , 107.6 , 103.3 , 61.0 , 56.2 . ms - ei : 370 ( m ) . hrms ( esi(+ ) ) calcd for c19h19n2o4s ( m + h ) : 371.1066 . found : 371.1052 . hrms ( esi(+ ) ) calcd for c19h19n2o4s ( m + h ) : 371.1066 . found : 371.1060 . h nmr ( 300 mhz , cdcl3 ) : 8.20 ( d , j = 6.9 hz , 2h ) , 8.09 ( d , j = 7.2 hz , 2h ) , 7.627.43 ( m , 6h ) , 2.51 ( s , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 171.3 , 165.2 , 157.1 , 144.4 , 133.7 , 133.3 , 132.8 , 131.9 , 130.3 , 130.1 , 128.8 , 128.4 , 128.3 , 128.1 , 127.9 , 122.0 , 12.0 . hrms ( esi(+ ) ) calcd for c17h15n2os ( m + h ) : 295.0905 . found : 295.0897 . h nmr ( 300 mhz , cdcl3 ) : 10.6 ( bs , 1h ) , 7.82 ( dd , j = 1.2 and 8.1 hz , 2h ) , 7.537.41 ( m , 5h ) , 7.01 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 164.7 , 155.1 , 144.2 , 132.7 , 132.0 , 130.7 , 130.0 , 129.9 , 128.8 , 127.4 , 122.2 , 115.4 , 115.2 , 12.1 . hrms ( esi(+ ) ) calcd for c17h14fn2os ( m + h ) : 313.0811 . found : 313.0802 . h nmr ( 300 mhz , cdcl3 ) : 10.01 ( bs , 1h ) , 7.92 ( d , j = 7.5 hz , 2h ) , 7.69 ( d , j = 8.1 hz , 2h ) , 7.577.45 ( m , 3h ) , 7.19 ( d , j = 8.1 hz , 2h ) , 7.14 ( s , 1h ) , 2.37 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 165.2 , 158.9 , 150.3 , 137.9 , 132.7 , 131.9 , 131.4 , 129.4 , 128.7 , 127.4 , 126.0 , 107.3 , 21.2 . hrms ( esi(+ ) ) calcd for c17h15n2os ( m + h ) : 295.0905 . found : 295.0899 . h nmr ( 300 mhz , cdcl3 ) : 10.92 ( bs , 1h ) , 7.80 ( d , j = 8.7 hz , 2h ) , 7.10 ( s , 1h ) , 6.94 ( s , 2h ) , 6.84 ( d , j = 8.7 hz , 2h ) , 3.86 ( s , 6h ) , 3.84(s , 3h ) , 3.83 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.6 , 163.1 , 159.1 , 153.2 , 149.9 , 137.9 , 130.0 , 129.4 , 124.0 , 114.0 , 107.5 , 103.3 , 60.8 , 56.0 , 55.4 . hrms ( esi(+ ) ) calcd for c20h21n2o5s ( m + h ) : 401.1171 . found : 401.1165 . h nmr ( 300 mhz , cdcl3 ) : 11.43 ( bs , 1h ) , 7.73 ( m , 2h ) , 7.30 ( m , 2h ) , 7.12 ( s , 1h ) , 6.88 ( s , 2h ) , 3.85 ( s , 6h ) , 3.83(s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.4 , 159.0 , 153.3 , 150.1 , 139.1 , 138.0 , 130.3 , 129.7 , 129.0 , 128.8 , 107.9 , 103.3 , 60.8 , 56.0 . hrms ( esi(+ ) ) calcd for c19h18cln2o4s ( m + h ) : 405.0676 . found : 405.0664 . h nmr ( 300 mhz , cdcl3 ) : 11.07 ( bs , 1h ) , 7.44 ( dd , j = 1.8 and 8.1 hz , 1h ) , 7.39 ( s , 1h ) , 7.11 ( s , 1h ) , 6.92 ( s , 2h ) , 6.78 ( d , j = 8.4 hz , 1h ) , 3.92 ( s , 3h ) , 3.86 ( s , 6h ) , 3.83(s , 3h ) , 3.82 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 159.2 , 153.3 , 152.8 , 150.1 , 148.9 , 138.0 , 129.9 , 124.2 , 120.8 , 110.4 , 110.3 , 107.6 , 103.3 , 60.9 , 56.0 , 55.8 . hrms ( esi(+ ) ) calcd for c21h23n2o6s ( m + h ) : 431.1277 . found : 431.1265 . h nmr ( 300 mhz , cdcl3 ) : 11.03 ( bs , 1h ) , 8.28 ( d , j = 9.0 hz , 1h ) , 7.10 ( s , 2h ) , 7.09 ( s , 1h ) , 6.68 ( dd , j = 2.1 and 9.0 hz , 1h ) , 6.56 ( d , j = 2.1 hz , 1h ) , 4.11 ( s , 3h ) , 3.96(s , 6h ) , 3.90 ( s , 3h ) , 3.89 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 162.6 , 159.2 , 158.1 , 153.4 , 149.9 , 138.0 , 134.5 , 130.4 , 112.0 , 107.5 , 106.1 , 103.4 , 98.7 , 60.9 , 56.4 , 56.2 , 55.7 . hrms ( esi(+ ) ) calcd for c21h23n2o6s ( m + h ) : 431.1277 . found : 431.1270 . h nmr ( 300 mhz , cdcl3 ) : 11.50 ( bs , 1h ) , 7.13 ( d , j = 0.3 hz , 1h ) , 7.06 ( s , 2h ) , 6.90 ( s , 2h ) , 3.87 ( s , 3h ) , 3.84(s , 6h ) , 3.82 ( s , 3h ) , 3.78 ( s , 6h ) . c nmr ( 75 mhz , cdcl3 ) : 165.1 , 159.3 , 153.3 , 153.1 , 150.2 , 141.9 , 138.1 , 129.6 , 126.8 , 107.8 , 104.8 , 103.2 , 60.9 , 60.8 , 56.1 , 56.0 . hrms ( esi(+ ) ) calcd for c22h25n2o7s ( m + h ) : 461.1382 . found : 461.1374 . h nmr ( 300 mhz , cdcl3 ) : 11.38 ( bs , 1h ) , 7.84 ( dd , j = 1.5 and 7.8 hz , 1h ) , 7.247.15 ( m , 3h ) , 7.12 ( s , 2h ) , 4.11 ( s , 3h ) , 3.96(s , 6h ) , 3.92 ( s , 3h ) , 3.89 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 162.6 , 157.5 , 153.4 , 152.6 , 150.1 , 148.0 , 138.1 , 130.3 , 124.8 , 124.0 , 123.0 , 117.0 , 107.6 , 103.4 , 61.9 , 60.9 , 56.2 . hrms ( esi(+ ) ) calcd for c21h23n2o6s ( m + h ) : 431.1277 . found : 431.1268 . h nmr ( 300 mhz , cdcl3 ) : 11.19 ( bs , 1h ) , 8.33 ( dd , j = 1.5 and 7.8 hz , 1h ) , 7.57 ( dt , j = 1.5 and hz , 1h ) , 7.197.07 ( m , 5h ) , 4.14 ( s , 3h ) , 3.96 ( s , 6h ) , 3.89 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 162.7 , 157.8 , 157.7 , 153.4 , 150.0 , 138.0 , 134.5 , 132.6 , 130.3 , 121.7 , 119.0 , 111.6 , 107.7 , 103.4 , 60.9 , 56.4 , 56.2 . hrms ( esi(+ ) ) calcd for c20h21n2o5s ( m + h ) : 401.1171 . h nmr ( 300 mhz , cdcl3 ) : 10.75 ( bs , 1h ) , 7.417.39 ( m , 2h ) , 7.31 ( m , 1h ) , 7.12 ( s , 1h ) , 7.05 ( m , 1h ) , 6.96 ( s , 2h ) , 3.88 ( s , 6h ) , 3.84 ( s , 3h ) , 3.77 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 159.8 , 158.5 , 153.3 , 150.1 , 138.0 , 133.1 , 129.9 , 129.8 , 119.4 , 119.2 , 112.2 , 107.6 , 103.3 , 60.9 , 56.0 , 55.3 . hrms ( esi(+ ) ) calcd for c20h21n2o5s ( m + h ) : 401.1171 . found : 401.1164 . to a cooled mixture of nah ( 0.26 g , 60% in oil , 6.5 mmol ) in thf ( 20 ml ) was added a solution of compound 14 ( 5 mmol ) in thf ( 10 ml ) dropwise . after that , the mixture was cooled to 0 c again and mei ( 6.5 mmol ) was added dropwise . the mixture was then warmed to room temperature and stirred for 2 h. water ( 5 ml ) was added to quench the reaction , and the mixture was further diluted with water ( 50 ml ) . after removal of all the solvent , the residue was purified by silica gel chromatography ( hexane / etoac = 4:1 ) to afford products 15 ( more polar ) and 16 as solids . h nmr ( 300 mhz , cdcl3 ) : 7.83 ( dd , j = 1.8 and 7.8 hz , 2h ) , 7.477.43 ( m , 3h ) , 6.29 ( d , j = 1.2 hz , 1h ) , 3.79 ( s , 3h ) , 2.31 ( d , j = 1.2 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 169.0 , 137.1 , 134.4 , 131.3 , 129.2 , 128.0 , 104.3 , 32.9 , 14.4 . hrms ( esi(+ ) ) calcd for c12h13n2os ( m + h ) : 233.0749 . found : 233.0741 . h nmr ( 300 mhz , cdcl3 ) : 8.38 ( dd , j = 1.8 and 7.8 hz , 2h ) , 7.547.39 ( m , 7h ) , 6.60 ( s , 1h ) , 3.63 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.3 , 168.4 , 136.9 , 136.3 , 134.8 , 132.3 , 131.53 , 131.49 , 130.0 , 129.9 , 129.2 , 128.1 , 127.3 , 108.1 , 34.0 . hrms ( esi(+ ) ) calcd for c17h14cln2os ( m + h ) : 329.0515 . found : 329.0505 . h nmr ( 300 mhz , cdcl3 ) : 7.83 ( dd , j = 1.8 and 8.1 hz , 2h ) , 7.64 ( m , 1h ) , 7.59 ( m , 1h ) , 7.517.36 ( m , 6h ) , 6.60 ( s , 1h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.9 , 137.8 , 136.8 , 132.8 , 132.5 , 132.2 , 131.6 , 130.5 , 129.3 , 128.1 , 127.8 , 123.0 , 107.8 , 34.9 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0006 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 1.5 and 8.1 hz , 2h ) , 7.65 ( d , j = 6.6 hz , 2h ) , 7.517.43 ( m , 4h ) , 7.28 ( d , j = 7.2 hz , 1h ) , 6.58 ( d , j = 0.9 hz , 1h ) , 3.74 ( d , j = 2.1 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.3 , 168.9 , 138.2 , 136.9 , 132.3 , 131.9 , 131.6 , 130.8 , 129.5 , 128.1 , 124.2 , 107.4 , 34.9 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0000 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( d , j = 7.8 hz , 2h ) , 7.517.43 ( m , 6h ) , 7.32 ( m , 1h ) , 6.59(s , 1h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.9 , 137.9 , 136.8 , 135.0 , 132.3 , 131.6 , 130.3 , 129.9 , 129.4 , 129.3 , 128.1 , 127.4 , 107.8 , 34.9 . hrms ( esi(+ ) ) calcd for c17h14cln2os ( m + h ) : 329.0515 . found : 329.0508 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 1.5 and 8.1 hz , 2h ) , 8.03 ( s , 1h ) , 7.91 ( s , 2h ) , 7.507.46 ( m , 3h ) , 6.73 ( s , 1h ) , 3.77 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.9 , 136.4 , 136.0 , 132.8 , 132.7 , 132.4 , 131.7 , 129.2 , 128.0 , 123.3 , 120.9 , 109.4 , 34.8 . hrms ( esi(+ ) ) calcd for c19h13f6n2os ( m + h ) : 431.0653 . found : 431.0646 . h nmr ( 300 hz , cdcl3 ) : 8.37 ( 2h , j = 7.8 hz , d ) , 7.507.43 ( 3h , m ) , 6.60 ( 2h , s ) , 6.56 ( 1h , s ) , 3.92 ( 3h , s ) , 3.90 ( 6h , s ) , 3.76 ( 3h , s ) . c nmr ( 75 hz , cdcl3 ) : 174.3 , 168.7 , 153.5 , 139.4 , 139.1 , 137.0 , 131.5 , 129.2 , 128.1 , 125.9 , 106.7 , 106.6 , 61.0 , 56.4 , 35.0 . hrms ( esi(+ ) ) calcd for c20h21n2o4s ( m + h ) : 385.1222 . found : 385.1215 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 1.8 and 8.1 hz , 2h ) , 7.537.42 ( m , 6h ) , 7.337.30 ( m , 2h ) , 3.61 ( s , 3h ) , 2.17 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 167.0 , 137.2 , 134.1 , 131.3 , 130.4 , 129.8 , 129.5 , 129.2 , 129.1 , 128.0 , 117.8 , 35.0 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . found : 309.1055 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 0.9 and 7.8 hz , 2h ) , 7.477.44 ( m , 3h ) , 7.327.22 ( m , 4h ) , 3.60 ( s , 3h ) , 2.15 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174 . 0 , 167.0 , 164.9 , 161.6 , 137.1 , 133.0 , 132.4 , 132.2 , 131.4 , 129.2 , 128.0 , 125.74 , 125.70 , 118.2 , 116.5 , 116.2 , 34.9 , 12.2 . . hrms ( esi(+ ) ) calcd for c18h16fn2os ( m + h ) : 327.0967 . found : 327.0957 . h nmr ( 300 mhz , cdcl3 ) : 8.38 ( dd , j = 1.5 and 7.8 hz , 2h ) , 7.487.44 ( m , 3h ) , 7.30 ( s , 4h ) , 6.53 ( s , 1h ) , 3.74 ( s , 3h ) , 2.44 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.2 , 168.9 , 139.8 , 139.6 , 137.1 , 131.4 , 129.6 , 129.23 , 129.17 , 128.0 , 127.7 , 106.6 , 34.9 , 21.4 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . found : 309.1054 . h nmr ( 300 mhz , cdcl3 ) : 8.33 ( d , j = 9.0 hz , 2h ) , 6.95 ( d , j = 9.0 hz , 2h ) , 6.60 ( s , 2h ) , 6.53 ( s , 1h ) , 3.92 ( s , 3h ) , 3.90(s , 6h ) , 3.88 ( s , 3h ) , 3.74 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 173.9 , 168.4 , 162.4 , 153.5 , 139.2 , 139.1 , 131.1 , 129.7 , 126.0 , 113.2 , 106.6 , 106.4 , 61.0 , 56.3 , 55.3 , 34.9 , 29.7 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1321 . h nmr ( 300 mhz , cdcl3 ) : 8.30 ( d , j = 8.7 hz , 2h ) , 7.42 ( d , j = 8.7 hz , 2h ) , 6.60 ( s , 2h ) , 6.58 ( s , 1h ) , 3.93 ( s , 3h ) , 3.90 ( s , 6h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 173.2 , 168.8 , 153.6 , 139.5 , 139.3 , 137.6 , 135.5 , 130.7 , 128.3 , 125.7 , 106.8 , 106.7 , 61.0 , 56.4 , 35.0 . hrms ( esi(+ ) ) calcd for c20h20cln2o4s ( m + h ) : 419.0832 . found : 419.0824 . h nmr ( 300 mhz , cdcl3 ) : 8.00 ( m , 1h ) , 7.91 ( m , 1h ) , 7.37 ( t , j = 7.8 hz , 1h ) , 7.05 ( m , 1h ) , 6.60 ( s , 2h ) , 6.57 ( s , 1h ) , 3.93 ( s , 3h ) , 3.90 ( s , 6h ) , 3.89 ( s , 3h ) , 3.76 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.8 , 159.5 , 153.5 , 139.4 , 139.2 , 138.5 , 129.0 , 125.8 , 121.8 , 118.0 , 113.6 , 106.7 , 106.6 , 61.0 , 56.4 , 55.4 , 35.0 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1320 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( dd , j = 1.8 and 7.8 hz , 2h ) , 7.497.41 ( m , 3h ) , 6.23 ( d , j = 1.2 hz , 1h ) , 3.95 ( s , 3h ) , 1.75 ( m , 1h ) , 1.02 ( m , 2h ) , 0.73 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 169.1 , 140.6 , 137.1 , 131.3 , 129.2 , 128.0 , 103.9 , 33.2 , 8.7 , 5.9 . hrms ( esi(+ ) ) calcd for c14h15n2os ( m + h ) : 259.0509 . found : 259.0519 . h nmr ( 300 mhz , cdcl3 ) : 7.68 ( s , 2h ) , 6.61 ( s , 2h ) , 6.57 ( s , 1h ) , 3.96 ( s , 6h ) , 3.93 ( s , 3h ) , 3.92(s , 3h ) , 3.91 ( s , 6h ) , 3.77 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 173.7 , 168.8 , 153.5 , 152.7 , 141.1 , 139.4 , 139.2 , 132.3 , 125.8 , 106.7 , 106.6 , 106.4 , 61.0 , 60.9 , 56.3 , 56.1 , 34.9 . hrms ( esi(+ ) ) calcd for c23h27n2o7s ( m + h ) : 475.1539 . found : 475.1526 . h nmr ( 300 mhz , cdcl3 ) : 7.97 ( dd , j = 1.8 and 7.8 hz , 1h ) , 7.607.46 ( m , 7h ) , 7.377.23 ( m , 2h ) , 3.74 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.4 , 159.0 , 146.0 , 134.5 , 133.5 , 132.1 , 131.3 , 130.9 , 130.6 , 128.73 , 128.66 , 127.6 , 126.9 , 114.6 , 38.5 . hrms ( esi(+ ) ) calcd for c17h14cln2os ( m + h ) : 329.0515 . found : 329.0515 . h nmr ( 300 mhz , cdcl3 ) : 8.1 ( s , 1h ) , 7.83 ( d , j = 7.8 hz , 1h ) , 7.597.44 ( m , 6h ) , 7.317.28 ( m , 2h ) , 3.76 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.5 , 160.2 , 147.9 , 136.7 , 134.4 , 131.0 , 130.7 , 130.2 , 129.1 , 128.7 , 127.6 , 124.5 , 122.9 , 110.2 , 38.5 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0001 . h nmr ( 300 mhz , cdcl3 ) : 7.80 ( d , j = 8.7 hz , 2h ) , 7.597.50 ( m , 7h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.4 , 160.2 , 148.3 , 134.5 , 133.7 , 131.8 , 131.0 , 128.7 , 127.6 , 121.8 , 109.6 , 38.5 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0005 . h nmr ( cdcl3 ) : 7.617.57 ( 2h , m ) , 7.557.50 ( 3h , m ) , 7.19 ( 1h , s ) , 7.15 ( 2h , s ) , 3.95 ( 6h , s ) , 3.89 ( 3h , s ) , 3.77 ( 3h , s ) . c nmr ( cdcl3 ) : 170.4 , 160.0 , 153.5 , 149.5 , 138.3 , 134.5 , 131.0 , 130.5 , 128.7 , 127.6 , 108.9 , 103.4 , 61.0 , 56.2 , 38.5 . ms - ei : 384 ( m ) . hrms ( esi(+ ) ) calcd for c20h21n2o4s ( m + h ) : 385.1222 . found : 385.1216 . h nmr ( 300 mhz , cdcl3 ) : 7.697.64 ( m , 2h ) , 7.577.48 ( m , 5h ) , 7.167.10 ( m , 2h ) , 3.67 ( s , 3h ) , 2.52 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.2 , 163.7 , 160.5 , 156.2 , 143.8 , 134.6 , 131.6 , 130.8 , 130.1 , 130.0 , 128.6 , 127.6 , 123.3 , 115.4 , 115.1 , 37.9 , 12.1 . hrms ( esi(+ ) ) calcd for c18h16fn2os ( m + h ) : 327.0967 . found : 327.0959 . h nmr ( 300 mhz , cdcl3 ) : 7.82 ( d , j = 8.1 hz , 2h ) , 7.597.49 ( m , 5h ) , 7.247.19 ( m , 3h ) , 3.76 ( s , 3h ) , 2.39 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.4 , 159.9 , 149.5 , 137.7 , 134.7 , 132.0 , 130.8 , 129.4 , 128.6 , 127.6 , 126.0 , 108.4 , 38.4 , 21.3 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . found : 309.1055 . h nmr ( 300 mhz , cdcl3 ) : 7.58 ( d , j = 8.4 hz , 2h ) , 7.16 ( s , 1h ) , 7.16 ( s , 2h ) , 7.00 ( d , j = 8.7 hz , 2h ) , 3.95 ( s , 6h ) , 3.89(s , 6h ) , 3.81 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.2 , 161.8 , 160.4 , 153.4 , 149.2 , 138.0 , 130.6 , 130.0 , 126.4 , 113.9 , 108.7 , 103.4 , 60.9 , 56.2 , 55.4 , 38.8 , 29.7 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1317 . h nmr ( 300 mhz , cdcl3 ) : 7.237.17 ( m , 3h ) , 7.16 ( s , 2h ) , 6.95 ( d , j = 8.4 hz , 1h ) , 3.96 ( s , 3h ) , 3.95(s , 6h ) , 3.93 ( s , 3h ) , 3.88 ( s , 3h ) , 3.82 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.1 , 160.4 , 153.4 , 151.4 , 149.3 , 149.0 , 138.1 , 130.5 , 126.5 , 121.3 , 111.3 , 110.4 , 108.7 , 103.4 , 61.0 , 56.2 , 56.0 , 38.8 , 29.7 . hrms ( esi(+ ) ) calcd for c22h25n2o6s ( m + h ) : 445.1433 . found : 445.1426 . h nmr ( 300 mhz , cdcl3 ) : 7.35 ( d , j = 8.4 hz , 1h ) , 7.16 ( s , 3h ) , 6.60 ( dd , j = 2.4 and 8.4 hz , 1h ) , 6.52 ( d , j = 2.1 hz , 1h ) , 3.94(s , 6h ) , 3.88 ( s , 3h ) , 3.87 ( s , 3h ) , 3.84 ( s , 3h ) , 3.64 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 168.9 , 162.8 , 159.9 , 157.1 , 153.4 , 149.0 , 138.0 , 130.7 , 129.9 , 117.2 , 108.5 , 105.0 , 103.4 , 98.5 , 60.9 , 56.2 , 55.6 , 55.5 , 36.7 . hrms ( esi(+ ) ) calcd for c22h25n2o6s ( m + h ) : 445.1433 . found : 445.1424 . h nmr ( 300 mhz , cdcl3 ) : 7.19 ( s , 1h ) , 7.16 ( s , 2h ) , 6.82 ( s , 2h ) , 3.95 ( s , 6h ) , 3.92 ( s , 3h ) , 3.90 ( s , 6h ) , 3.89(s , 3h ) , 3.80 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.1 , 160.2 , 153.5 , 153.3 , 149.4 , 140.3 , 138.2 , 130.5 , 129.5 , 108.9 , 105.1 , 103.4 , 61.01 , 60.98 , 56.3 , 56.2 , 38.7 . hrms ( esi(+ ) ) calcd for c23h27n2o7s ( m + h ) : 475.1539 . found : 475.1530 . h nmr ( 300 mhz , cdcl3 ) : 7.207.18 ( m , 2h ) , 7.15 ( s , 2h ) , 7.05 ( dd , j = 1.5 and 8.1 hz , 1h ) , 6.93 ( dd , j = 1.5 and 7.8 hz , 1h ) , 3.95(s , 6h ) , 3.93 ( s , 3h ) , 3.88 ( s , 3h ) , 3.86 ( s , 3h ) , 3.63 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 168.4 , 159.4 , 153.4 , 152.7 , 149.1 , 145.2 , 138.0 , 130.5 , 130.0 , 124.9 , 119.0 , 114.0 , 108.7 , 103.4 , 61.7 , 60.9 , 56.2 , 55.9 , 36.7 . hrms ( esi(+ ) ) calcd for c22h25n2o6s ( m + h ) : 445.1433 . found : 445.1428 . h nmr ( 300 mhz , cdcl3 ) : 7.47 ( m , 1h ) , 7.38 ( dd , j = 1.8 and 7.5 hz , 1h ) , 7.18 ( s , 1h ) , 7.16 ( s , 2h ) , 7.08 ( dt , j = 0.9 and 7.5 hz , 1h ) , 7.00 ( d , j = 8.4 hz , 1h ) , 3.95 ( s , 6h ) , 3.88 ( s , 3h ) , 3.86 ( s , 3h ) , 3.63 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 168.9 , 155.5 , 153.4 , 149.1 , 138.0 , 131.7 , 130.6 , 128.3 , 124.5 , 121.1 , 111.0 , 108.7 , 103.4 , 60.9 , 56.2 , 55.6 , 36.5 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1319 . h nmr ( 300 mhz , cdcl3 ) : 7.41 ( m , 1h ) , 7.19 ( s , 1h ) , 7.15 ( s , 2h ) , 7.157.05 ( m , 3h ) , 3.95 ( s , 6h ) , 3.89 ( s , 3h ) , 3.86 ( s , 3h ) , 3.76 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.2 , 159.7 , 153.4 , 149.3 , 138.1 , 135.7 , 130.5 , 129.8 , 119.5 , 116.8 , 112.8 , 108.9 , 103.4 , 60.9 , 56.2 , 55.4 , 38.4 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1322 . these cancer cell lines were routinely cultured in dmem medium supplemented with 10% fbs , 4 mm glutamine , 1 mm sodium pyruvate , 100 iu / ml penicillin , 100 g / ml streptomycin , and 0.25 g / ml amphotericin . briefly , mda - mb-231 , a549 , or hela cells were seeded at a density of 2.5 10 in 500 l of serum - free and phenol red - free media in the upper chamber of a 24-well transwell system . dmem supplemented with fbs ( 5% ) was used as a chemoattractant in the lower wells . after 24 h , membranes were scrubbed , fixed with 10% phospho - buffered formalin , permeabilized with 100% ice - cold methanol , and stained with 0.1% crystal violet in 20% methanol . membranes were removed and mounted on glass slides for visualization by light microscopy . for dose dependent migration assays , mda - mb-231 cells were seeded in the transwell migration chambers as described above and treated with 10 , 10 , 10 , and 10 m concentrations of each thiazole compound for measurement of resulting cell migration ; 0.1% dmso was used as vehicle control . data are represented as % of migrated cells treated with vehicle per 100 field of view ( 100 ) . for growth assay in the presence of 10 m individual thiazole compound , mda - mb-231 cells were plated in six - well plates at a density of 50 000 per well in dmem medium supplemented with 10% fbs . the cells were then cultured for 5 days , while equal treatment volumes of dmso were used as vehicle control . the ratio of thiazole compound treated cell numbers to vehicle treated cell numbers was defined as survival ratio . matrigel - coated invasion chambers ( bd biosciences , nj ) were used to determine the inhibitory effect of the thiazole derivatives on mda - mb-231 cells . briefly , cells in exponential phase of growth were serum - starved for 24 h prior to seeding , detached by brief trypsinization , and resuspended in medium containing dmso or thiazole derivatives . mda - mb-231 cells ( 2.5 10 cells / well ) were seeded in the upper chamber , and medium supplemented with 10% fbs as chemoattractant containing dmso or 10 m thiazole derivatives was added to the bottom well . after incubation for 24 h , the noninvasive cells were removed from the upper surface of the membrane by scrubbing , and the invasive cells on the under surface of the membrane were fixed with 4% formaldehyde for 10 min at room temperature and stained with 0.04% crystal violet , counted microscopically at 100 magnification . five fields per membrane the relative invasion of cells was calculated as the percentage invasion through the matrigel membrane relative to that of dmso treated cells . the fascin total rna samples were extracted from hela cells using a purelink total rna purification system ( invitrogen ) and quantitatively analyzed using a nanodrop spectrophotometer ( thermo scientific ) . the fascin cdna was generated using a superscript iii one - step rt - pcr system ( invitrogen ) with the following primers : fascin1-f ( sense ) 5-gaa ttc atg acc gcc aac ggc aca gc-3 and fascin1-r ( antisense ) 5-aag ctt cta gta ctc cca gag cga ggc-3. the rt - pcr reaction was carried out as follows : step 1 , 45 c for 30 min and 94 c for 2 min ; step 2 , for 35 cycles 94 c for 15 s , 51 c for 30 s , and 72 c for 1 min and 30 s ; step 3 , 72 c for 5 min and hold at 4 c . human fascin cdna ( 1.5 kilobases [ kb ] ) samples were then cloned into a pcdna 3.1 vector ( invitrogen ) . for fascin overepression , the mda - mb-231 cells were seeded at a density of 5 10 cells par well in six - well plates . at 7080% confluence , the cells were transiently tranfected with 4 g of the fascin - expressing plasmid pcdna - fascin . the transfection was achieved by incubating the cells overnight at 37 c in a co2 incubator with lipofectamine 2000 reagent ( invitrogen ) . the next day , cell medium was replaced with 1000 g / m g418 followed by medium replacement every 23 days for a total 1012 days . stable clones were isolated after selection with 500 g / ml g418 for 23 weeks , and fascin expression levels were determined by western blot . fertilized embryos ( charles river laboratories , charleston , sc ) were incubated at 37.5 c for 3 days , removed from their shell using a dremel tool , and placed into a covered weighing boat for 7 further days of incubation . solidified 30 l onplants containing 2.1 mg / ml rat tail collagen ( bd biosciences , bedford , ma ) and 10 ng of bfgf and 30 ng of vegf in the presence or absence of thiazole analogues 5o and 5p were placed on the cam over two pieces of nylon mesh approximately 0.5 cm . after 3 additional days of incubation , images were taken of each plug on surviving embryos using a mini - vid camera ( lw scientific ; lawrenceville , ga ) and quantified in a masked fashion on a scale from 0 to 3 with 0 representing no angiogenesis and 3 representing extreme angiogenesis . data from one scorer ( confirmed by a second masked scorer ) are presented as the mean standard error of the mean . statistical significance was determined by one - way analysis of variance ( anova ) followed by dunnett s multiple comparison test ( graphpad prism 6 , la jolla , ca ) . vehicle or thiazole analogues treated hela cells were fixed for 15 min with 3.7% paraformaldehyde in pbs . cells were washed with pbs and permeabilized in pemeabilization buffer ( 0.5% triton x-100 ( w / v ) in pbs ) for 10 min and washed again with pbs . the cells were then treated for 30 min with 200 l of 100 nm acti - stain 488 phalloidin in the dark . the coverslips were rinsed with pbs and inverted on a drop of antifade mounting media on a glass slide . cells were observed under a fluorescence microscope equipped with a 480ex/535em filter set , a digital ccd camera , and 40 objective .

fascin has recently emerged as a potential therapeutic target , as its expression in cancer cells is closely associated with tumor progression and metastasis . following the initial discovery of a series of thiazole derivatives that demonstrated potent antimigration and antiinvasion activities via possible inhibition of fascin function , we report here the design and synthesis of 63 new thiazole derivatives by further structural modifications in search of more potent fascin inhibitors . the 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs . the most potent analogue , 5p , inhibited 50% of cell migration at 24 nm . moreover , the thiazole analogues showed strong antiangiogenesis activity , blocking new blood vessel formation in a chicken embryo membrane assay . finally , a functional study was conducted to investigate the mechanism of action via interaction with the f - actin bundling protein fascin .

Introduction Results and Discussion Conclusion Experimental Section

the actin - bundling protein fascin has been linked to tumor progression , invasion , and metastasis , a fatal development of the disease . fascin has recently emerged as a novel therapeutic target for treatment of cancer metastasis . however , no follow - up studies of biological activities were reported to confirm that the compounds indeed target fascin to inhibit migration , invasion , and metastasis . from shape - based molecular modeling and subsequent rational design and synthesis , we have recently identified a thiazole lead compound 1 ( figure 1 ) that effectively blocked cell migration and invasion via interactions with the protein fascin . the finding of thiazole compounds as antimigration and antiinvasion agents opened up new possibilities of fascin - targeting molecules that can be further optimized for greater potency and bioavailability while maintaining minimal cytotoxicity . to further explore and optimize the structure activity relationships , we have designed and synthesized 63 additional thiazole derivatives where we sought to ( 1 ) homologize the two lead structures by varying the substitution on the thiazole nitrogen , ( 2 ) change the linker structure between thiazole and phenyl groups , and ( 3 ) modify other substitution groups on both the thiazole ring and the phenyl rings ( figure 1 ) . these thiazole analogues were then biologically evaluated to determine their cytotoxicity , antimigration , and antiinvasion activities . for the most potent thiazole derivatives , an in vivo assay utilizing chick embryo chorioallantoic membrane ( cam ) finally we investigated their mode of action by overexpressing the protein fascin in cancer cell lines to determine if the activities of the compounds can abrogate the enhanced migration and invasion of the transfected cell lines . as shown in scheme 1 , compounds 5 and 7 and their isomers 6 and 8 were respectively obtained from the n - alkylation of the amides 3 and 4 which were prepared following the literature procedure . a transwell migration assay was used to determine the effects of the synthesized thiazole derivatives on the migratory capacity of mda - mb-231 , an invasive and metastatic breast cancer cell line . in the presence of varying concentrations of the thiazole derivatives , the cells ability to migrate to the lower chamber with media containing 5% fbs was measured by counting the total number of cells in the lower chamber after 24 h. the concentration of individual thiazole compounds at which inhibition of migration is observed at 50% is defined as the ic50 value listed in table 1 . to evaluate possible contributions of cell viability loss to reduced migration , the thiazole derivatives were also subjected to cell survival assays to determine their cytotoxicities . the 63 newly synthesized thiazole derivatives displayed a wide range of antimigration activities as reflected in the ic50 values going from a low of 24 nm to greater than 50 m or no apparent activity . such variations in activity appears to be dependent on minor structural modifications , revealing some interesting trends that may help our understanding of the structure activity - relationships for further optimization of pharmacological index of thiazole derivatives . compounds 5l r are homologues from the lead structure 1 that were designed and prepared to investigate the effect of thiazole - n substitution on gain or loss of the antimigration activity . for comparison , 1 , the most potent thiazole derivative from our previous study , was also included in the assay . when the thiazole n - methyl is changed to an ethyl group ( 5l ) , there was a marked decrease of activity from 0.218 to 1.945 m . this trend continues as the alkyl chain length increased to n - butyl ( 5n , ic50 = 0.196 m ) , n - hexyl ( 5o , ic50 = 0.045 m ) , and n - dodecyl ( 5p , ic50 = 0.024 m ) . in addition , allyl substitution at the thiazole nitrogen ( 5q ) yielded activity comparable to n - propyl ( 5 m ) , but changing the substitution ( 5r ) to propynyl leads to a significant loss of the activity , resulting in an ic50 value of 3.89 m . on another note , putting the propynyl group on the amide nitrogen ( 6 ) appears to restore the activity . the lead compound 2 was a potent thiazole derivative discovered in a previous study , which also displayed significant cytotoxicity in several cancer cell lines at 10 m . thus , when the alkyl substitution at the thiazole nitrogen varied from methyl ( 5j ) , ethyl ( 7a ) , propyl ( 7b ) to allyl ( 7c ) and propynyl ( 7d ) , the cytotoxicity of the compounds remained significant , with relative survival ratio after 5-day treatment at 10 m ranging from 20.4% to 70.9% . however , the cytotoxicity is largely abrogated when the methyl and ethyl substitution is on the amide nitrogen ( 4e , 8a , 8b ) while strong antimigration activity was retained . it was also noted that these compounds ( 7a d ) exhibited moderate to strong inhibition of cell growth , suggesting that , in addition to blocking fascin - mediated migration , these compounds may have other molecular targets that regulate cell survival and growth . next we examine how modifications on the thiazole ring affect antimigration activity . a methyl substitution on the 5-position of the thiazole ring ( 14h ) results in a near complete loss of activity . an attempt to move the thiazole phenyl ring to the 5-position results in a more linear analogue that has stronger growth inhibitory effect but also more antimigratory ( 14j ) . interestingly , adding a thiazole n - methyl group to the above three analogues gives a more potent 15h ( compared to 14h ) and 15i ( compared to 15i ) but an inactive 15j ( compared to 14j ) . modifications on the thiazole phenyl ring by halogen and trifluoromethyl substitutions afforded six analogues 14b , 14c , 14d , 14e , 14f , 14 g , of which only 14e and 14f showed strong antimigration activities with moderate inhibition of cell growth at the same time . adding a methyl group on the thiazole nitrogen to some of these analogues appears to endow more potent cytotoxicity while increasing the overall inhibitory effect on cell motility . 15c , 15d , 15e , 15f , and 15 g all displayed potent antimigration activities with ic50 values ranging from 0.096 m ( 15f ) to 1.07 m ( 15 g ) . these compounds showed almost no antimigration activities except 14o with an ic50 value of 1.03 m . on the other hand , if the methyl substitution is on the amide nitrogen ( 16 g through 16 m ) , the results are mixed . also , from 15o to 16j , the position change of methyl substitution from thiazole nitrogen to amide nitrogen resulted in an increase of antimigration activity by 4 times . to determine the antiinvasion activities of the thiazole compounds , the most potent antimigration analogues were selected for a matrigel invasion assay . as shown in figure 2 , at 10 m , all tested compounds demonstrate 50% or greater inhibition of cell invasion through the matrigel . it is also noted that the antiinvasion activities of the selected thiazole derivatives do not always correlate with their antimigration activities . given the association of cell migration with angiogenesis and the possible involvement of fascin in angiogenesis , we next investigated if the thiazole compounds might have any antiangiogenic properties using the chick chorioallantoic membrane ( cam ) assay . in this test the vascular system of a fertilized chicken embryo is used as a model . figure 3 demonstrates the effects of 5o and 5p on the development of embryonal blood vessels compared to a negative control ( pbs ) and a positive control ( 10 ng / plug basic fibroblast growth factor ( bfgf ) and 30 ng / plug vascular endothelial growth factor ( vegf ) ) without the thiazole compounds . the antiangiogenesis activity of the three thiazole compounds was determined by the suppression of angiogenic action of bv ( bfgf + vegf ) when each compound was added to a collagen containing bv and placed on the chorioallantoic membrane of 10-day old chick embryos for 3 days . as shown in figure 3 , the analogues 5o and 5p were seen to potently block the angiogenic action of bv . angiogenesis was scored by two scorers in a blinded fashion on a scale from 0 to 3 where 0 contained no new vessels and 3 exhibited extreme angiogenesis determined by the presence of new vessels radiating out from the plug in addition to the regular repeating pattern of the normal cam vasculature . in these experiments , the thiazole analogues 5o and 5p significantly inhibited the induction of angiogenesis by bfgf and vegf ( bv ) after 3 days . our previous study found that the thiazole derivatives blocked migration and invasion of cancer cells by impairing the cytoskeleton dynamics accompanied by reduced colocalization of the actin - bundling protein fascin . to provide further evidence that fascin might be involved in the antimigratory action of the newly synthesized thiazole analogues , we overexpressed fascin in mda - mb-231 cells to investigate the resulting cell motility and the effect of the thiazole compounds on the cells overexpressing fascin . figure 4 shows the higher level of fascin expression in the transfected cells than the native level of fascin in the control cells ( figure 4a ) . consistent with previous findings , the fascin+ cells exhibited significantly higher ( 2.4-fold increase ) migration than the control cells ( vector ) . treatment of the fascin+ cells with our most potent thiazole analogues , 5o and 5p , nearly completely abrogated the enhanced migration as illustrated in figure 5 . at 10 m 5o and 5p , the control mda - mb-231 cells lost about 80% migratory capacity . the thiazole compounds also inhibited the fascin+ mda - mb-231 cells by about the same percentage ( figure 5 ) . these results provided additional evidence that the thiazole derivatives likely acted through interaction with fascin . enhanced cell migration induced by fascin - overexpression is abrogated by thiazole analogues 5l , 5o , and 5p . to investigate if the thiazole analogues inhibit cell migration and invasion by impairing the f - actin network , actin filaments of hela cells treated with selected thiazole analogues were stained with phalloidin tagged with fluorescent agent ( acti - stain 488 fluorescent phalloidin ) . as shown in figure 6 , the control cells showed a distinct presence of f - actin filaments along the cells and had more evenly distributed f - actin structures in the cytoplasm . in contrast , after treatment with the three thiazole analogues 5o , 5p , and 8b each at 10 m , the cells exhibited various degrees of disorganization of actin filaments and a decrease of f - actin staining intensity near the cell membranes . these results suggest that the antimigration and antiinvasion activities of the thiazole analogues may be mediated by interfering with the actin filament network . effects of thiazole analogues 5o , 5p , and 8b on the f - actin structure of hela cells . cells were plated on glass coverslips and incubated with vehicle ( dmso ) , 5o , 5p , or 8b for 24 h and were fixed and stained with acti - stain 488 phalloidin . cells were observed under a fluorescent microscope , a digital ccd camera , and 40 objective . in summary , the 63 new thiazole derivatives designed and prepared by further structural modifications of the previously discovered lead compounds 1 and 2 produced more potent fascin inhibitors . among the active compounds , the 5 series of analogues with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other structural motifs . the most potent analogue , 5p , inhibited 50% of cell migration at a concentration of 24 nm . as expected , all thiazole derivatives that were selected for matrigel invasion assays were found to have potent antiinvasion activities . in addition to antimigration and antiinvasion properties , two representative thiazole derivatives , 5o and 5p , also showed strong antiangiogenesis activity , blocking new blood vessel formation in a chicken embryo membrane assay . a functional study in which enhanced cell motility due to fascin overexpression was nearly completely abrogated by the most potent analogues 5o and 5p provided additional evidence that the action of the thiazole derivatives involved targeting the f - actin bundling protein fascin . this proposed mechanism of action is further supported by the observation that cellular f - actin structures were significantly impaired by the treatment of the thiazole analogues . for all products , the purity was ascertained to be greater than 95% by the hplc method using a shimadzu ( columbia , md ) 2010 hplc uv / ms system with a c-18 reverse phase column and by gc ms analyses using an agilent technologies 5975c inert msd mass spectrometer . after that , the mixture was cooled to 0 c again and mei or rbr ( 6.5 mmol ) was added dropwise . after being stirred at room temperature for 2 h under nitrogen atmosphere , the reaction mixture was concentrated in vacuo . the residue was purified by flash chromatography to give product 10 ( 0.57 g , 55% yield ) as a solid . after being stirred at room temperature for 2 h under nitrogen atmosphere , the saturated na2co3 solution was added to quench the reaction , and the solution was extracted with ethyl acetate , dried over mgso4 , and concentrated in vacuo . the residue was purified by flash chromatography to give product 14 as a solid . after that , the mixture was cooled to 0 c again and mei ( 6.5 mmol ) was added dropwise . after removal of all the solvent , the residue was purified by silica gel chromatography ( hexane / etoac = 4:1 ) to afford products 15 ( more polar ) and 16 as solids . dmem supplemented with fbs ( 5% ) was used as a chemoattractant in the lower wells . for dose dependent migration assays , mda - mb-231 cells were seeded in the transwell migration chambers as described above and treated with 10 , 10 , 10 , and 10 m concentrations of each thiazole compound for measurement of resulting cell migration ; 0.1% dmso was used as vehicle control . the ratio of thiazole compound treated cell numbers to vehicle treated cell numbers was defined as survival ratio . matrigel - coated invasion chambers ( bd biosciences , nj ) were used to determine the inhibitory effect of the thiazole derivatives on mda - mb-231 cells . mda - mb-231 cells ( 2.5 10 cells / well ) were seeded in the upper chamber , and medium supplemented with 10% fbs as chemoattractant containing dmso or 10 m thiazole derivatives was added to the bottom well . after incubation for 24 h , the noninvasive cells were removed from the upper surface of the membrane by scrubbing , and the invasive cells on the under surface of the membrane were fixed with 4% formaldehyde for 10 min at room temperature and stained with 0.04% crystal violet , counted microscopically at 100 magnification . the fascin cdna was generated using a superscript iii one - step rt - pcr system ( invitrogen ) with the following primers : fascin1-f ( sense ) 5-gaa ttc atg acc gcc aac ggc aca gc-3 and fascin1-r ( antisense ) 5-aag ctt cta gta ctc cca gag cga ggc-3. for fascin overepression , the mda - mb-231 cells were seeded at a density of 5 10 cells par well in six - well plates . at 7080% confluence , the cells were transiently tranfected with 4 g of the fascin - expressing plasmid pcdna - fascin . solidified 30 l onplants containing 2.1 mg / ml rat tail collagen ( bd biosciences , bedford , ma ) and 10 ng of bfgf and 30 ng of vegf in the presence or absence of thiazole analogues 5o and 5p were placed on the cam over two pieces of nylon mesh approximately 0.5 cm . after 3 additional days of incubation , images were taken of each plug on surviving embryos using a mini - vid camera ( lw scientific ; lawrenceville , ga ) and quantified in a masked fashion on a scale from 0 to 3 with 0 representing no angiogenesis and 3 representing extreme angiogenesis . vehicle or thiazole analogues treated hela cells were fixed for 15 min with 3.7% paraformaldehyde in pbs . cells were observed under a fluorescence microscope equipped with a 480ex/535em filter set , a digital ccd camera , and 40 objective .

primary sjgren 's syndrome ( pss ) is a systemic autoimmune disease characterized by autoimmune damage of salivary glands ( sg ) and lacrimal glands that leads to dry - mouth and dry - eye symptoms . at least one - third of patients developed extraglandular cutaneous , pulmonary , renal , or neurological manifestations , which severely affected the quality of life and could cause lethal consequences [ 1 , 2 ] . comprehensive effects of immunological , genetical , and environmental factors are involved in the pathogenesis of pss , whose exact etiology remains elusive . it has been shown that various immune cell populations such as macrophages , dendritic cells , t cells , and b cells are involved in the pathogenesis of pss [ 35 ] . a predominant presence of cd4 + t cells in inflammatory infiltrates in sg implied that t cell subsets might contribute to glandular damage in pss [ 6 , 7 ] . th17 cells , which are characterized by the production of proinflammatory cytokine il-17 , have been proved to be involved in inflammation , autoimmunity , and glandular tissue damage in pss [ 810 ] . it has been found that adoptive transfer of th17 cells into the inducible il-17a ko mice which are resistant to the induction of pss could rapidly acquire a pss profile . in patients with pss , presence of th17 cells has been observed in inflamed tissues [ 12 , 13 ] . in the salivary glands of pss patients , cd161 , also known as klrb1 ( killer cell lectin - like receptor subfamily b , member 1 ) , interacts with the ceramide - generating enzyme acid sphingomyelinase and is involved in cellular signaling and activation [ 14 , 15 ] . recently , cosmi et al . [ 16 , 17 ] have shown that cd161 is the marker of all human il-17-producing t cell subsets and human th17 cells originate from the cd4+cd161 + t cell subset . subsequent studies have highlighted a pathogenic role of cd4+cd161 + t cells in rheumatoid arthritis ( ra ) , juvenile idiopathic arthritis ( jia ) , crohn 's disease - associated perianal fistulas , and asthma [ 1821 ] . the frequency of circulating cd4+cd161 + t cells increases in ra patients and is positively correlated with the 28-joint disease activity score ( das28 ) , crp , vas score , and so forth . the accumulation of cd161 + t lymphocytes was associated with cd patients with complex fistulizing perianal disease . according to cd25 expression , cd4+cd161 + t cells can be further divided into the regulatory ( cd4+cd25+cd161 + t cell ) and effector ( cd4+cd25cd161 + t cell ) subpopulations . recent studies in inflammatory arthritis have identified cd4+cd25+cd161 + t cells as the il-17-producing th17-like treg cells [ 22 , 23 ] , which showed decreased suppressive activity and might contribute to autoimmunity in inflammatory sites . the cd4+cd25cd161 + t effector cells do not possess suppressive function and might be pathogenic in autoimmune conditions due to their il-17 production ; however , there is no clinical and experimental data directly addressing this point . in pss , the clinical relevance of circulating cd4+cd161 + t cells and their subpopulations remain elusive . in this study , we evaluated the production of il-17 of circulating cd4+cd161 + t cells in pss and determined the levels of circulating cd4+cd161 + t cells and their two subsets ( cd4+cd25+cd161 + t and cd4+cd25cd161 + t ) in pss patients . we evaluated the levels of these cell populations and analyzed the clinical significance of these cell subsets . our study showed that cd4+cd161 + t cell subsets are positively correlated with the disease activity and severity of pss and may be potential therapeutic targets for the treatment of pss . a total of 58 pss patients of the department of rheumatology and immunology , peking university people 's hospital , were recruited in the study . all patients diagnosed as pss fulfilled the 2002 american - european consensus group criteria and individuals combined with other rheumatic diseases were excluded . there were no significant differences in the ages or sex ratios between the patient and control groups . written consent was provided by all the patients recruited to donate their blood samples and clinical information in this study . peripheral blood mononuclear cells ( pbmcs ) pbmcs were stained for 30 min in the dark at 4c with the monoclonal antibodies pe - cf594 mouse anti - human cd4 ( bd biosciences , san diego , ca , usa ) , pe anti - human cd25 ( biolegend , san diego , ca , usa ) , fitc anti - human cd161 ( biolegend ) , 7aad ( bd pharmingen , san diego , ca , usa ) , and cd45ro pe - cy7 ( bd biosciences ) or with fitc mouse igg1 isotype control ( biolegend ) and mouse igg1 isotype control pe ( ebioscience , san diego , ca , usa ) . for intracellular staining of cytokines , incubate pbmcs in rpmi 1640 medium ( gibco , life technologies , shanghai , china ) in 5% co2 at 37c , then stimulate these cells for 5 h , pbmcs were stimulated for 5 h with 50 ng / ml phorbol 12-myristate 13-acetate ( pma ; sigma - aldrich , steinheim , germany ) and 1 g / ml ionomycin ( bd pharmingen ) in the presence of 10 g / ml brefeldin - a ( bfa ; biolegend ) and subsequently fixed and permeabilized by foxp3/transcription factor staining buffer set ( ebioscience ) . then the cells were stained for 30 min away from the light at 4c with anti - human il-17a apc ( ebioscience ) or mouse igg1 isotype control apc ( ebioscience ) . cd4+cd25 + and cd4+cd25 t cells were enriched from pbmcs by magnetic cell sorting ( stemcell technologies , vancouver , bc , canada ) and then stained with fitc anti - human cd161 ( biolegend ) and sorted further into the cd161 + and cd161 fractions using a bd aria ii flow cytometer . the cd4+cd25+cd161 + or cd4+cd25cd161 + t cells or cd4+cd25 t cells were cocultured with effector t cells ( teff , cd4+cd25 t ) from the third - party healthy donors stained with cfse ( invitrogen , ca , usa ) together with the treg suppression inspector beads ( miltenyi biotec gmbh , bergisch gladbach , germany ) . the following features of pss were included in this study : xerostomia , xerophthalmia , parotid gland enlargement , swollen and/or tender joints , interstitial lung diseases , anaemia ( hb < 115 g / l ) , leucopenia ( white blood cell count < 3,500/l ) , and thrombocytopenia ( platelet count < 125,000/l ) . all patients underwent extensive serological examinations , including tests of antinuclear antibody ( ana ) , anti - ro / ssa antibody ( anti - ssa ) , anti - la / ssb antibody ( anti - ssb ) , anti--fodrin antibody , rheumatoid factor ( rf ) , complement component c3 , complement component c4 , erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) , immunoglobulin a ( iga ) , immunoglobulin g ( igg ) , immunoglobulin m ( igm ) , and -globulin . anti--fodrin antibody , c3 , c4 , iga , igg , and igm were tested by elisa with normal ranges of 018 u / ml , 0.791.52 anti - ssa and anit - ssb were measured by elisa , ana was measured by indirect immunofluorescence , and rf was measured by immune turbidimetry . positive rfs were defined as values equal or more than 20 iu / ml . crp was examined by immunonephelometry method and values equal or more than 8 mg / l were considered positive . the fractional percentage of -globulin was checked by performing serum protein electrophoresis with a normal range of 11.118.8% . the disease activity was evaluated using essdai , a clinical index of disease activity measurement based on the assessment of 12 domains ( constitutional , lymphadenopathy , glandular , articular , cutaneous , pulmonary , renal , muscular , peripheral nervous system , central nervous system , hematological , and biological ) . data were presented as mean standard deviation and statistical significance between two groups was assessed with the nonparametric mann - whitney test , paired t - test , test , logistic regression , and analysis of covariance ( ancova ) . spearman 's rank correlation coefficient was applied to calculate the correlations . a value of p < 0.05 was considered to be significant . the cut - off values of t cell subsets were determined by receiver operating characteristics ( roc ) curve . demographic , clinical , and laboratory characteristics of pss patients and healthy controls are shown in table 1 . 58 pss patients and 16 healthy controls with matched age and gender were recruited in this study ( age : 57.84 13.01 versus 51.59 18.58 , p = 0.158 ; gender : p = 0.524 ) . the pss patients had a mean disease duration of 7.51 years ranging from 1 to 30.67 and the mean essdai score of these patients was 3.86 ranging from 1 to 9 ( table 1 ) . we assessed the intracellular il-17 expression in circulating cd4 + t cells of pss patients . both of the cd161 + and cd161 subsets of cd4 + t cells expressed il-17 , while the percentage of il-17-producing cells was significantly higher in the cd161 + fraction than in cd161 one ( 5.76 2.21 versus 2.24 0.94 , p = 0.0025 , figure 1(a ) ) . for the effector or regulatory subpopulation of cd4+cd161 + t cells , higher frequency of il-17-producing cells was also detected when compared with their cd4+cd161 counterpart in pss patients ( 5.52 2.28 versus 2.30 0.99 , p = 0.0058 ; 14.15 7.95 versus 3.33 1.96 , p = 0.0169 , figure 1(a ) ) . similar to il-17 , ifn- was expressed by both of the cd161 + and cd161 subsets . although ifn- expression was increased in cd161 + subset compared to the cd161 subsets , there was no statistical significance between the ifn- production level of these two subsets ( 28.88 9.04 versus 43.02 15.67 , p > 0.05 , figure 1(b ) ) . we also compared il-17 and ifn - gamma production of cd4+cd161 + t cells in pss patients and healthy controls . the il-17 expression in both cd4+cd161 + and cd4+cd161 t cells was higher in pss patients than in healthy controls , but it did not reach statistical significance ( il-17 , pss versus hc : cd161 2.53 1.29 versus 1.86 1.39 , p = 0.4641 ; cd161 + 5.29 2.37 versus 3.98 2.61 , p = 0.4318 ) . for ifn - gamma production we did not detect any difference between pss and hc patients in these two t cell subsets ( ifn - r , pss versus hc : cd161 28.78 10.23 versus 29.18 8.64 , p = 1.0000 ; cd161 + 47.82 11.80 versus 43.18 15.59 , p = 0.8413 ) . according to the study by cosmi et al . , th17 cells mainly originated from the cd4+cd161 + t cell precursors . to further identify whether the cd4+cd161 + t cell subset actively producing il-17 in this study has already attained the memory phenotype or not , we analyzed the cell - surface expression of cd45ro on these cells from pss patients . both of the cd161 + and cd161 subsets expressed cd45ro , while most of the cd4+cd161 + t cells were cd45ro+ cells , compared with the cd161 ones ( 92.74 4.28 versus 55.98 12.80 , p = 0.0079 , figure 2 ) , which clearly showed that nearly all the cd4+cd161 + t cells were memory t cells . we evaluated the levels of cd4+cd161 + t cells and their two subpopulations in pbmcs of pss patients and hcs by flow cytometry ( figures 3(a)3(c ) ) . compared with hcs , the pss patients showed a significant increase of circulating cd4+cd25+cd161 + t cell subset ( 13.04 7.32 versus 4.60 1.05 , p < 0.0001 , figure 3(d ) ) , while the levels of cd4+cd25cd161 + t and the overall cd4+cd161 + t in pbmcs were not significantly different between the pss patients and hcs ( both p > 0.05 , figures 3(e ) and 3(f ) ) . three cut - off values were determined by roc analysis to distinguish pss patients with overelevated cd161 + t cell subsets from those bearing normal cd161 + t cell subset levels comparable to the healthy controls ( 6.70% for cd4+cd25+cd161 + t cells , 17.79% for cd4+cd25cd161 + t cells , and 17.66% for overall cd4+cd161 + t cells ; table 2 ) . according to the cut - off value , cd4+cd161 + t cells and their two subpopulations were divided into the elevated group and the normal group , respectively . for the overall cd4+cd161 + t cells , it was found that their level was associated with increased esr ( = 5,346 , p = 0.021 ) even with further multivariate analysis ( p = 0.020 ) . we subsequently analyzed the correlation between cd4+cd161 + t cells and laboratory features of pss . as shown in table 3 and figure 4 , cd4+cd161 + t was positively correlated with esr ( r = 0.2776 , p = 0.0349 ) or platelet reduction ( r = 0.2736 , p = 0.0413 ) . this subset also showed a tendency of correlation with anti - ssb levels ( r = 0.2498 , p = 0.0586 ) at the border of statistical significance . the percentages of cd4+cd161 + t cells were then compared between patient groups with or without autoimmune clinical and laboratory features . patients with thrombocytopenia , ana positivity , increased esr , elevated -globulin , or essdai 4 showed higher proportions of cd4+cd161 + t ( 22.21 10.69% versus 15.95 6.95% , p = 0.013 ; 18.56 8.71% versus 12.03 4.33% , p = 0.043 ; 20.53 9.35% versus 14.86 6.48% , p = 0.012 ; 19.20 9.49% versus 14.73 5.36% , p = 0.026 ; 19.71 9.77% versus 15.30 6.18% , p = 0.049 ; table 4 , figures 5(a)5(e ) ) . further multivariate analysis still showed that patients with increased esr had significantly higher proportion of cd4+cd161 + t cell ( p = 0.005 ) , which implicated the proinflammatory role of this cell subset . the clinical relevance of the effector and regulatory subsets of cd4+cd161 + t cells was evaluated by the same analysis . although the regulatory subset ( cd4+cd25+cd161 + t cells ) of cd4+cd161 + t cells was significantly elevated in pss patients , the clinical relevance analysis did not show any significant association of this subset with the clinical and laboratory features of pss ( tables 2 , 3 , and 4 ) . the effector subset ( cd4+cd25cd161 + t cell ) showed remarkably similar clinical relevance pattern to the whole cd4+cd161 + t cells . associations were also found between the effector subset and increased esr ( p = 0.010 ) . furthermore , a positive correlation was verified between this subpopulation and esr ( r = 0.3145 , p = 0.0162 ) . a tendency of mild positive correlation between this subset and anti - ssb levels ( r = 0.2562 , p = 0.0523 ) or platelet reduction ( r = 0.2562 , p = 0.0566 ) was also observed ( table 3 , figure 4 ) . patients with thrombocytopenia , increased esr , elevated -globulin , or essdai 4 possessed higher proportions of effector subset than those without the characteristic above in univariate analysis ( 22.43 11.37% versus 16.09 7.30% , p = 0.018 ; 20.83 9.66% versus 14.74 6.96% , p = 0.010 ; 19.54 10.00% versus 14.47 5.07% , p = 0.013 ; 20.11 10.37% versus 15.13 6.02% , p = 0.032 ; table 4 , figures 5(f)5(i ) ) . foxp3 is the master regulator of the treg lineage , and helios is the specific transcription factor and marker for natural tregs which develop in thymus and keep a more stable regulatory phenotype than the induced tregs developing in the peripheral blood . the expression of the two functional regulators was evaluated in both cd4+cd25+cd161 + and cd4+cd25+cd161 t cells of pss patients . it was showed that cd4+cd25+cd161 + t cells expressed significantly lower foxp3 and helios than cd4+cd25+cd161 subpopulation ( 49.20 14.23 versus 19.75 6.77 , 41.04 15.33 versus 13.17 6.64 , p < 0.05 , figures 6(a ) and 6(b ) ) , which implicated that the cd4+cd25+cd161 + t cells might process decrease of immunoregulatory functions compared with cd4+cd25+cd161 t cells . we further compared the expression levels of foxp3 and helios in cd4+cd25+cd161 + and cd4+cd25+cd161 t cells from pss patients and healthy controls ( supplementary figures 1 and 2 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/307453 ) and found that in comparison to their cd161 + or cd161 counterparts from healthy controls , the cd4+cd25+cd161 + or cd4+cd25+cd161 t cells from pss patients had significantly elevated foxp3 or helios expression levels ( supplementary figures 1 and 2 ) . this finding suggested an increase of treg frequency took place in pss conditions , which might reflect the negative feedback to t cell responses to downregulate the autoimmunity . to directly assess the regulatory function of these t cell subsets , in vitro suppression assay although cd4+cd25+cd161 + t cells from pss patients retained suppressive activity on cd4+cd25 effector t cell proliferation , they showed decreased suppression on cd4+cd25 effector t cell proliferation compared with the cd4+cd25+cd161 t cells , which confirmed the impaired regulatory function of this inflammatory cytokine producing treg subset ( figure 7 ) . th17 cells have been proved to contribute to autoimmune responses and tissue destruction in rheumatic diseases [ 24 , 25 ] . recent studies have highlighted the pathogenic role of th17 cells in pss [ 2628 ] . il-17 might launch local inflammatory responses and further induce production of a variety of proinflammatory cytokines in pss . apart from this , th17 cells have been proved to be involved in pathogenesis of pss by producing il21 [ 2932 ] and il22 and upregulate matrix metalloproteinases ( mmps ) [ 33 , 34 ] . circulating il22 was significantly elevated in pss and showed significant correlations with major characteristics such as xerostomia , anti - ssb , rheumatoid factor , and hypergammaglobulinemia . th17 cells are also highly effective b cell helpers not only inducing a strong proliferative response of b cells in vitro but also triggering antibody production with class switch recombination in vivo , via the b cell stimulating functions of il-17 and il21 . as the il-17-producing cells , clinical relevance of cd4+cd161 + t cells has been addressed in several rheumatic diseases . cosmi et al . have found the frequencies of cd4+cd161 + cells in the jia synovial fluid are positively correlated with esr and levels of crp . maggi et al . have proved that cd4+cd161 + t lymphocytes infiltrate crohn 's disease - associated perianal fistulas and are reduced by anti - tnf local therapy . besides the roles of pathogenic cell subsets such as th17 in pss development , abnormal cytokine production in pss has been investigated for years . early studies revealed that ifn- elevation will cause epithelium destruction in targeted glands in pss , and pss was considered as a th1 driven disease for a long time . the elevation of baff was also proved to be one of the main reasons for the overactivation of b cells in pss [ 38 , 39 ] . recent studies identified the high expression of th17 related cytokines in peripheral blood or salivary glands from pss patients , which included il-17 , il-6 , il-21 , il-22 , and il-23 [ 2628 , 37 ] . the concurrent presence of these th17 cytokines stabilized th17 phenotype and will trigger the downstream inflammatory events . in this study , we found that most of the cd4+cd161 + t cells are cd45ro+ memory t cells . this suggested that cd4+cd161 + t cells had gone through antigen activation in pss and could respond to the antigens released by target organs of pss . this study examined the intracellular il-17 and ifn- expression in pss patients and verified that cd161 + subpopulation was more capable of producing il-17 than its cd161 counterparts . this observation was in agreement with previous studies that cd161 + t cells contain the majority of human th17 cells [ 1821 ] . it was also shown that cd4+cd161 + t cells expressed more ifn- than the cd4+cd161 t cells , though the difference did not reach statistical significance , which was in accordance with the previous observation that cd161 + t produces significantly more ifn- than the cd161 subset in asthma . since both ifn- and il-17 could promote the development of pss [ 13 , 28 , 37 , 41 ] , cd4+cd161 + t cells would contribute to pss pathogenesis at least by expressing these proinflammatory cytokines . besides proinflammatory cytokine expression , the migratory capacity of cd161 + t cells had been reported by previous studies . these cells could migrate to inflammatory tissues due to expression of specific chemokines like lectin - like transcript 1 or chemokine receptors such as cxcr16 or cd161 itself . once the cd161 + t cells located in the inflammatory tissues , they may function like th17 or th1 to enhance autoimmunity or take part in tissue damage by direct interactions with local tissue components . a recent study also reported the cd161 + th17 lineage cells are resistant to regulatory t cell - mediated suppression in the context of autoimmunity . the exact pathogenic roles they play in targeted tissues are still elusive now and need to be exploited in the future studies . we further revealed the clinical significance of cd4+cd161 + t cell subsets in pss for the first time , especially focusing on the correlation between these subsets and pss disease activity parameters . the circulating cd4+cd161 + t cell levels were positively correlated with esr , thrombocytopenia , and anti - ssb in pss . patients with ana or elevated -globulin also showed higher levels of cd4+cd161 + t cells . more circulating cd4+cd161 + t cells were found in patients with higher disease activity ( essdai 4 ) than patients with lower disease activity . further multivariate analysis confirmed the association of cd4+cd161 + t cells with inflammatory markers like increased esr . these results suggest that il-17-producing cd4+cd161 + t cells might play a role in the inflammation development and b cell activation in pss , at least by the effects of il-17 , though no significant difference of their levels was observed between patients with pss and hcs . cd25 has been identified as the key surface marker of regulatory t cells ( tregs ) . our recent finding has reported that cd161 expression defined an il-17-producing treg subset which might be pathogenic in inflammatory articular sites in rheumatoid arthritis . therefore , in this study we further analyzed two subsets of cd4+cd161 + t cells ( we named them effector and regulatory subpopulation ) , which were characterized by the absence or presence of cd25 expression on cell surfaces . in both two fractions , cd4+cd25cd161 + t , the effector subpopulation , the majority of cd4+cd161 + t cells , showed similar clinical relevance to the overall cd4+cd161 + t cells in pss , either in distribution in peripheral blood between patients and hcs or in correlations with disease activity parameters . t cells , the regulatory subset of cd4+cd161 + t cells , significantly increased in pss patients and the ratio of il-17-producing cells of this subset was much higher than that in the overall cd4+cd161 + t cells ( 14.15 7.95% versus 5.76 2.21% , p = 0.034 ) and cd4+cd25cd161 + subset ( 14.15 7.95% versus 5.52 2.28% , p = 0.032 ) , which might implicate that abnormal treg functions could take place in pss . the regulatory function of the regulatory subset of cd4+cd161 + t cells was also evaluated in this study . cd4+cd25+cd161 + t cells expressed significant lower foxp3 and helios than the cd4+cd25+cd161 subset , and in vitro suppression assay directly showed decreased suppression activity of the cd161 + treg subset . these results indicated that cd4+cd25+cd161 + t cells processed impaired regulatory function when they actively produced more proinflammatory cytokines like il-17 , which implicated that they might play pathogenic roles in pss . since the cd4+cd25+cd161 + t cells are rare , their overall contribution to pss disease development might not be so obvious . on the other hand , the limited sample number recruited in this study might be another reason for the lack of statistical significance . further studies are needed to elucidate the exact roles of this il-17-producing treg subset in pss . in this study , we determined the levels of circulating cd4+cd161 + t cells and their regulatory ( cd4+cd25+cd161 + t cell ) and effector ( cd4+cd25cd161 + t cell ) subpopulations in patients with pss and analyzed the clinical significance of the cd4+cd161 + t cell subsets in pss for the first time . this study showed that cd4+cd161 + t cell subpopulations were more capable of producing il-17 than their cd161 counterparts . cd4+cd25+cd161 + t cells , the regulatory subset of cd4+cd161 + t cells , significantly increased in pss patients , which showed abnormality in treg functions . the circulating cd4+cd161 + t cell levels were positively associated with disease activity parameters and autoantibody presence . these results suggest that il-17-producing cd4+cd161 + t cells might play a role in the inflammation development and b cell activation in pss .

objective . th17 cells have been demonstrated to play an important role in the onset and development of primary sjgren 's syndrome ( pss ) . in this study , we evaluated the expansion and clinical significance of circulating cd4+cd161 + t cell and its effector ( cd4+cd25cd161 + t cell ) and regulatory ( cd4+cd25+cd161 + t cell ) subpopulations . methods . fifty - eight pss patients and 16 healthy controls ( hcs ) were recruited in our study . the cell populations and intracellular il-17 expression were analyzed by flow cytometry . the disease activity was evaluated by the eular - ss disease activity index ( essdai ) . autoantibodies were measured by elisa or indirect immunofluorescence assay . results . the cd161 + t cell fractions showed higher proportions of il-17-producing cells . the frequencies of the overall cd4+cd161 + t cell population and its effector subset were positively correlated with disease activity parameters and more severe disease manifestations . a significant elevation of the cd4+cd25+cd161 + t cell subpopulation was observed in the peripheral blood of pss patients compared to hcs and this subset showed decreased regulatory functions compared with the cd4+cd25+cd161 population . conclusion . circulating cd4+cd161 + t cell populations associated with pss disease activity and severity . these cells might be involved in the development of pss and could be potential therapeutic targets in the treatment of pss .

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

primary sjgren 's syndrome ( pss ) is a systemic autoimmune disease characterized by autoimmune damage of salivary glands ( sg ) and lacrimal glands that leads to dry - mouth and dry - eye symptoms . it has been shown that various immune cell populations such as macrophages , dendritic cells , t cells , and b cells are involved in the pathogenesis of pss [ 35 ] . th17 cells , which are characterized by the production of proinflammatory cytokine il-17 , have been proved to be involved in inflammation , autoimmunity , and glandular tissue damage in pss [ 810 ] . in the salivary glands of pss patients , cd161 , also known as klrb1 ( killer cell lectin - like receptor subfamily b , member 1 ) , interacts with the ceramide - generating enzyme acid sphingomyelinase and is involved in cellular signaling and activation [ 14 , 15 ] . [ 16 , 17 ] have shown that cd161 is the marker of all human il-17-producing t cell subsets and human th17 cells originate from the cd4+cd161 + t cell subset . the frequency of circulating cd4+cd161 + t cells increases in ra patients and is positively correlated with the 28-joint disease activity score ( das28 ) , crp , vas score , and so forth . according to cd25 expression , cd4+cd161 + t cells can be further divided into the regulatory ( cd4+cd25+cd161 + t cell ) and effector ( cd4+cd25cd161 + t cell ) subpopulations . recent studies in inflammatory arthritis have identified cd4+cd25+cd161 + t cells as the il-17-producing th17-like treg cells [ 22 , 23 ] , which showed decreased suppressive activity and might contribute to autoimmunity in inflammatory sites . in pss , the clinical relevance of circulating cd4+cd161 + t cells and their subpopulations remain elusive . in this study , we evaluated the production of il-17 of circulating cd4+cd161 + t cells in pss and determined the levels of circulating cd4+cd161 + t cells and their two subsets ( cd4+cd25+cd161 + t and cd4+cd25cd161 + t ) in pss patients . we evaluated the levels of these cell populations and analyzed the clinical significance of these cell subsets . our study showed that cd4+cd161 + t cell subsets are positively correlated with the disease activity and severity of pss and may be potential therapeutic targets for the treatment of pss . a total of 58 pss patients of the department of rheumatology and immunology , peking university people 's hospital , were recruited in the study . peripheral blood mononuclear cells ( pbmcs ) pbmcs were stained for 30 min in the dark at 4c with the monoclonal antibodies pe - cf594 mouse anti - human cd4 ( bd biosciences , san diego , ca , usa ) , pe anti - human cd25 ( biolegend , san diego , ca , usa ) , fitc anti - human cd161 ( biolegend ) , 7aad ( bd pharmingen , san diego , ca , usa ) , and cd45ro pe - cy7 ( bd biosciences ) or with fitc mouse igg1 isotype control ( biolegend ) and mouse igg1 isotype control pe ( ebioscience , san diego , ca , usa ) . for intracellular staining of cytokines , incubate pbmcs in rpmi 1640 medium ( gibco , life technologies , shanghai , china ) in 5% co2 at 37c , then stimulate these cells for 5 h , pbmcs were stimulated for 5 h with 50 ng / ml phorbol 12-myristate 13-acetate ( pma ; sigma - aldrich , steinheim , germany ) and 1 g / ml ionomycin ( bd pharmingen ) in the presence of 10 g / ml brefeldin - a ( bfa ; biolegend ) and subsequently fixed and permeabilized by foxp3/transcription factor staining buffer set ( ebioscience ) . the cd4+cd25+cd161 + or cd4+cd25cd161 + t cells or cd4+cd25 t cells were cocultured with effector t cells ( teff , cd4+cd25 t ) from the third - party healthy donors stained with cfse ( invitrogen , ca , usa ) together with the treg suppression inspector beads ( miltenyi biotec gmbh , bergisch gladbach , germany ) . the following features of pss were included in this study : xerostomia , xerophthalmia , parotid gland enlargement , swollen and/or tender joints , interstitial lung diseases , anaemia ( hb < 115 g / l ) , leucopenia ( white blood cell count < 3,500/l ) , and thrombocytopenia ( platelet count < 125,000/l ) . anti--fodrin antibody , c3 , c4 , iga , igg , and igm were tested by elisa with normal ranges of 018 u / ml , 0.791.52 anti - ssa and anit - ssb were measured by elisa , ana was measured by indirect immunofluorescence , and rf was measured by immune turbidimetry . the disease activity was evaluated using essdai , a clinical index of disease activity measurement based on the assessment of 12 domains ( constitutional , lymphadenopathy , glandular , articular , cutaneous , pulmonary , renal , muscular , peripheral nervous system , central nervous system , hematological , and biological ) . demographic , clinical , and laboratory characteristics of pss patients and healthy controls are shown in table 1 . 58 pss patients and 16 healthy controls with matched age and gender were recruited in this study ( age : 57.84 13.01 versus 51.59 18.58 , p = 0.158 ; gender : p = 0.524 ) . we assessed the intracellular il-17 expression in circulating cd4 + t cells of pss patients . both of the cd161 + and cd161 subsets of cd4 + t cells expressed il-17 , while the percentage of il-17-producing cells was significantly higher in the cd161 + fraction than in cd161 one ( 5.76 2.21 versus 2.24 0.94 , p = 0.0025 , figure 1(a ) ) . for the effector or regulatory subpopulation of cd4+cd161 + t cells , higher frequency of il-17-producing cells was also detected when compared with their cd4+cd161 counterpart in pss patients ( 5.52 2.28 versus 2.30 0.99 , p = 0.0058 ; 14.15 7.95 versus 3.33 1.96 , p = 0.0169 , figure 1(a ) ) . we also compared il-17 and ifn - gamma production of cd4+cd161 + t cells in pss patients and healthy controls . the il-17 expression in both cd4+cd161 + and cd4+cd161 t cells was higher in pss patients than in healthy controls , but it did not reach statistical significance ( il-17 , pss versus hc : cd161 2.53 1.29 versus 1.86 1.39 , p = 0.4641 ; cd161 + 5.29 2.37 versus 3.98 2.61 , p = 0.4318 ) . , th17 cells mainly originated from the cd4+cd161 + t cell precursors . to further identify whether the cd4+cd161 + t cell subset actively producing il-17 in this study has already attained the memory phenotype or not , we analyzed the cell - surface expression of cd45ro on these cells from pss patients . both of the cd161 + and cd161 subsets expressed cd45ro , while most of the cd4+cd161 + t cells were cd45ro+ cells , compared with the cd161 ones ( 92.74 4.28 versus 55.98 12.80 , p = 0.0079 , figure 2 ) , which clearly showed that nearly all the cd4+cd161 + t cells were memory t cells . we evaluated the levels of cd4+cd161 + t cells and their two subpopulations in pbmcs of pss patients and hcs by flow cytometry ( figures 3(a)3(c ) ) . compared with hcs , the pss patients showed a significant increase of circulating cd4+cd25+cd161 + t cell subset ( 13.04 7.32 versus 4.60 1.05 , p < 0.0001 , figure 3(d ) ) , while the levels of cd4+cd25cd161 + t and the overall cd4+cd161 + t in pbmcs were not significantly different between the pss patients and hcs ( both p > 0.05 , figures 3(e ) and 3(f ) ) . three cut - off values were determined by roc analysis to distinguish pss patients with overelevated cd161 + t cell subsets from those bearing normal cd161 + t cell subset levels comparable to the healthy controls ( 6.70% for cd4+cd25+cd161 + t cells , 17.79% for cd4+cd25cd161 + t cells , and 17.66% for overall cd4+cd161 + t cells ; table 2 ) . for the overall cd4+cd161 + t cells , it was found that their level was associated with increased esr ( = 5,346 , p = 0.021 ) even with further multivariate analysis ( p = 0.020 ) . as shown in table 3 and figure 4 , cd4+cd161 + t was positively correlated with esr ( r = 0.2776 , p = 0.0349 ) or platelet reduction ( r = 0.2736 , p = 0.0413 ) . patients with thrombocytopenia , ana positivity , increased esr , elevated -globulin , or essdai 4 showed higher proportions of cd4+cd161 + t ( 22.21 10.69% versus 15.95 6.95% , p = 0.013 ; 18.56 8.71% versus 12.03 4.33% , p = 0.043 ; 20.53 9.35% versus 14.86 6.48% , p = 0.012 ; 19.20 9.49% versus 14.73 5.36% , p = 0.026 ; 19.71 9.77% versus 15.30 6.18% , p = 0.049 ; table 4 , figures 5(a)5(e ) ) . further multivariate analysis still showed that patients with increased esr had significantly higher proportion of cd4+cd161 + t cell ( p = 0.005 ) , which implicated the proinflammatory role of this cell subset . the clinical relevance of the effector and regulatory subsets of cd4+cd161 + t cells was evaluated by the same analysis . although the regulatory subset ( cd4+cd25+cd161 + t cells ) of cd4+cd161 + t cells was significantly elevated in pss patients , the clinical relevance analysis did not show any significant association of this subset with the clinical and laboratory features of pss ( tables 2 , 3 , and 4 ) . the effector subset ( cd4+cd25cd161 + t cell ) showed remarkably similar clinical relevance pattern to the whole cd4+cd161 + t cells . foxp3 is the master regulator of the treg lineage , and helios is the specific transcription factor and marker for natural tregs which develop in thymus and keep a more stable regulatory phenotype than the induced tregs developing in the peripheral blood . the expression of the two functional regulators was evaluated in both cd4+cd25+cd161 + and cd4+cd25+cd161 t cells of pss patients . it was showed that cd4+cd25+cd161 + t cells expressed significantly lower foxp3 and helios than cd4+cd25+cd161 subpopulation ( 49.20 14.23 versus 19.75 6.77 , 41.04 15.33 versus 13.17 6.64 , p < 0.05 , figures 6(a ) and 6(b ) ) , which implicated that the cd4+cd25+cd161 + t cells might process decrease of immunoregulatory functions compared with cd4+cd25+cd161 t cells . we further compared the expression levels of foxp3 and helios in cd4+cd25+cd161 + and cd4+cd25+cd161 t cells from pss patients and healthy controls ( supplementary figures 1 and 2 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/307453 ) and found that in comparison to their cd161 + or cd161 counterparts from healthy controls , the cd4+cd25+cd161 + or cd4+cd25+cd161 t cells from pss patients had significantly elevated foxp3 or helios expression levels ( supplementary figures 1 and 2 ) . to directly assess the regulatory function of these t cell subsets , in vitro suppression assay although cd4+cd25+cd161 + t cells from pss patients retained suppressive activity on cd4+cd25 effector t cell proliferation , they showed decreased suppression on cd4+cd25 effector t cell proliferation compared with the cd4+cd25+cd161 t cells , which confirmed the impaired regulatory function of this inflammatory cytokine producing treg subset ( figure 7 ) . apart from this , th17 cells have been proved to be involved in pathogenesis of pss by producing il21 [ 2932 ] and il22 and upregulate matrix metalloproteinases ( mmps ) [ 33 , 34 ] . have found the frequencies of cd4+cd161 + cells in the jia synovial fluid are positively correlated with esr and levels of crp . in this study , we found that most of the cd4+cd161 + t cells are cd45ro+ memory t cells . this suggested that cd4+cd161 + t cells had gone through antigen activation in pss and could respond to the antigens released by target organs of pss . this study examined the intracellular il-17 and ifn- expression in pss patients and verified that cd161 + subpopulation was more capable of producing il-17 than its cd161 counterparts . it was also shown that cd4+cd161 + t cells expressed more ifn- than the cd4+cd161 t cells , though the difference did not reach statistical significance , which was in accordance with the previous observation that cd161 + t produces significantly more ifn- than the cd161 subset in asthma . since both ifn- and il-17 could promote the development of pss [ 13 , 28 , 37 , 41 ] , cd4+cd161 + t cells would contribute to pss pathogenesis at least by expressing these proinflammatory cytokines . once the cd161 + t cells located in the inflammatory tissues , they may function like th17 or th1 to enhance autoimmunity or take part in tissue damage by direct interactions with local tissue components . a recent study also reported the cd161 + th17 lineage cells are resistant to regulatory t cell - mediated suppression in the context of autoimmunity . we further revealed the clinical significance of cd4+cd161 + t cell subsets in pss for the first time , especially focusing on the correlation between these subsets and pss disease activity parameters . the circulating cd4+cd161 + t cell levels were positively correlated with esr , thrombocytopenia , and anti - ssb in pss . patients with ana or elevated -globulin also showed higher levels of cd4+cd161 + t cells . more circulating cd4+cd161 + t cells were found in patients with higher disease activity ( essdai 4 ) than patients with lower disease activity . these results suggest that il-17-producing cd4+cd161 + t cells might play a role in the inflammation development and b cell activation in pss , at least by the effects of il-17 , though no significant difference of their levels was observed between patients with pss and hcs . therefore , in this study we further analyzed two subsets of cd4+cd161 + t cells ( we named them effector and regulatory subpopulation ) , which were characterized by the absence or presence of cd25 expression on cell surfaces . in both two fractions , cd4+cd25cd161 + t , the effector subpopulation , the majority of cd4+cd161 + t cells , showed similar clinical relevance to the overall cd4+cd161 + t cells in pss , either in distribution in peripheral blood between patients and hcs or in correlations with disease activity parameters . t cells , the regulatory subset of cd4+cd161 + t cells , significantly increased in pss patients and the ratio of il-17-producing cells of this subset was much higher than that in the overall cd4+cd161 + t cells ( 14.15 7.95% versus 5.76 2.21% , p = 0.034 ) and cd4+cd25cd161 + subset ( 14.15 7.95% versus 5.52 2.28% , p = 0.032 ) , which might implicate that abnormal treg functions could take place in pss . the regulatory function of the regulatory subset of cd4+cd161 + t cells was also evaluated in this study . cd4+cd25+cd161 + t cells expressed significant lower foxp3 and helios than the cd4+cd25+cd161 subset , and in vitro suppression assay directly showed decreased suppression activity of the cd161 + treg subset . since the cd4+cd25+cd161 + t cells are rare , their overall contribution to pss disease development might not be so obvious . on the other hand , the limited sample number recruited in this study might be another reason for the lack of statistical significance . in this study , we determined the levels of circulating cd4+cd161 + t cells and their regulatory ( cd4+cd25+cd161 + t cell ) and effector ( cd4+cd25cd161 + t cell ) subpopulations in patients with pss and analyzed the clinical significance of the cd4+cd161 + t cell subsets in pss for the first time . this study showed that cd4+cd161 + t cell subpopulations were more capable of producing il-17 than their cd161 counterparts . cd4+cd25+cd161 + t cells , the regulatory subset of cd4+cd161 + t cells , significantly increased in pss patients , which showed abnormality in treg functions . the circulating cd4+cd161 + t cell levels were positively associated with disease activity parameters and autoantibody presence . these results suggest that il-17-producing cd4+cd161 + t cells might play a role in the inflammation development and b cell activation in pss .

protozoan parasitic diseases remain a major concerned public health problem , especially in tropical regions . the major death toll is due to malaria , african and american trypanosomiasis , and leishmaniasis , whose high mortality rates in underdeveloped developing countries are associated to poor hygienic conditions and lack of efficient prophylactic measures . for many years , the public health impacts of the parasitic diseases have been grossly underestimated , mainly due to lack of awareness of its serious impact on health . protozoan parasites of the genus leishmania cause severe diseases that threaten human beings , both for the high death rates involved and the economic loss resulting from morbidity , primarily in the tropical and subtropical areas . it ranks the second only to malaria , and the control of leishmaniasis remains a serious problem with ever increasing cases worldwide . it has become a major focus of concern and a serious third world problem affecting the poorer sections of the society . leishmaniasis is included in the list of neglected tropical diseases ( ntds ) and has a strong link to poverty . the disease has been reported in 88 countries in five continents africa , asia , europe , north america , and south america out of the seven continents ( 22 in the new world and 66 in the old world ) , out of which 16 are developed countries , 72 are developing , and 13 of them are among the least developed . approximately , 350 million individuals are at risk of leishmaniasis and 20 million people are infected worldwide , and an estimated 2.0 million new cases occur each year with an incidence of 1.5 million cases per annum of the disfiguring cutaneous leishmaniasis ( cl ) and 0.5 million cases per annum of the potentially fatal visceral leishmaniasis ( vl ) [ 9 , 10 ] . however , with increasing travel to and from endemic regions , there is an increase in the number of patients suffering from leishmaniasis [ 1012 ] . the importance of this parasitic disease is further stressed out by the rise of leishmania / hiv coinfection in many parts of the world including european countries such as spain , italy , france , and portugal where up to 9% of the aids patients suffer from fatal visceral leishmaniasis . however , due to underreporting and misdiagnosis , the number of actual cases is expected to be higher . no effective vaccines are available against leishmania infections as yet and the treatment relies solely on chemotherapy , with pentavalent antimonials as first - line drugs and amphotericin b and pentamidine as second - line agents [ 10 , 11 ] . miltefosine is the first recognized oral treatment for leishmaniasis , but resistance to miltefosine may emerge easily during treatment due to single point mutation . there is a pressing need for the identification of novel drug targets , virulence factors , and development of vaccines to expand our understanding of the prevention and treatment of leishmaniasis . the majority of infections are originally zoonotic , although some cases of transmission of l. donovani from human to human are also known . the different epidemiological cycles are ( i ) a primitive or sylvatic cycle ( human infection is accidental , transmission occurring in wild foci ) , for example , l. braziliensis ; ( ii ) a secondary or peridomestic cycle ( the reservoir is a peridomestic or domestic animal , the parasite being transmitted to humans by anthropophilic sand flies ) , for example , l. infantum ; ( iii ) a tertiary , strictly anthroponotic cycle ( in which the animal reservoir has disappeared or has not yet been identified , and the sand fly vectors are totally anthroponotic ) , for example , l. donovani l. tropica was considered to be a strict anthroponosis , but several cases of canine infection have been described [ 1618 ] . leishmaniasis is a complex disease caused by haemoflagellate obligate intracellular protozoa belonging to the genus leishmania , family trypanosomatidae of the order kinetoplastida . visceral leishmaniasis ( vl ) , cutaneous leishmaniasis ( cl ) , and rarer manifestations such as mucosal leishmaniasis and post - kala - azar dermal leishmaniasis ( pkdl ) are the major forms of this disease . theparasites have a dimorphic lifecycle ; flagellated promastigotes develop in the gut of female phlebotomine sandflies to infectious forms that are transmitted to mammalian hosts [ 20 , 21 ] . inside the host , the parasites survive and multiply as amastigotes within parasitophorous vacuoles ( pvs ) of macrophages [ 22 , 23 ] . depending on the transmission cycle , vl and cl are considered to be either anthroponotic ( avl / acl ) or zoonotic ( zvl / zcl ) . anthroponotic vl is caused by leishmania donovani and is mainly distributed in the indian subcontinent where it accounts for 70% of the burden of vl with estimated annual incidence of 500,000 and 50,000 deaths each year , a death toll that is surpassed among the parasitic diseases only by malaria . both figures are approximations as vl is frequently not recognized or not reported [ 25 , 26 ] . the majority ( > 90% ) of cases occur in just six countries bangladesh , india , nepal , sudan , ethiopia , and brazil ( figure 1 ) . severe vl epidemics have been reported in the past in southern sudan ; in context of civil war and famine , vl killed an estimated 100,000 people out of a population of 280,000 between 1984 and 1998 . as india , nepal , and bangladesh harbor an estimated 67% of the global vl disease burden , the commitment of the government of these countries to launch regional vl elimination programme is welcome . the target of this programme is to eliminate vl as a public health problem by 2015 , by using a local approach to reduce the annual incidence of vl to less than one case per ten thousand individuals . visceral leishmaniasis ( vl ) results in death if not treated , the majority of leishmaniasis deaths go unrecognized , and even with treatment access , vl may result in case - fatality rates of 1020% [ 2931 ] . in east africa , it causes around 50,000 annual cases , in the form of epidemic outbreaks distributed in scattered displaced populations with a high death rate . post - kala - azar dermal leishmaniasis ( pkdl ) , which is developed in 550% of avl patients depending on geographical areas , requires lengthy and costly treatment with a low efficacy [ 32 , 33 ] . the most relevant factors behind the spread of avl are the increasing transmission in urban areas with large numbers of immigrants living in poor conditions , the breakdown of social and health structures , malnutrition inducing weakening of the immune system , and finally hiv - leishmania coinfection . the hiv - vl coinfection is characterized by frequent relapses and a high fatality rate , and cases are considered to constitute an important infectious reservoir . zoonotic vl is caused by l. infantum and is widely distributed in central asia , middle east , the mediterranean , and brazil . up to 50,000 annual cases may be caused by this form worldwide , with a scattered distribution . its causative agents are leishmania major , l. tropica , l. aethiopica , and l infantum in old world and l. mexicana , l. venezuelensis , l. amazonensis , l. braziliensis , l. panamensis , l. guyanensis , l. peruviana , and l. chagasi in new world . it is frequently self - healing in the old world but when the lesions are multiple and disabling with disfiguring scars , it creates a lifelong aesthetic stigma . in central asia , the middle east , north africa , and some sub - saharan countries , zcl caused by l. major accounts for 500,000 cases every year . acl caused by l. tropica is transmitted in urban zones and affects around 400,000 patients annually . zcl caused by l. aethiopica is present in ethiopia and is the most neglected form of cl despite 50,000 annual cases and a potential serious clinical progression , including diffuse cl and to a lesser degree mucocutaneous leishmaniasis . in south america , around 300,000 new cases of zcl species belonging to the subgenus viannia ( l. braziliensis , l. panamensis , l. peruviana , and l. guyanensis ) are capable of causing mucocutaneous leishmaniasis . no or incomplete treatment of cl is associated with the subsequent development of mucocutaneous leishmaniasis . the subgenus leishmania groups comprise two main species , l. mexicana , causing a form of cl that heals spontaneously but can sometimes cause necrosis of the external ear ( the chiclero ulcer ) , and l. amazonensis , which can in some cases manifest as diffuse cl in patients with weak immune systems . although leishmaniasis affect 98 countries in the world , it should be stressed that 90% of vl cases occur in india , bangladesh , sudan , brazil , nepal , and ethiopia , and 90% of cl cases occur in afghanistan , algeria , ethiopia , sudan , iran , iraq , saudi arabia , syria , brazil , and peru . apart from vl and cl , diffused cutaneous leishmaniasis is also a type which is difficult to treat due to disseminated lesions that resemble leprosy and do not heal spontaneously . this form is especially related to a defective immune system and it is often characterized by relapses after treatment . causative agents of mcl in old world are leishmania aethiopica ( rare ) l. major and in new world are l. mexicana , l. amazonensis , l. braziliensis , l. guyanensis , and l. panamensis . the parasite invades the mucocutaneous region of the body and spreads to the oronasal / pharyngeal mucosa . suffering and mutilation are severe , and death occurs as a result of bronchopneumonia or malnutrition . the classification of leishmania was initially based on ecobiological criteria such as vectors , geographical distribution , tropism , antigenic properties , and clinical manifestations [ 3841 ] . however , biochemical and molecular analysis showed that pathological and geographical criteria were often inadequate and thus , other criteria such as the patterns of polymorphism exhibited by kinetoplastid dna ( k - dna ) markers , proteins , or antigens came to be used to classify leishmania [ 4249 ] . initially , species classification was based on various extrinsic criteria such as clinical , geographical , and biological characteristics , for example , l. guyanensis ( isolated in guyana ) , l. peruviana ( isolated in peru ) , l. infantum ( isolated from a child in tunisia ) , and l. gerbilli ( isolated from gerbils ) . since the 1970s , intrinsic criteria such as immunological , biochemical , and genetic data have been used to define species of leishmania . use of these molecular techniques led to the publication of a taxonomic scheme by the world health organization . new methods of detection , isolation , and genetic identification resulted in a massive increase in the number of species are described . today these species generally present different epidemiological and clinical characteristics related to different genetic and phenotypic profiles . the validity of the classification scheme , considered by some workers as too arbitrary , has been questioned several times . debate has centered on l. panamensis , l. peruviana , l. chagasi , l. infantum , l. archibaldi , l. garnhami , l. pifanoi , l. venezuelensis , and l. forattinii [ 15 , 51 , 52 ] . different studies have already clarified the status of some of these species ; for example , l. chagasi is accepted as a synonym of l. infantum and l. peruviana has been validated as an independent species . very recently , fraga and his team used sequences of the highly conserved 70-kda heat shock protein ( hsp70 gene ) to analyze isolates and strains of different geographic origin , showing that only eight monophyletic groups were detectable against the 17 examined ( table 1 ) . the reason behind the diagnostic challenges for leishmaniasis is the wide spectrum of clinical manifestations that they present : ulcerative skin lesions developing at the site of the sand fly bite ( localized cutaneous leishmaniasis ) ; multiple nonulcerative nodules ( diffuse cutaneous leishmaniasis ) ; destructive mucosal inflammation ( mucosal leishmaniasis ( ml ) ) ; disseminated , potentially fatal , and visceral infection ( visceral leishmaniasis ( vl ) ) . however , differential diagnosis is important because diseases of other etiologies with a clinical spectrum similar to that of the leishmaniases ( e.g. , leprosy , skin cancers , and tuberculosis for cl and malaria and schistosomiasis for vl ) are often present in areas of endemicity . cutaneous leishmaniasis ( cl ) lesions , for example , may vary in severity ( e.g. , in lesion size ) , clinical appearance ( e.g. , open ulcer versus flat plaques versus wart - like lesions ) , and duration ( e.g. , in time of evolution or in time to spontaneous cure ) . patient management , screening of asymptomatic infections , surveillance including verification of elimination , and epidemiological studies are some of the areas where diagnostic tests play a major role . in all cases , it is desirable to have the diagnosis of leishmaniasis confirmed by the finding of the etiological agent or its antigen or molecule in the sample obtained from the lesion . when these approaches fail , immunological tests are used to provide indirect parameters for the diagnosis . ideally , a test should make the distinction between acute disease and asymptomatic infection , as most of the antileishmanial drugs are toxic . moreover , such tests should be highly sensitive and specific , simple and affordable , but unfortunately some commonly used serological tests like dat carry some significant drawbacks : the inability to differentiate between clinically active and asymptomatic infections and showing positive long after cure . molecular diagnostic tools like pcr and real - time pcr are quite sensitive and specific but are cumbersome to perform and have a high cost . dna - based tests are available in strip formats but these can not be used in the field . compared to other diagnostic techniques available , the molecular approaches remain expensive and require technological expertise , and efforts should be made to make pcr platforms more user - friendly and cost - effective , especially in remote areas where leishmaniasis is endemic . demonstration of the amastigote form of the parasite by light microscopic examination of tissue aspirates from spleen , bone marrow , or lymph nodes is regarded as the most suitable diagnostic instrument . in preparations stained with giemsa or leishman stain , the cytoplasm appears to be pale blue , with a relatively large nucleus that stains with red . in the same plane as the nucleus , but at a right angle to it , is a deep red or violet rod - like body called a kinetoplast . the sensitivity of the direct examination is low , in case of cutaneous and mucocutaneous leishmaniasis , with a range of approximately 1570% in the old and the new world [ 57 , 58 ] . in case of visceral leishmaniasis , the specificity of this technique is high , although the sensitivity varies depending on the tissue used , being higher for spleen ( 9399% ) than for bone marrow ( 5386% ) or lymph node ( 5365% ) aspirates . however , the procedure for splenic aspiration is risky for fatal internal bleeding , so the results are totally dependent on technical expertise and quality of prepared slides / reagents , both of which are often not available in field settings . the microculture method ( mcm ) is a good method for the diagnosis of visceral leishmaniasis ( vl ) with samples from both the bone marrow ( bm ) and peripheral blood ( pb ) . the mcm is superior to the traditional culture method ( tcm ) as determined by its higher sensitivity in the detection of promastigotes and the more rapid time for emergence of promastigotes . the sensitivity of mcm ( 100% in bms and 77.8100% in pb ) is considerably higher than that of the tcm ( 37.5100% in bms and 0100% in pb ) according to decreasing parasite density ( p < 0.05 ) . the serological diagnosis is based on the presence of specific humoral response for which identification of antibodies in the sera of patients is done . serum - based direct agglutination tests ( dats ) using lyophilized promastigotes or urine - based latex agglutination tests ( lats ) are used for determining antileishmanial antibodies or antigens in leishmaniasis patients . dat has a high sensitivity ( 90100% ) and specificity ( 95100% ) [ 62 , 63 ] . however , the major disadvantage of dat is the need of multiple pipetting , relatively long incubation time , high cost of antigen , and limited production facility of quality controlled antigen . as with any antibody - based test , dat remains positive for a long time after the disease is cured , thus can not be used as a test of cure or for diagnosis of relapses . detecting antigen directly is an excellent method of diagnosing an infection and is more specific than antibody - based immunodiagnostic tests . agglutination test to detect the antigen has been evaluated extensively in clinical trials , using urine collected from well - defined cases and controls from endemic and nonendemic regions . several surface antigens , ribosomal proteins , nuclear proteins , histones , and kinesin - related proteins elicit specific humoral immune responses in vl patients . recombinant antigens have considerably improved the sensitivity and specificity of immunological diagnosis over crude / total antigens . they are more preferred than other antigens both in immunoblotting as well as elisa . immunochromatographic strips using k39 antigen have been a quite promising method that has been tested widely . k39 ( recombinant ) antigen contains 39 amino acids encoded in the highly conserved kinesin region of l. chagasi . using its recombinant product , an immunochromatographic - based strip test is used in which rk39 is fixed on a nitrocellulose paper , and colloidal gold - protein a is used for detection . in the initial clinical evaluation , 100% sensitivity and 98% specificity was observed . ict suffers from the same disadvantage as dat , being positive in a significant proportion of healthy individuals in endemic regions and for long periods after cure of vl . elisa has also been used in the serodiagnosis of all types of leishmaniasis but the sensitivity and specificity of elisa depends upon the antigen used . most promising results are shown by antigen rk39 with sensitivity and specificity of 100% and 96% , respectively . the antibody titres to this antigen directly correlate with active disease and have potential in monitoring the chemotherapy and in predicting the clinical relapse . due to the requirement of skilled personnel , sophisticated equipment , and electricity , elisa is not used in the endemic regions for the diagnosis of vl . pcr - based assays form the mainstay of molecular diagnosis of leishmanial infection as they enhance sensitivity , reliability , and rapidity for the benefit of researchers and health professionals . the primers target several multicopy sequences for the diagnosis of human and canine vl which include ribosomal rna genes ; kinetoplast dna ( kdna ) ; miniexon - derived rna ( med rna ) genes and genomic repeats , the -tubulin gene region , glycoprotein 63 ( gp63 ) gene locus , and internal transcribed spacer ( its ) regions . thepcr - elisahas shown promising results for diagnosing visceral leishmaniasis ( vl ) in blood samples . however , the method has been validated mostly with hiv - positive patients who are known to have high levels of parasitaemia . as far as blood samples are concerned , pcr - elisais more sensitive ( 83.9% ) than conventional pcr ( 73.2% ) and demonstrated 100% and 87.2% specificity when healthy controls who had never travelled to a vl - endemic area and controls from a vl - endemic area as references , respectively , were used . rt - pcr simply refers to amplification of dna ( by pcr ) that is monitored while the amplification is occurring . the benefit of this rt - pcr is that it allows the researcher to better determine the amount of starting dna in the sample before the amplification by pcr . also real - time pcr can distinguish specific sequences from a complex mixture of dna . however , its application requires the availability of primers and probes that must be selected according to very rigid conditions , which can not always be easily applied . table 2 depicts the properties of few generally used diagnostic tools for leishmaniasis and pkdl . pentavalent antimonials were developed in 1945 ; the generic sodium stibogluconate ( pentosan ) and branded meglumine antimoniate , and so forth , are the first choice in the treatment of both visceral and cutaneous leishmaniases over more than five decades where resistance is not reported . to overcome the challenge of clinical resistance to antimony , pentamidine has been tried for the treatment of visceral leishmaniasis and was the first drug to be used for patients refractory to pentavalent antimony ( sbv ) . this drug is associated with serious adverse events like insulin - dependent diabetes mellitus , shock , and hypoglycaemia and death in significant proportion of patients . the declining efficacy , resistance , and serious toxicity associated with the drugs have made it unsuitable as a viable alternative to sbv for kala - azar patients [ 93 , 94 ] . amphotericin b is one of the most effective antileishmanial drug , which induces high cure rates . use of formulation of amphotericin b , a polyene antifungal drug for treatment of leishmaniasis is biochemically rational because the target of the drug is ergosterol , which is the major membrane sterol of leishmania species . due to high affinity of amphotericin b for sterols , aqueous pores are formed in the membrane leading to increased membrane permeability and killing of leishmania . the need to develop less toxic , more effective formulation of amphotericin b has led to new clinical formulation of amphotericin b in which deoxycholate has been replaced by other lipids . miltefosine , an alkyl phospholipids developed as an antitumor agent , has an excellent antileishmanial activity.paromomycin is an aminoglycoside antibiotic with unique antileishmanials activity . sitamaquine , a primaquine analogue ( 8-aminoquinolene ) , is another orally administrable compound ( table 3 ) . to date , little is known about its efficacy and toxicity . however , there is a need to go for combitorial chemotherapies which are under process and provide better outputs . the irony of the disease leishmaniasis is that it is the only tropical disease , which is being treated by nonleishmanial drugs . much of the focus till recent times was being made only on drug trials / combination therapy of available nonleishmanial drugs , evaluation of diagnostic and prognostic capability of available tools , and very little emphasis was being paid on other aspects by leishmanial biologists and researchers . recently much emphasis has been given on novel control strategies in terms of new drug targets and vaccine candidates . the route to drug target identification has been through comparative biochemistry of host and parasite enzymes , metabolites or protein identified in parasite . biochemical analysis , genome sequencing of three leishmania species ( l. major , l. braziliensis , and l. infantum ) has identified potentially useful target enzymes , transporters , metabolites , and hypothetical proteins that are distinct to parasite and their mammalian host . the genome mining will also aid in large scale proteomics studies generating expression profiles of leishmania parasites and gene targets for treatment development . search of new potential drug targets mainly focuses on biochemical and metabolic pathways essential for parasite survival . the target enzymes of these pathways should have significant structural and functional differences from their mammalian counterparts for selective inhibition of target sites . further , strategies to target more than one enzyme of a metabolic pathway simultaneously may prove more usefulness and effectiveness . biological studies for the function of 50% of leishmania genes are lacking . the comparative genome study would provide a route to find those that might be essential to each species . the enzymes of thiol metabolism / thiols , of parasitic protozoa , are different from those of mammals in many ways . trypanosoma and leishmania are most remarkable in that they have trypanothione reductase ( tr ) instead of glutathione reductase ( gr ) . this enzyme is responsible for maintaining the parasites , reducing intracellular milieu by keeping trypanothione [ n , n8-bis-(glutathionyl ) spermidine ] in the dithiol state . the crucial role of tr for thiol homeostasis and its absence from mammalian cells suggest that it might be well suited as a target molecule for rational drug development . the trypanothione system , which replaces the nearly ubiquitous glutathione / glutathione reductase ( gr ) system , protects the parasites from oxidative damage and toxic heavy metals and delivers the reducing equivalents for dna synthesis . the parasite system is far less efficient than mammalian glutathione peroxidases in detoxifying hydroperoxide , but has the advantage of much broader substrate specificity , with lipid hydroperoxides also being reduced . the relatively low activity of the tryparedoxin peroxidase system is in accordance with the high sensitivity of the parasites to oxidative stress . trypanosomes and leishmania have superoxide dismutase ( sod ) , but lack catalase and glutathione peroxidase . thus , the trypanothione system seems to be the only mechanism to detoxify hydrogen peroxides . several reverse genetic approaches have undoubtedly shown that tr is essential in different leishmania species as well as in bloodstream of t. brucei and is thus an attractive target molecule for structure - based drug design . have reported the cloning , sequencing , and expression of the tr encoding gene from l. ( l. ) amazonensis . a 3d homology model for l. amazonensis tr was constructed based on the previously reported crithidia fasciculata structure . within the past 15 years , numerous compounds have been elucidated that inhibit tr , but not human gr , which is the closest related host enzyme . despite knowledge of the three - dimensional structure of the protein and of complexes with its substrates and an inhibitor , as well as several high - throughput and virtual screening approaches , inhibitors of tr the extremely wide active site of the parasite enzyme represents an obstacle for a structure - based drug design . in addition , the pharmacokinetic properties of the potential inhibitors are crucial because insufficient uptake , rapid extrusion , or metabolism plays significant roles in determining the in vivo efficacy of a drug . recently , the biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives were evaluated to determine their effect on trypanothione reductase ( tryr ) activity in leishmania sp . and trypanosoma cruzi . mi-4-no(2 ) showed enzyme inhibition effect on extracts from different cultures of parasites , which was confirmed using the recombinant enzyme from t. cruzi ( tctryr ) and leishmania infantum ( litryr ) thioredoxin reductase ( trxr ) is a pyridine nucleotide - disulphide oxidoreductase as gr , tr , and lipoamide dehydrogenase . trxr maintains the levels of reduced thioredoxin , a protein involved in the activity of ribonucleotide reductase , transcription factors , and cell signaling and the detoxification of reactive oxygen species . most studies to date on trxr of parasitic protozoa have concerned the enzyme of p. falciparum . current evidence suggests that it is a promising drug target , although validation is awaited . the absence of this pathway in mammalian host and trypanosomatids sensitivity towards oxidative stress , trypanothione reductase , and enzymes of trypanothione metabolism is an attractive drug target for antileishmanial drug designing . homology modeling of leishmania infantum , tr , and mammalian glutathione reductase shows a remarkable difference in their three - dimensional and catalytic active sites . hence , specific inhibitors designed against tr may be an ideal drug that will stop parasite growth without altering host glutathione reductase ( gr ) activity . the enzymes of this pathway are attractive targets for the specific treatment of leishmaniasis , because the etiological agents for the disease , that is , the leishmanial parasites have a strict requirement for specific endogenous sterols ( ergosterol and analogs ) for survival and growth and can not use the abundant supply of cholesterol present in their mammalian host . a number of enzymes in the ergosterol biosynthetic pathway have been investigated as potential drug targets for these organisms and have shown great promise . thus , c14-demethylase , sterol 24-methyltransferase , 3-hydroxy-3-methylglutaryl coa reductase , squalene epoxide , squalene synthase , and farnesyl pyrophosphate synthase have been studied both individually and in combination , with varying degrees of success [ 103 , 104 ] . ergosterol biosynthesis inhibitors with potent in vitro activity and special pharmacokinetic properties in mammals can induce radical parasitological cure in animal models of several forms of leishmaniasis . trypanosomatids contain predominantly ergostane - based sterols , which differ from cholesterol , the main sterol in mammalian cells , in the presence of a methyl group in the 24 position . the methylation is initiated by s - adenosyl - l - methionine : delta ( 24 ( 25))-sterol methenyltransferase , an enzyme present in protozoa , but absent in mammals . the importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniasis . the c-24 transmethylation reactions involving s - adenosyl - l - methionine as the methyl donor and a -sterol or -sterol substrate can be inhibited by various azasterols with a nitrogen substitution in the side chain and such compounds have been tested against trypanosomatids . polyamines are ubiquitous organic cations found in all eukaryotic cells and play critical role in key cellular processes such as growth , differentiation , and macromolecular biosynthesis . during the course of inhibition of any of the polyamine , the parasite can not synthesize trypanothione , a conjugate of spermidine and glutathione that is unique to trypanosoma and leishmania . trypanothione is a reducing agent with many protective and regulatory functions and consequently its depletion proves detrimental to the parasites . recent studies on polyamine supplementation show that l. donovani lacks an intact back conversion pathway , thus the pathways operating in promastigote stage of parasite differ crucially from that in the host . adomet dc inhibitor cures animal leishmanises but has not been tested on humans and seeks further experimental studies . arginase provides a building block for production of polyamines so it has been touted as a potential antileishmanial drug target , because n(omega)-hydroxyarginine , an inhibitor of arginase that is produced by the macrophages during the formation of nitric oxide , can reduce polyamine levels in leishmania amastigotes and lowers parasitic loads [ 109 , 110 ] . other enzymes of this pathway which are under study as antileishmanial target are ornithine decarboxylases , s - adenosylmethionione , and spermidine synthase . in all kinetoplastida studied so far the majority of the glycolytic enzymes are localized in organelles called glycosomes , whereas in other organisms these are cytosolic . as a result of this compartmentation , many regulatory mechanisms operating in other cell types can not work in trypanosomes as reflected by the insensitivity of the glycosomal hexokinase ( hk ) and phosphofructokinase ( pfk ) to compounds that act as activity regulators in other cell types [ 111 , 112 ] . several approaches have been considered(1 ) exploitation of metabolic differences ; ( 2 ) exploitation of differences in 3d structure ; ( 3 ) exploitation of unique reactive residue in or near the active site of the parasite enzyme leishmania like other trypanosomatids depends solely on its host for carbon source to fulfill its energy requirements . the amastigotes uptake blood glucose from mammalian blood stream and receive other essential components like fatty acids , amino acids from phagolysosome of macrophages . due to the result of the metabolic activities of glycosomes , superoxide radicals are generated as side products in large amount . to protect glycosomal enzymes from superoxide radical toxicity , fe - superoxide dismutases ( fesods ) are evolved in leishmania species . more importantly , fesod is absent in mammalian counterpart , so it could be used as effective drug target . cyclin - dependent kinases ( cdks ) play crucial role in cell division cycle , transcription , apoptosis , and differentiation . in leishmania mexicana , disruption of cdk - related kinase 3 ( crk3 ) leads to change ploidy level of the cell , though it was avoided when extra copy of crk3 was expressed from episome , ensuring that crk3 is essential . the chemical inhibitors of class indirubin of crk3 hampers the parasite viability within macrophage , proving the validity of crk3 as potential drug target . in leishmania donovani , it was recently shown that glycogen synthase kinase ( ldgsk3 ) is also involved in cell cycle control and apoptosis as validations based on indirubin test , exploiting the ldgsk3 as potential drug target in combination with crk3 . dihydrofolate reductase ( dhfr ) is a key enzyme in folate metabolism , linked to the production of thymidine . therefore , inhibition of dhfr prevents biosynthesis of thymidine and as a consequence , dna biosynthesis . fortunately , this enzyme from leishmania major and trypanosoma cruzi has been crystallized and the structural data can be used to exploit structural difference between parasite and human enzymes that may help to design selective dhfr inhibitors [ 116 , 117 ] . an approach to discover novel parasite dhfr inhibitors using database mining has also been made to search the cambridge structural database but dhfr as drug target requires more attention [ 118 , 119 ] . in addition , it has been shown that the enzyme dihydrofolate reductase - thymidylate ( dhfr - ts ) that catalyzes conversion of dihydrofolate from methyhylene tetrahydrofolate ( m - thf ) and thymidine , which is related to parasite survival and the parasite lacking this enzyme is not able to survive in animals . the unique mitochondrial features of leishmania make this organelle an ideal drug target while minimizing toxicity.leishmania has a single large mitochondrion which is distributed in branches under the subpelicullar microtubes and a specialized region rich in dna called the kinetoplast . fonseca - silva et al . demonstrated that the effect of the quercetin is associated with ros production leading to mitochondrial dysfunction , ultimately causing parasite death . the effects of several drugs that interfere directly with mitochondrial physiology in parasites such as leishmania are under study . the unique mitochondrial features of leishmania make this organelle an ideal drug target while minimizing toxicity . all the neglected tropical diseases together take the toll of millions of lives every year . lack of efficiency , high price , and growing resistance of the current antileishmanials imply the importance of search for new targets to be focused for making drugs against leishmania . the challenge is to convert such studies in effective strategic programmes aimed to control and eradicate the disease .

leishmaniasis ranks the third in disease burden in disability - adjusted life years caused by neglected tropical diseases and is the second cause of parasite - related deaths after malaria ; but for a variety of reasons , it is not receiving the attention that would be justified seeing its importance . leishmaniasis is a diverse group of clinical syndromes caused by protozoan parasites of the genus leishmania . it is estimated that 350 million people are at risk in 88 countries , with a global incidence of 11.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis . improvements in diagnostic methods for early case detection and latest combitorial chemotherapeutic methods have given a new hope for combating this deadly disease . the cell biology of leishmania and mammalian cells differs considerably and this distinctness extends to the biochemical level . this provides the promise that many of the parasite 's proteins should be sufficiently different from hosts and can be successfully exploited as drug targets . this paper gives a brief overview of recent developments in the diagnosis and approaches in antileishmanial drug discovery and development .

1. Introduction 2. Diagnosis 3. New Potential Drug Targets 4. Thiol Metabolism 5. Mitochondria 6. Conclusions

for many years , the public health impacts of the parasitic diseases have been grossly underestimated , mainly due to lack of awareness of its serious impact on health . protozoan parasites of the genus leishmania cause severe diseases that threaten human beings , both for the high death rates involved and the economic loss resulting from morbidity , primarily in the tropical and subtropical areas . it ranks the second only to malaria , and the control of leishmaniasis remains a serious problem with ever increasing cases worldwide . leishmaniasis is included in the list of neglected tropical diseases ( ntds ) and has a strong link to poverty . the disease has been reported in 88 countries in five continents africa , asia , europe , north america , and south america out of the seven continents ( 22 in the new world and 66 in the old world ) , out of which 16 are developed countries , 72 are developing , and 13 of them are among the least developed . approximately , 350 million individuals are at risk of leishmaniasis and 20 million people are infected worldwide , and an estimated 2.0 million new cases occur each year with an incidence of 1.5 million cases per annum of the disfiguring cutaneous leishmaniasis ( cl ) and 0.5 million cases per annum of the potentially fatal visceral leishmaniasis ( vl ) [ 9 , 10 ] . however , with increasing travel to and from endemic regions , there is an increase in the number of patients suffering from leishmaniasis [ 1012 ] . the importance of this parasitic disease is further stressed out by the rise of leishmania / hiv coinfection in many parts of the world including european countries such as spain , italy , france , and portugal where up to 9% of the aids patients suffer from fatal visceral leishmaniasis . there is a pressing need for the identification of novel drug targets , virulence factors , and development of vaccines to expand our understanding of the prevention and treatment of leishmaniasis . the different epidemiological cycles are ( i ) a primitive or sylvatic cycle ( human infection is accidental , transmission occurring in wild foci ) , for example , l. braziliensis ; ( ii ) a secondary or peridomestic cycle ( the reservoir is a peridomestic or domestic animal , the parasite being transmitted to humans by anthropophilic sand flies ) , for example , l. infantum ; ( iii ) a tertiary , strictly anthroponotic cycle ( in which the animal reservoir has disappeared or has not yet been identified , and the sand fly vectors are totally anthroponotic ) , for example , l. donovani l. tropica was considered to be a strict anthroponosis , but several cases of canine infection have been described [ 1618 ] . leishmaniasis is a complex disease caused by haemoflagellate obligate intracellular protozoa belonging to the genus leishmania , family trypanosomatidae of the order kinetoplastida . visceral leishmaniasis ( vl ) , cutaneous leishmaniasis ( cl ) , and rarer manifestations such as mucosal leishmaniasis and post - kala - azar dermal leishmaniasis ( pkdl ) are the major forms of this disease . theparasites have a dimorphic lifecycle ; flagellated promastigotes develop in the gut of female phlebotomine sandflies to infectious forms that are transmitted to mammalian hosts [ 20 , 21 ] . anthroponotic vl is caused by leishmania donovani and is mainly distributed in the indian subcontinent where it accounts for 70% of the burden of vl with estimated annual incidence of 500,000 and 50,000 deaths each year , a death toll that is surpassed among the parasitic diseases only by malaria . severe vl epidemics have been reported in the past in southern sudan ; in context of civil war and famine , vl killed an estimated 100,000 people out of a population of 280,000 between 1984 and 1998 . as india , nepal , and bangladesh harbor an estimated 67% of the global vl disease burden , the commitment of the government of these countries to launch regional vl elimination programme is welcome . the target of this programme is to eliminate vl as a public health problem by 2015 , by using a local approach to reduce the annual incidence of vl to less than one case per ten thousand individuals . in east africa , it causes around 50,000 annual cases , in the form of epidemic outbreaks distributed in scattered displaced populations with a high death rate . the most relevant factors behind the spread of avl are the increasing transmission in urban areas with large numbers of immigrants living in poor conditions , the breakdown of social and health structures , malnutrition inducing weakening of the immune system , and finally hiv - leishmania coinfection . zoonotic vl is caused by l. infantum and is widely distributed in central asia , middle east , the mediterranean , and brazil . up to 50,000 annual cases may be caused by this form worldwide , with a scattered distribution . it is frequently self - healing in the old world but when the lesions are multiple and disabling with disfiguring scars , it creates a lifelong aesthetic stigma . in central asia , the middle east , north africa , and some sub - saharan countries , zcl caused by l. major accounts for 500,000 cases every year . acl caused by l. tropica is transmitted in urban zones and affects around 400,000 patients annually . zcl caused by l. aethiopica is present in ethiopia and is the most neglected form of cl despite 50,000 annual cases and a potential serious clinical progression , including diffuse cl and to a lesser degree mucocutaneous leishmaniasis . in south america , around 300,000 new cases of zcl species belonging to the subgenus viannia ( l. braziliensis , l. panamensis , l. peruviana , and l. guyanensis ) are capable of causing mucocutaneous leishmaniasis . although leishmaniasis affect 98 countries in the world , it should be stressed that 90% of vl cases occur in india , bangladesh , sudan , brazil , nepal , and ethiopia , and 90% of cl cases occur in afghanistan , algeria , ethiopia , sudan , iran , iraq , saudi arabia , syria , brazil , and peru . apart from vl and cl , diffused cutaneous leishmaniasis is also a type which is difficult to treat due to disseminated lesions that resemble leprosy and do not heal spontaneously . the parasite invades the mucocutaneous region of the body and spreads to the oronasal / pharyngeal mucosa . use of these molecular techniques led to the publication of a taxonomic scheme by the world health organization . the validity of the classification scheme , considered by some workers as too arbitrary , has been questioned several times . the reason behind the diagnostic challenges for leishmaniasis is the wide spectrum of clinical manifestations that they present : ulcerative skin lesions developing at the site of the sand fly bite ( localized cutaneous leishmaniasis ) ; multiple nonulcerative nodules ( diffuse cutaneous leishmaniasis ) ; destructive mucosal inflammation ( mucosal leishmaniasis ( ml ) ) ; disseminated , potentially fatal , and visceral infection ( visceral leishmaniasis ( vl ) ) . however , differential diagnosis is important because diseases of other etiologies with a clinical spectrum similar to that of the leishmaniases ( e.g. patient management , screening of asymptomatic infections , surveillance including verification of elimination , and epidemiological studies are some of the areas where diagnostic tests play a major role . in all cases , it is desirable to have the diagnosis of leishmaniasis confirmed by the finding of the etiological agent or its antigen or molecule in the sample obtained from the lesion . when these approaches fail , immunological tests are used to provide indirect parameters for the diagnosis . ideally , a test should make the distinction between acute disease and asymptomatic infection , as most of the antileishmanial drugs are toxic . compared to other diagnostic techniques available , the molecular approaches remain expensive and require technological expertise , and efforts should be made to make pcr platforms more user - friendly and cost - effective , especially in remote areas where leishmaniasis is endemic . demonstration of the amastigote form of the parasite by light microscopic examination of tissue aspirates from spleen , bone marrow , or lymph nodes is regarded as the most suitable diagnostic instrument . in preparations stained with giemsa or leishman stain , the cytoplasm appears to be pale blue , with a relatively large nucleus that stains with red . in the same plane as the nucleus , but at a right angle to it , is a deep red or violet rod - like body called a kinetoplast . the sensitivity of the direct examination is low , in case of cutaneous and mucocutaneous leishmaniasis , with a range of approximately 1570% in the old and the new world [ 57 , 58 ] . in case of visceral leishmaniasis , the specificity of this technique is high , although the sensitivity varies depending on the tissue used , being higher for spleen ( 9399% ) than for bone marrow ( 5386% ) or lymph node ( 5365% ) aspirates . the microculture method ( mcm ) is a good method for the diagnosis of visceral leishmaniasis ( vl ) with samples from both the bone marrow ( bm ) and peripheral blood ( pb ) . the mcm is superior to the traditional culture method ( tcm ) as determined by its higher sensitivity in the detection of promastigotes and the more rapid time for emergence of promastigotes . the serological diagnosis is based on the presence of specific humoral response for which identification of antibodies in the sera of patients is done . as with any antibody - based test , dat remains positive for a long time after the disease is cured , thus can not be used as a test of cure or for diagnosis of relapses . detecting antigen directly is an excellent method of diagnosing an infection and is more specific than antibody - based immunodiagnostic tests . several surface antigens , ribosomal proteins , nuclear proteins , histones , and kinesin - related proteins elicit specific humoral immune responses in vl patients . due to the requirement of skilled personnel , sophisticated equipment , and electricity , elisa is not used in the endemic regions for the diagnosis of vl . the primers target several multicopy sequences for the diagnosis of human and canine vl which include ribosomal rna genes ; kinetoplast dna ( kdna ) ; miniexon - derived rna ( med rna ) genes and genomic repeats , the -tubulin gene region , glycoprotein 63 ( gp63 ) gene locus , and internal transcribed spacer ( its ) regions . thepcr - elisahas shown promising results for diagnosing visceral leishmaniasis ( vl ) in blood samples . the benefit of this rt - pcr is that it allows the researcher to better determine the amount of starting dna in the sample before the amplification by pcr . pentavalent antimonials were developed in 1945 ; the generic sodium stibogluconate ( pentosan ) and branded meglumine antimoniate , and so forth , are the first choice in the treatment of both visceral and cutaneous leishmaniases over more than five decades where resistance is not reported . to overcome the challenge of clinical resistance to antimony , pentamidine has been tried for the treatment of visceral leishmaniasis and was the first drug to be used for patients refractory to pentavalent antimony ( sbv ) . amphotericin b is one of the most effective antileishmanial drug , which induces high cure rates . use of formulation of amphotericin b , a polyene antifungal drug for treatment of leishmaniasis is biochemically rational because the target of the drug is ergosterol , which is the major membrane sterol of leishmania species . due to high affinity of amphotericin b for sterols , aqueous pores are formed in the membrane leading to increased membrane permeability and killing of leishmania . however , there is a need to go for combitorial chemotherapies which are under process and provide better outputs . the irony of the disease leishmaniasis is that it is the only tropical disease , which is being treated by nonleishmanial drugs . much of the focus till recent times was being made only on drug trials / combination therapy of available nonleishmanial drugs , evaluation of diagnostic and prognostic capability of available tools , and very little emphasis was being paid on other aspects by leishmanial biologists and researchers . the genome mining will also aid in large scale proteomics studies generating expression profiles of leishmania parasites and gene targets for treatment development . biological studies for the function of 50% of leishmania genes are lacking . the enzymes of thiol metabolism / thiols , of parasitic protozoa , are different from those of mammals in many ways . this enzyme is responsible for maintaining the parasites , reducing intracellular milieu by keeping trypanothione [ n , n8-bis-(glutathionyl ) spermidine ] in the dithiol state . the parasite system is far less efficient than mammalian glutathione peroxidases in detoxifying hydroperoxide , but has the advantage of much broader substrate specificity , with lipid hydroperoxides also being reduced . the relatively low activity of the tryparedoxin peroxidase system is in accordance with the high sensitivity of the parasites to oxidative stress . several reverse genetic approaches have undoubtedly shown that tr is essential in different leishmania species as well as in bloodstream of t. brucei and is thus an attractive target molecule for structure - based drug design . have reported the cloning , sequencing , and expression of the tr encoding gene from l. ( l. ) amazonensis . within the past 15 years , numerous compounds have been elucidated that inhibit tr , but not human gr , which is the closest related host enzyme . despite knowledge of the three - dimensional structure of the protein and of complexes with its substrates and an inhibitor , as well as several high - throughput and virtual screening approaches , inhibitors of tr the extremely wide active site of the parasite enzyme represents an obstacle for a structure - based drug design . in addition , the pharmacokinetic properties of the potential inhibitors are crucial because insufficient uptake , rapid extrusion , or metabolism plays significant roles in determining the in vivo efficacy of a drug . mi-4-no(2 ) showed enzyme inhibition effect on extracts from different cultures of parasites , which was confirmed using the recombinant enzyme from t. cruzi ( tctryr ) and leishmania infantum ( litryr ) thioredoxin reductase ( trxr ) is a pyridine nucleotide - disulphide oxidoreductase as gr , tr , and lipoamide dehydrogenase . current evidence suggests that it is a promising drug target , although validation is awaited . homology modeling of leishmania infantum , tr , and mammalian glutathione reductase shows a remarkable difference in their three - dimensional and catalytic active sites . a number of enzymes in the ergosterol biosynthetic pathway have been investigated as potential drug targets for these organisms and have shown great promise . thus , c14-demethylase , sterol 24-methyltransferase , 3-hydroxy-3-methylglutaryl coa reductase , squalene epoxide , squalene synthase , and farnesyl pyrophosphate synthase have been studied both individually and in combination , with varying degrees of success [ 103 , 104 ] . trypanosomatids contain predominantly ergostane - based sterols , which differ from cholesterol , the main sterol in mammalian cells , in the presence of a methyl group in the 24 position . the importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniasis . the c-24 transmethylation reactions involving s - adenosyl - l - methionine as the methyl donor and a -sterol or -sterol substrate can be inhibited by various azasterols with a nitrogen substitution in the side chain and such compounds have been tested against trypanosomatids . during the course of inhibition of any of the polyamine , the parasite can not synthesize trypanothione , a conjugate of spermidine and glutathione that is unique to trypanosoma and leishmania . trypanothione is a reducing agent with many protective and regulatory functions and consequently its depletion proves detrimental to the parasites . recent studies on polyamine supplementation show that l. donovani lacks an intact back conversion pathway , thus the pathways operating in promastigote stage of parasite differ crucially from that in the host . arginase provides a building block for production of polyamines so it has been touted as a potential antileishmanial drug target , because n(omega)-hydroxyarginine , an inhibitor of arginase that is produced by the macrophages during the formation of nitric oxide , can reduce polyamine levels in leishmania amastigotes and lowers parasitic loads [ 109 , 110 ] . several approaches have been considered(1 ) exploitation of metabolic differences ; ( 2 ) exploitation of differences in 3d structure ; ( 3 ) exploitation of unique reactive residue in or near the active site of the parasite enzyme leishmania like other trypanosomatids depends solely on its host for carbon source to fulfill its energy requirements . due to the result of the metabolic activities of glycosomes , superoxide radicals are generated as side products in large amount . in leishmania mexicana , disruption of cdk - related kinase 3 ( crk3 ) leads to change ploidy level of the cell , though it was avoided when extra copy of crk3 was expressed from episome , ensuring that crk3 is essential . the chemical inhibitors of class indirubin of crk3 hampers the parasite viability within macrophage , proving the validity of crk3 as potential drug target . dihydrofolate reductase ( dhfr ) is a key enzyme in folate metabolism , linked to the production of thymidine . fortunately , this enzyme from leishmania major and trypanosoma cruzi has been crystallized and the structural data can be used to exploit structural difference between parasite and human enzymes that may help to design selective dhfr inhibitors [ 116 , 117 ] . in addition , it has been shown that the enzyme dihydrofolate reductase - thymidylate ( dhfr - ts ) that catalyzes conversion of dihydrofolate from methyhylene tetrahydrofolate ( m - thf ) and thymidine , which is related to parasite survival and the parasite lacking this enzyme is not able to survive in animals . demonstrated that the effect of the quercetin is associated with ros production leading to mitochondrial dysfunction , ultimately causing parasite death . all the neglected tropical diseases together take the toll of millions of lives every year .

these molecules are implicated in several processes , including adult hippocampal neurogenesis , neuronal firing activity , the formation of the synaptic cleft , the enhancement of neuronal plasticity , and the regulation of mitochondrial dynamics [ 1 , 2 ] . in this context , the ligand wnt5a has been described as an important regulator of several neuronal processes , including protection against amyloid aggregation , dendritic spine formation , expression of micrornas , and regulation of synaptic currents . together , these results highlight the importance of wnt5a in neuronal function [ 36 ] . deregulation of wnt signaling by either loss or gain of function is associated with the progression of various diseases , including cancer , diabetes mellitus type ii , and neurodegenerative diseases , such as parkinson 's and alzheimer 's disease ( ad ) [ 7 , 8 ] . in recent years , several reports have suggested a new role for wnt signaling as a regulator of metabolic pathways [ 9 , 10 ] . this idea has been proposed based on the indirect effects of wnt signaling on other regulators / sensors of glucose metabolism , including phosphoinositide 3-kinase ( pi3k ) and amp - activated protein kinase ( ampk ) , which are both involved in sensing the general metabolic state [ 11 , 12 ] . in brain tissue , glucose is oxidized through glycolysis / oxidative phosphorylation to produce atp , most of which is consumed by neurons during the restoration of ion gradients that are disrupted by synaptic transmission [ 13 , 14 ] . decreased glucose utilization by brain cells has been directly correlated with several brain pathologies , including ad [ 15 , 16 ] . by contrast , enhancing glucose utilization in vivo induces significant improvements in cognitive functions , including memory and learning [ 1719 ] . despite the importance of glucose metabolism in brain function , no studies have described the effect of the wnt5a pathway on glucose metabolism in cortical neurons . in the present study , we demonstrate that wnt5a stimulates glucose uptake , increases glycolytic rate , and stimulates the pentose phosphate pathway ( ppp ) in neurons . additionally , wnt5a treatment increases the activity of regulatory enzymes , such as hexokinase ( hk ) and glucose-6-phosphate dehydrogenase ( g6pdh ) . the effect of wnt5a was dependent on the production of nitric oxide ( no ) . these results suggest that the activation of wnt signaling plays a central role in the regulation of neuronal metabolism and that this effect could be important for the correct function of the neuronal network . the dissection was performed on samples immersed in dissection buffer containing 10 mm hepes ( ph 7.4 , 320 mosm / l ) . the tissues were incubated with 0.25% trypsin-0.2% edta ( w / v ) for 15 min at 37c and then triturated to homogeneity with a fire - polished pasteur pipette . the cells were seeded in poly - d - lysine - coated culture dishes at a density of 510 cells / cm and cultured in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen , usa ) containing 10% ( v / v ) fetal bovine serum ( thermo fisher scientific inc . after 30 min , the culture medium was changed to neurobasal ( neub ) medium supplemented with b27 ( invitrogen , usa ) , 2 mm l - glutamine , 100 u / ml penicillin , 100 mg / ml streptomycin , and 2.5 mg / ml fungizone ( thermo fisher scientific inc . for all the experiments , the cells were used after 7 days in vitro [ 20 , 21 ] . control and wnt5a - conditioned media were prepared from control l cells ( american type culture collection ( atcc ) : crl-2648 ) and l - wnt5a cells ( atcc : crl2814 ) , respectively ; the cells were cultured according to the protocol described by atcc . briefly , the cells were grown to 70% confluence in mem supplemented with 10% fbs . the medium containing the wnt ligand was recovered ( batch 1 ) , and this process was repeated with the same cells after an additional 4 days in culture ( batch 2 ) . then , batches 1 and 2 of the conditioned media were combined [ 20 , 2226 ] . the neurons were cultured in neub supplemented with b27 until the day of the experiments . before the treatment with the wnt ligand , the cells were maintained in neub without b27 for 1 h ( to avoid the possible effects of the growth factors present in b27 ) . then , the cells were incubated with control or wnt5a media ( 1 - 2 h ) . the neurons were also treated with inhibitors of wnt signaling ; the inhibitors were coincubated with the control media or wnt5a media . the inhibitors used were fzcrd-2 ( 5 ng / ml , antagonist of wnt ligands ) , kn-93 ( 10 m , inhibitor of cam kinase ii ) , g6976 ( 200 nm , inhibitor of pkc ) , and 7-nitroindazole ( 7-ni ; 5 m , inhibitor of nitric oxide synthases ) . all the wnt inhibitors were obtained from sigma - aldrich , usa . the neurons were also treated with inhibitors of glucose metabolism , such as cytochalasin b ( cyt b , 20 m , inhibitor of glucose transporters ( gluts ) ) , cytochalasin e ( cyt e , 20 m , control of the action of cyt b ) , 2-deoxy - d - glucose ( 2-dg , 7 mm , competitive inhibitor of hk ) , and dichloroacetate ( dca , 5 mm , inhibitor of glycolysis that blocks the activity of the pyruvate dehydrogenase complex ) . moreover , we treated the neurons with sodium nitroprusside ( snp , 0800 nm ) , a no donor . in all cases , the neurons were treated with the inhibitors for 30 min prior to the experiment with or without the wnt5a media . after the treatment with wnt5a in the presence or absence of wnt inhibitors , the neurons were carefully selected using a microscope to ensure that only plates containing cultures of uniformly growing neurons were used . following incubation with the wnt ligand , the cells were washed with incubation buffer ( 15 mm hepes , 135 mm nacl , 5 mm kcl , 1.8 mm cacl2 , and 0.8 mm mgcl2 ) supplemented with 0.5 mm glucose . uptake was measured at room temperature by the addition of 11.2 ci of 2-deoxy - d-[1,2-(n)h ] glucose ( [ 2-h]-dg ) at a final specific activity of 13 disintegrations / min / pmol ( approximately 1 mci / mmol ) . uptake was stopped by washing the cells with ice - cold pbs supplemented with 1 mm hgcl2 . the cells were incubated with the glucose metabolism inhibitors for 30 min in the presence of a radioactive substrate . for the slice experiments , the slices were incubated with ( [ 2-h]-dg ) for a specific period , and then a standard methodology was used . the kinetic parameters were determined using a single rectangular hyperbola of the form vmax[glc]/(km + [ glc ] ) , which was adjusted to the data by nonlinear regression using sigmaplot 12 . the ( [ 2-h]-dg ) was purchased from perkinelmer , usa . after treatment with the wnt ligand , the neurons were washed with pbs , treated with trypsin / edta , and centrifuged at 500 g for 5 min at 4c . then , the cells were resuspended at a 1 : 3 dilution in isolation medium ( 250 mm sucrose , 20 mm hepes , 10 mm kcl , 1.5 mm mgcl2 , 1 mm edta , 1 mm dtt , 2 mg / ml aprotinin , 1 mg / ml pepstatin a , and 2 mg / ml leupeptin ) , sonicated at 4c , and then centrifuged at 1,500 g for 5 min at 4c . the purified fraction was mixed with the reaction medium ( 25 mm tris - hcl , 1 mm dtt , 0.5 mm nadp / na , 2 mm mgcl2 , 1 mm atp , 2 u / ml g6pdh , and 10 mm glucose ) and incubated for 30 min at 37c . the reaction was stopped by the addition of 10% trichloroacetic acid ( tca ) , and nadph production was measured at 340 nm . after treatment with wnt5a , the cells were detached from the culture plates using trypsin / edta ( sigma - aldrich , usa ) . next , the neurons were placed in tubes containing 5 mm glucose and then washed twice in krebs henseleit solution ( 11 mm na2hpo4 , 122 mm nacl , 3.1 mm kcl , 0.4 mm kh2po4 , 1.2 mm mgso4 , and 1.3 mm cacl2 , ph 7.4 ) , containing the appropriate concentration of glucose . after equilibration in 0.5 ml of hank 's balanced salt solution / glucose at 37c for 10 min , 0.5 ml of hank 's balanced salt solution containing various concentrations of [ 3-h ] glucose was added , with a final specific activity of 13 disintegrations / min / pmol ( approximately 1 mci / mmol ) . aliquots of 100 l were then transferred to another tube , placed inside a capped scintillation vial containing 0.5 ml of water , and incubated at 45c for 48 h. after this vapor - phase equilibration step , the tube was removed from the vial , a scintillation mixture was added , and the h2o content was measured by scintillation counting over a 5 min period . the [ 3-h ] glucose was obtained from perkinelmer , usa . the cell viability after the experiment was approximately 90% . after treatment with the respective compound , the cells were washed with pbs , collected by scraping in 0.25% trypsin-0.2% edta ( w / v ) , and pelleted . subsequently , the pellet was discarded , and the supernatant was further separated by centrifugation at 13,000 g for 30 min at 4c . finally , the supernatant was used to quantify the g6pdh activity in a reaction buffer consisting of 1 mm atp and 10 mm glucose-6-phosphate ( g6p ) after a 30-min incubation at 37c . the reaction was stopped by the addition of 10% tca , and the nadph production was measured at 340 nm . glucose oxidation via the ppp was measured using a previously described method , which is based on the difference in co2 production from [ 1-c ] glucose ( decarboxylated in the 6-phosphogluconate dehydrogenase - catalyzed reaction and in the krebs cycle ) and [ 6-c ] glucose ( only decarboxylated in the krebs cycle ) . after the treatment with wnt5a in the presence or absence of inhibitors , the medium was removed , and the neurons were washed with ice - cold pbs and collected by trypsinization . the cell pellets were resuspended in o2-saturated krebs henseleit buffer ( 11 mm na2hpo4 , 122 mm nacl , 3.1 mm kcl , 0.4 mm kh2po4 , 1.2 mm mgso4 , and 1.3 mm cacl2 , ph 7.4 ) , and 500 l of this suspension ( ~10 cells ) was placed in erlenmeyer flasks with 0.5 ml of the krebs henseleit solution containing 0.5 ci d-[1-c ] glucose or 2 ci d-[6-c ] glucose and 5.5 mm d - glucose ( final concentration ) . the erlenmeyer flasks were equipped with a central well containing an eppendorf tube with 500 l of benzethonium hydroxide . the flasks were flushed with o2 for 20 s , sealed with rubber caps , and incubated for 60 min in a 37c water bath with shaking . the incubations were stopped by the injection of 0.2 ml of 1.75 m hclo4 into the main well , although shaking was continued for an additional 20 min to facilitate co2 trapping by benzethonium hydroxide . both [ 1-c ] glucose and [ 6-c ] glucose were purchased from perkinelmer , usa . after the treatment with wnt5a , we measured the atp levels in whole - cell lysates of primary neurons using an atp determination kit ( invitrogen / molecular probes ) . the animals were housed at the animal house facility of the facultad de ciencias biolgicas , pontificia universidad catlica de chile , in accordance with the guide for the care and use of laboratory animals ( nih - usa publication 86 - 23 ) . transverse slices ( 350 m ) from the dorsal hippocampus were cut in cold artificial cerebrospinal fluid ( acsf , 119 mm nacl , 26.2 mm nahco3 , 2.5 mm kcl , 1 mm nah2po4 , 1.3 mm mgcl2 , and 10 mm glucose ) using a vibroslice microtome ( world precision instruments ) and incubated in acsf for 1 h at room temperature . the experiments were performed in a recording chamber at room temperature ( 2022c ) [ 38 , 39 ] . all the experiments were performed with an n of 5 ; we used triplicates for each condition of each experiment . the data were analyzed by one - way or two - way analysis of variance ( anova ) followed by bonferroni 's post hoc test ; p 0.05 and p 0.01 were considered significant . the activation of the noncanonical wnt pathway was determined by monitoring the p - pkc levels . we observed a 1.5-fold increase in the p - pkc / pkc ratio after 2 h of wnt5a treatment , and this increase was blocked by the wnt scavenger fzcrd-2 ( data not shown ) . the first step of glucose metabolism is the uptake of glucose from the extracellular media into the cell . we used 2-deoxyglucose ( 2-dg ) , a radioactive analog of glucose , to study glucose uptake . in the brain , glucose is taken up by glucose transporters ( gluts ) and phosphorylated by the enzyme hk , but it is not further metabolized . under control conditions , we observed a time - dependent uptake of 2-dg , with a maximum of 19.2 3.5 nmol 10 cells at 90 sec . after the wnt5a treatment , the 2-dg uptake increased , with a maximum of 39.4 5.3 nmol 10 cells at 90 sec . wnt pathway activation triggered a 2-fold increase in 2-dg uptake , with a significant difference at 15 sec . treatment with the glut inhibitor cyt b ( 20 m ) decreased 2-dg uptake to 1.56 0.4 nmol 10 cells at 90 sec ( figure 1(a ) ) . incubation with wnt5a during the initial phase of monitoring induced an increase in uptake from 9.9 1.5 nmol 10 cells to 20.8 2.0 nmol 10 cells ; this increase was blocked by the coincubation with fzcrd-2 . interestingly , we observed that 7-ni , an inhibitor of neuronal no synthase ( nnos ) [ 41 , 42 ] , blocked the effect of wnt5a , suggesting that the effect of this ligand was mediated by the intracellular release of ca followed by the downstream generation of no . we treated the cells with inhibitors of several downstream targets of noncanonical wnt signaling to further investigate this effect . neither kn-93 ( inhibitor of calcium / calmodulin - dependent protein kinase ii ( camkii ) ) nor g6976 ( inhibitor of pkc ) blocked the effect of wnt5a . by contrast , treatment with the no donor snp alone increased the 2-dg uptake to 17.8 4.3 nmol 10 cells ( figure 1(b ) ) . the kinetic parameters of glucose uptake were estimated for a more comprehensive analysis of the effect of wnt signaling . under control conditions , we observed a km value of 7.1 0.8 mm and a vmax of 9.4 0.4 pmol10 cells / min ( figure 1(c ) ) . in the wnt5a - treated cells , the km and vmax values were 2.5 0.1 mm and 8.7 0.2 pmol10 cells / min , respectively ( figure 1(d ) ) . figure 1(e ) illustrates how exposure to the wnt5a ligand increased the apparent affinity of glucose in the neuronal transport pathway . after being transported into the cell , glucose is converted to g6p by hk , the first regulatory enzyme of glycolysis . we observed that the wnt5a treatment induced a robust increase in activity from 2.2 0.17 units / mg to 4.4 0.8 units / mg after 2 h of treatment ( figure 2(a ) , ( i ) ) . the effect of wnt5a was prevented by the coincubation with fzcrd-2 and 7-ni ( figure 2(a ) , ( ii ) ) . our results thus far show that treating neurons with wnt5a stimulates the uptake of 2-dg and hk activity . the next step was to determine whether this increased uptake was correlated with an increase in the glucose utilization through glycolysis . first , we measured the glycolytic rate at several time points after treatment with wnt5a . under control conditions , we observed rates of 1.1 0.15 pmol / mg protein . wnt5a increased the glycolytic rate in a time - dependent manner , with a maximum rate of 2.9 0.3 pmol / mg protein at 2 h versus a control rate of 1.3 0.19 pmol / mg protein . this effect was partially blocked by the coincubation with fzcrd-2 ( figure 2(b ) , ( i ) ) . using a pharmacological approach , we studied the effects of several inhibitors at 2 h and observed that the effect of wnt5a was blocked by the coincubation with fzcrd-2 and 7-ni ( figure 2(b ) , ( ii ) ) . our previous results suggest that the downstream generation of no following the activation of noncanonical wnt signaling may be important for the effect of wnt5a on the glycolytic rate . therefore , we analyzed the effect of snp , a no donor , by treating cells with several concentrations of snp for 2 h ; we observed a strong increase in the glycolytic rate , with a maximum of 150 12 pmol / mg protein in the cells that were treated with 700 nm snp . the effect of snp was partially abolished by the coincubation with 7-ni . as a control , the cyt b treatment markedly decreased the glycolytic rate in neurons ( figure 2(b ) , ( iii ) ) . our results indicate that the activation of wnt signaling stimulates glucose uptake and the utilization of this molecule by glycolysis , suggesting an increase in the levels of atp . we measured the neuronal atp levels following a 2 h treatment with wnt5a to confirm this prediction . under control conditions , we measured a value of 36.02 10.03 nmol atp / mg of protein . after the treatment with wnt5a , the atp levels increased to 61.78 6.11 nmol atp / mg of protein ; this increase in the atp levels was blocked when cells were coincubated with wnt5a and the antagonist fzdcrd-2 ( figure 2(c ) ) . as described above , several metabolic pathways could use the g6p generated by hk , including the ppp . we measured the effect of wnt5a on g6pdh activity and observed an increase in the enzymatic activity after 1 h of wnt5a treatment ( figure 3(a ) , ( i ) ) . the effect of wnt5a on g6pdh activity was blocked by the coincubation with fzcrd-2 and 7-ni ( figure 3(a ) , ( ii ) ) . then , we measured the ppp activity using radioactive glucose and observed that the wnt5a treatment increased the ppp activity from 0.33 0.02 nmol / min mg protein in the control condition to 0.77 0.07 nmol / min mg protein . the effect of wnt5a on the ppp activity was blocked by the coincubation with fzdcrd-2 ( figure 3(b ) ) . we studied whether the effects of the wnt5a ligand could be recapitulated in a more complex model to further analyze the effect of wnt pathway activation on glucose metabolism . for this experiment , we used cortical - hippocampal slices from mouse brains ( figure 4(a ) ) . after a 30 min treatment , we observed significant time - dependent differences in the accumulation of 2-dg , with a maximum of 6.9 0.9 nmol 10 after 2 h of wnt5a treatment compared with 3.4 0.4 nmol 10 for the control condition ( figure 4(b ) , ( i ) ) . the effect of a 1 h wnt5a treatment was blocked by treatments with fzdcrd-2 and 7-ni in a similar manner to that observed in neuronal cultures ( figure 4(b ) , ( ii ) ) . after the 1 h incubation with wnt5a , we observed an increase in the glycolytic rate in the slices from 1.07 0.11 pmol / mg protein to 2.12 0.08 pmol / mg protein ; this effect was blocked by the coincubation with 2-dg and fzdcrd-2 ( figure 4(c ) ) . finally , we measured the ppp activity in slices and observed an activity of 0.32 0.01 nmol / min mg protein in the controls . the wnt5a treatment induced an increase in the ppp activity to 0.78 0.02 01 nmol / min mg protein ; this effect was blocked by the coincubation with fzdcrd-2 and 7-ni ( figure 4(d ) ) . in the present work , we studied the effect of wnt5a on glucose metabolism in cortical neurons . we report that wnt5a treatment stimulates glucose uptake in a time - dependent manner ; this increase was correlated with an increase in both the hk activity and the glycolytic rate . moreover , the treatment with wnt5a increased the activity of the g6pdh enzyme and the ppp . together , these results suggest that the activation of wnt signaling by wnt5a regulates cellular glucose metabolism in neurons . wnt signaling can basically be divided into two pathways : the canonical pathway ( wnt/-catenin ) and the noncanonical pathway . wnt5a has been described as a noncanonical wnt ligand , and the noncanonical pathway includes the planar cell polarity ( wnt / pcp ) and wnt / ca pathways . in the wnt / pcp pathway , the wnt ligand binds to the fzd receptor and activates small gtpases that subsequently induce the expression of genes related to the reorganization of the cytoskeleton . in the wnt / ca pathway , ligand binding to the fzd receptor activates the enzyme phospholipase c ( plc ) , which increases the production of inositol triphosphate ( ip3 ) , generating an increase in the intracellular ca concentration that leads to the activation of calcium - dependent proteins [ 4547 ] . subsequently , wnt5a signaling increases the intracellular levels of several second messengers , including ca and no , which are both considered regulators of glucose metabolism , a critical process required for brain function [ 4851 ] . the importance of the wnt pathway in regulating glucose metabolism has been increasingly recognized in recent years due to studies in humans , where several components of the wnt pathway have been identified as risk factors for metabolic diseases , including age - related dementia ; however , the final effect depends on whether the canonical or noncanonical wnt pathway is affected [ 52 , 53 ] . activation of the wnt/-catenin pathway promotes a decrease in the plasma glucose levels in vivo , and this decrease modulates the localization and expression of glut4 in adipocytes and increases glucose uptake in these cells . similarly , patients with diabetes mellitus type ii have been shown to exhibit increased expression of sfrp5 , a wnt inhibitor , which has been correlated with a decrease in wnt5a levels . furthermore , activation of the wnt / ca pathway has been reported to modulate mitochondrial dynamics , which may affect atp generation [ 56 , 57 ] . approximately 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to cerebral functions , even though the brain only accounts for 2% of the total body mass . the atp generated by glucose oxidation is used to maintain and restore the ion gradients dissipated by signaling processes , such as postsynaptic stimulation and action potentials , and for neurotransmitter uptake and recycling . glucose is the main energy substrate of the adult brain , and its metabolism is mainly divided into two stages : glucose uptake and intracellular oxidative metabolism . our data also showed that wnt5a increases the apparent affinity of the glut3 transporter , the main glut isoform expressed in neurons . because we did not observe changes in the vmax value , we can disregard the expression of other glut isoforms in our experiments . the effect of wnt5a on glucose uptake will increase the bioavailability of intracellular glucose , with a subsequent increase in the g6p levels generated by hk , another target of the wnt5a pathway . the generated g6p is mainly used by three different metabolic pathways : glycolysis , the ppp , and the glycogen synthesis pathway . glycolysis is coupled to the krebs cycle and oxidative phosphorylation to generate substrates for atp production in mitochondria through oxidative phosphorylation . by contrast , the ppp is required to protect neurons from oxidative stress through the generation of nadph [ 61 , 62 ] . however , the neuronal utilization of glucose for glycogen synthesis has been accepted for years ; neurons have recently been shown to use glucose for glycogen synthesis , a function that is typically attributed to astrocytes . however , this glycogen synthesis could induce neuronal cell death through the formation of glycogen aggregates called lafora bodies [ 64 , 65 ] . neuronal glycogen may be important for promoting neuronal survival under pathological conditions , such as oxidative stress and hypoxia [ 6668 ] . in the present work , we did not evaluate the effect of wnt5a on glycogen synthesis , but we can not discard this possibility because the importance of glycogen in neurons has recently been reported [ 67 , 69 ] . we observed that wnt5a increases the glycolytic rate and ppp activity , thus promoting atp generation and increasing the levels of the nadph cofactor . the increase in the nadph levels resulting from ppp activity could be a protective mechanism against oxidative stress ( generated by the increased ros levels produced in the mitochondria ) , because this molecule is critical for the recycling of antioxidant agents , such as ascorbic acid and glutathione [ 69 , 70 ] . additionally , we observed that the effects of wnt5a on 2-dg uptake and the glycolytic rate were blocked by an inhibitor of nnos . this finding suggests a role for intracellular no , a molecule generated downstream of the wnt / ca pathway , because ca release activates calcineurin , a phosphatase that leads to nnos activation and no production ( figure 5 ) [ 41 , 71 ] . no has been previously shown to stimulate glucose metabolism in neurons ; however , the specific mechanism underlying this effect remains unknown [ 72 , 73 ] . because the brain includes other cells , such as astrocytes , which play critical roles in the regulation of neuronal metabolism [ 7476 ] , we tested the effects of wnt5a on the more complex system of hippocampal slices . here , we observed results similar to those of the in vitro studies , suggesting that the effects of wnt5a are mainly localized to neurons ; however , further studies are required to confirm this hypothesis . our results suggest a novel function for wnt signaling as an activator of glucose metabolism in neurons . this novel role of wnt signaling in neuronal physiology might be an interesting topic in the search for new treatments for neurological disorders .

in the last few years , several reports have proposed that wnt signaling is a general metabolic regulator , suggesting a role for this pathway in the control of metabolic flux . wnt signaling is critical for several neuronal functions , but little is known about the correlation between this pathway and energy metabolism . the brain has a high demand for glucose , which is mainly used for energy production . neurons use energy for highly specific processes that require a high energy level , such as maintaining the electrical potential and synthesizing neurotransmitters . moreover , an important metabolic impairment has been described in all neurodegenerative disorders . despite the key role of glucose metabolism in the brain , little is known about the cellular pathways involved in regulating this process . we report here that wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway ; the effects of wnt5a require the intracellular generation of nitric oxide . our data suggest that wnt signaling stimulates neuronal glucose metabolism , an effect that could be important for the reported neuroprotective role of wnt signaling in neurodegenerative disorders .

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

these molecules are implicated in several processes , including adult hippocampal neurogenesis , neuronal firing activity , the formation of the synaptic cleft , the enhancement of neuronal plasticity , and the regulation of mitochondrial dynamics [ 1 , 2 ] . in this context , the ligand wnt5a has been described as an important regulator of several neuronal processes , including protection against amyloid aggregation , dendritic spine formation , expression of micrornas , and regulation of synaptic currents . deregulation of wnt signaling by either loss or gain of function is associated with the progression of various diseases , including cancer , diabetes mellitus type ii , and neurodegenerative diseases , such as parkinson 's and alzheimer 's disease ( ad ) [ 7 , 8 ] . in recent years , several reports have suggested a new role for wnt signaling as a regulator of metabolic pathways [ 9 , 10 ] . this idea has been proposed based on the indirect effects of wnt signaling on other regulators / sensors of glucose metabolism , including phosphoinositide 3-kinase ( pi3k ) and amp - activated protein kinase ( ampk ) , which are both involved in sensing the general metabolic state [ 11 , 12 ] . in brain tissue , glucose is oxidized through glycolysis / oxidative phosphorylation to produce atp , most of which is consumed by neurons during the restoration of ion gradients that are disrupted by synaptic transmission [ 13 , 14 ] . despite the importance of glucose metabolism in brain function , no studies have described the effect of the wnt5a pathway on glucose metabolism in cortical neurons . in the present study , we demonstrate that wnt5a stimulates glucose uptake , increases glycolytic rate , and stimulates the pentose phosphate pathway ( ppp ) in neurons . additionally , wnt5a treatment increases the activity of regulatory enzymes , such as hexokinase ( hk ) and glucose-6-phosphate dehydrogenase ( g6pdh ) . the effect of wnt5a was dependent on the production of nitric oxide ( no ) . these results suggest that the activation of wnt signaling plays a central role in the regulation of neuronal metabolism and that this effect could be important for the correct function of the neuronal network . then , batches 1 and 2 of the conditioned media were combined [ 20 , 2226 ] . before the treatment with the wnt ligand , the cells were maintained in neub without b27 for 1 h ( to avoid the possible effects of the growth factors present in b27 ) . the neurons were also treated with inhibitors of wnt signaling ; the inhibitors were coincubated with the control media or wnt5a media . the inhibitors used were fzcrd-2 ( 5 ng / ml , antagonist of wnt ligands ) , kn-93 ( 10 m , inhibitor of cam kinase ii ) , g6976 ( 200 nm , inhibitor of pkc ) , and 7-nitroindazole ( 7-ni ; 5 m , inhibitor of nitric oxide synthases ) . the neurons were also treated with inhibitors of glucose metabolism , such as cytochalasin b ( cyt b , 20 m , inhibitor of glucose transporters ( gluts ) ) , cytochalasin e ( cyt e , 20 m , control of the action of cyt b ) , 2-deoxy - d - glucose ( 2-dg , 7 mm , competitive inhibitor of hk ) , and dichloroacetate ( dca , 5 mm , inhibitor of glycolysis that blocks the activity of the pyruvate dehydrogenase complex ) . moreover , we treated the neurons with sodium nitroprusside ( snp , 0800 nm ) , a no donor . in all cases , the neurons were treated with the inhibitors for 30 min prior to the experiment with or without the wnt5a media . after the treatment with wnt5a in the presence or absence of wnt inhibitors , the neurons were carefully selected using a microscope to ensure that only plates containing cultures of uniformly growing neurons were used . uptake was measured at room temperature by the addition of 11.2 ci of 2-deoxy - d-[1,2-(n)h ] glucose ( [ 2-h]-dg ) at a final specific activity of 13 disintegrations / min / pmol ( approximately 1 mci / mmol ) . the cells were incubated with the glucose metabolism inhibitors for 30 min in the presence of a radioactive substrate . the kinetic parameters were determined using a single rectangular hyperbola of the form vmax[glc]/(km + [ glc ] ) , which was adjusted to the data by nonlinear regression using sigmaplot 12 . next , the neurons were placed in tubes containing 5 mm glucose and then washed twice in krebs henseleit solution ( 11 mm na2hpo4 , 122 mm nacl , 3.1 mm kcl , 0.4 mm kh2po4 , 1.2 mm mgso4 , and 1.3 mm cacl2 , ph 7.4 ) , containing the appropriate concentration of glucose . after equilibration in 0.5 ml of hank 's balanced salt solution / glucose at 37c for 10 min , 0.5 ml of hank 's balanced salt solution containing various concentrations of [ 3-h ] glucose was added , with a final specific activity of 13 disintegrations / min / pmol ( approximately 1 mci / mmol ) . glucose oxidation via the ppp was measured using a previously described method , which is based on the difference in co2 production from [ 1-c ] glucose ( decarboxylated in the 6-phosphogluconate dehydrogenase - catalyzed reaction and in the krebs cycle ) and [ 6-c ] glucose ( only decarboxylated in the krebs cycle ) . after the treatment with wnt5a in the presence or absence of inhibitors , the medium was removed , and the neurons were washed with ice - cold pbs and collected by trypsinization . the cell pellets were resuspended in o2-saturated krebs henseleit buffer ( 11 mm na2hpo4 , 122 mm nacl , 3.1 mm kcl , 0.4 mm kh2po4 , 1.2 mm mgso4 , and 1.3 mm cacl2 , ph 7.4 ) , and 500 l of this suspension ( ~10 cells ) was placed in erlenmeyer flasks with 0.5 ml of the krebs henseleit solution containing 0.5 ci d-[1-c ] glucose or 2 ci d-[6-c ] glucose and 5.5 mm d - glucose ( final concentration ) . the animals were housed at the animal house facility of the facultad de ciencias biolgicas , pontificia universidad catlica de chile , in accordance with the guide for the care and use of laboratory animals ( nih - usa publication 86 - 23 ) . we observed a 1.5-fold increase in the p - pkc / pkc ratio after 2 h of wnt5a treatment , and this increase was blocked by the wnt scavenger fzcrd-2 ( data not shown ) . the first step of glucose metabolism is the uptake of glucose from the extracellular media into the cell . we used 2-deoxyglucose ( 2-dg ) , a radioactive analog of glucose , to study glucose uptake . in the brain , glucose is taken up by glucose transporters ( gluts ) and phosphorylated by the enzyme hk , but it is not further metabolized . wnt pathway activation triggered a 2-fold increase in 2-dg uptake , with a significant difference at 15 sec . incubation with wnt5a during the initial phase of monitoring induced an increase in uptake from 9.9 1.5 nmol 10 cells to 20.8 2.0 nmol 10 cells ; this increase was blocked by the coincubation with fzcrd-2 . interestingly , we observed that 7-ni , an inhibitor of neuronal no synthase ( nnos ) [ 41 , 42 ] , blocked the effect of wnt5a , suggesting that the effect of this ligand was mediated by the intracellular release of ca followed by the downstream generation of no . we treated the cells with inhibitors of several downstream targets of noncanonical wnt signaling to further investigate this effect . neither kn-93 ( inhibitor of calcium / calmodulin - dependent protein kinase ii ( camkii ) ) nor g6976 ( inhibitor of pkc ) blocked the effect of wnt5a . the kinetic parameters of glucose uptake were estimated for a more comprehensive analysis of the effect of wnt signaling . in the wnt5a - treated cells , the km and vmax values were 2.5 0.1 mm and 8.7 0.2 pmol10 cells / min , respectively ( figure 1(d ) ) . figure 1(e ) illustrates how exposure to the wnt5a ligand increased the apparent affinity of glucose in the neuronal transport pathway . we observed that the wnt5a treatment induced a robust increase in activity from 2.2 0.17 units / mg to 4.4 0.8 units / mg after 2 h of treatment ( figure 2(a ) , ( i ) ) . the next step was to determine whether this increased uptake was correlated with an increase in the glucose utilization through glycolysis . first , we measured the glycolytic rate at several time points after treatment with wnt5a . wnt5a increased the glycolytic rate in a time - dependent manner , with a maximum rate of 2.9 0.3 pmol / mg protein at 2 h versus a control rate of 1.3 0.19 pmol / mg protein . using a pharmacological approach , we studied the effects of several inhibitors at 2 h and observed that the effect of wnt5a was blocked by the coincubation with fzcrd-2 and 7-ni ( figure 2(b ) , ( ii ) ) . our previous results suggest that the downstream generation of no following the activation of noncanonical wnt signaling may be important for the effect of wnt5a on the glycolytic rate . therefore , we analyzed the effect of snp , a no donor , by treating cells with several concentrations of snp for 2 h ; we observed a strong increase in the glycolytic rate , with a maximum of 150 12 pmol / mg protein in the cells that were treated with 700 nm snp . our results indicate that the activation of wnt signaling stimulates glucose uptake and the utilization of this molecule by glycolysis , suggesting an increase in the levels of atp . after the treatment with wnt5a , the atp levels increased to 61.78 6.11 nmol atp / mg of protein ; this increase in the atp levels was blocked when cells were coincubated with wnt5a and the antagonist fzdcrd-2 ( figure 2(c ) ) . as described above , several metabolic pathways could use the g6p generated by hk , including the ppp . we measured the effect of wnt5a on g6pdh activity and observed an increase in the enzymatic activity after 1 h of wnt5a treatment ( figure 3(a ) , ( i ) ) . then , we measured the ppp activity using radioactive glucose and observed that the wnt5a treatment increased the ppp activity from 0.33 0.02 nmol / min mg protein in the control condition to 0.77 0.07 nmol / min mg protein . the effect of wnt5a on the ppp activity was blocked by the coincubation with fzdcrd-2 ( figure 3(b ) ) . we studied whether the effects of the wnt5a ligand could be recapitulated in a more complex model to further analyze the effect of wnt pathway activation on glucose metabolism . for this experiment , we used cortical - hippocampal slices from mouse brains ( figure 4(a ) ) . after a 30 min treatment , we observed significant time - dependent differences in the accumulation of 2-dg , with a maximum of 6.9 0.9 nmol 10 after 2 h of wnt5a treatment compared with 3.4 0.4 nmol 10 for the control condition ( figure 4(b ) , ( i ) ) . after the 1 h incubation with wnt5a , we observed an increase in the glycolytic rate in the slices from 1.07 0.11 pmol / mg protein to 2.12 0.08 pmol / mg protein ; this effect was blocked by the coincubation with 2-dg and fzdcrd-2 ( figure 4(c ) ) . finally , we measured the ppp activity in slices and observed an activity of 0.32 0.01 nmol / min mg protein in the controls . the wnt5a treatment induced an increase in the ppp activity to 0.78 0.02 01 nmol / min mg protein ; this effect was blocked by the coincubation with fzdcrd-2 and 7-ni ( figure 4(d ) ) . in the present work , we studied the effect of wnt5a on glucose metabolism in cortical neurons . we report that wnt5a treatment stimulates glucose uptake in a time - dependent manner ; this increase was correlated with an increase in both the hk activity and the glycolytic rate . moreover , the treatment with wnt5a increased the activity of the g6pdh enzyme and the ppp . together , these results suggest that the activation of wnt signaling by wnt5a regulates cellular glucose metabolism in neurons . wnt5a has been described as a noncanonical wnt ligand , and the noncanonical pathway includes the planar cell polarity ( wnt / pcp ) and wnt / ca pathways . in the wnt / pcp pathway , the wnt ligand binds to the fzd receptor and activates small gtpases that subsequently induce the expression of genes related to the reorganization of the cytoskeleton . in the wnt / ca pathway , ligand binding to the fzd receptor activates the enzyme phospholipase c ( plc ) , which increases the production of inositol triphosphate ( ip3 ) , generating an increase in the intracellular ca concentration that leads to the activation of calcium - dependent proteins [ 4547 ] . subsequently , wnt5a signaling increases the intracellular levels of several second messengers , including ca and no , which are both considered regulators of glucose metabolism , a critical process required for brain function [ 4851 ] . the importance of the wnt pathway in regulating glucose metabolism has been increasingly recognized in recent years due to studies in humans , where several components of the wnt pathway have been identified as risk factors for metabolic diseases , including age - related dementia ; however , the final effect depends on whether the canonical or noncanonical wnt pathway is affected [ 52 , 53 ] . activation of the wnt/-catenin pathway promotes a decrease in the plasma glucose levels in vivo , and this decrease modulates the localization and expression of glut4 in adipocytes and increases glucose uptake in these cells . similarly , patients with diabetes mellitus type ii have been shown to exhibit increased expression of sfrp5 , a wnt inhibitor , which has been correlated with a decrease in wnt5a levels . furthermore , activation of the wnt / ca pathway has been reported to modulate mitochondrial dynamics , which may affect atp generation [ 56 , 57 ] . approximately 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to cerebral functions , even though the brain only accounts for 2% of the total body mass . the atp generated by glucose oxidation is used to maintain and restore the ion gradients dissipated by signaling processes , such as postsynaptic stimulation and action potentials , and for neurotransmitter uptake and recycling . glucose is the main energy substrate of the adult brain , and its metabolism is mainly divided into two stages : glucose uptake and intracellular oxidative metabolism . our data also showed that wnt5a increases the apparent affinity of the glut3 transporter , the main glut isoform expressed in neurons . because we did not observe changes in the vmax value , we can disregard the expression of other glut isoforms in our experiments . the effect of wnt5a on glucose uptake will increase the bioavailability of intracellular glucose , with a subsequent increase in the g6p levels generated by hk , another target of the wnt5a pathway . the generated g6p is mainly used by three different metabolic pathways : glycolysis , the ppp , and the glycogen synthesis pathway . however , the neuronal utilization of glucose for glycogen synthesis has been accepted for years ; neurons have recently been shown to use glucose for glycogen synthesis , a function that is typically attributed to astrocytes . neuronal glycogen may be important for promoting neuronal survival under pathological conditions , such as oxidative stress and hypoxia [ 6668 ] . in the present work , we did not evaluate the effect of wnt5a on glycogen synthesis , but we can not discard this possibility because the importance of glycogen in neurons has recently been reported [ 67 , 69 ] . we observed that wnt5a increases the glycolytic rate and ppp activity , thus promoting atp generation and increasing the levels of the nadph cofactor . the increase in the nadph levels resulting from ppp activity could be a protective mechanism against oxidative stress ( generated by the increased ros levels produced in the mitochondria ) , because this molecule is critical for the recycling of antioxidant agents , such as ascorbic acid and glutathione [ 69 , 70 ] . additionally , we observed that the effects of wnt5a on 2-dg uptake and the glycolytic rate were blocked by an inhibitor of nnos . this finding suggests a role for intracellular no , a molecule generated downstream of the wnt / ca pathway , because ca release activates calcineurin , a phosphatase that leads to nnos activation and no production ( figure 5 ) [ 41 , 71 ] . no has been previously shown to stimulate glucose metabolism in neurons ; however , the specific mechanism underlying this effect remains unknown [ 72 , 73 ] . because the brain includes other cells , such as astrocytes , which play critical roles in the regulation of neuronal metabolism [ 7476 ] , we tested the effects of wnt5a on the more complex system of hippocampal slices . here , we observed results similar to those of the in vitro studies , suggesting that the effects of wnt5a are mainly localized to neurons ; however , further studies are required to confirm this hypothesis . our results suggest a novel function for wnt signaling as an activator of glucose metabolism in neurons . this novel role of wnt signaling in neuronal physiology might be an interesting topic in the search for new treatments for neurological disorders .

chronic pelvic pain ( cpp ) in women is commonly described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or to lead to medical care.1 - 2 cpp may originate from one or more organ systems or pathologies and may have multiple contributing factors.3 the prevalence may vary from country to country . in primary care , the prevalence of cpp is found to be comparable to that of asthma and back pain , with values of 3.7% , 3.8% , and 4.1% , respectively.4 in a us study , the prevalence among women of reproductive age reached 24%,4 - 8 whereas in new zealand , grace , and zondervan identified a prevalence of 25.4% when considering a three - month duration of symptoms.9 this elevated prevalence was confirmed by latthe et al . in a recent review of this condition.10 the direct and indirect costs of this condition amount to over two billion dollars per year , and the condition is responsible for 10% of all gynecologic visits , 40 to 50% of all gynecologic laparoscopies and 12% of all hysterectomies.5 cpp has a direct impact on marital status as well as on the social and professional lives of women , and it thus constitutes an important public health problem . in brazil , international studies have demonstrated a high prevalence of persistent pain in women from brazil , including approximately 36% in rio de janeiro.11 according to the health ministry of brazil , in 1997 there were 1.8 million gynecologic visits and approximately 300,000 hospital admissions of women aged 15 to 69 with complaints associated with cpp.12 in a survey of diseases and/or health problems among working women in so paulo , gomes & tanaka13 reported that 13% of them mentioned abdominal and pelvic pain among their major complaints . recently , some studies have concluded that drug or alcohol abuse , abortions , inflammatory pelvic disease , cesarean sections , and psychological diseases are associated with cpp.14 the etiology of cpp in women is not clear , and cpp usually involves a complex interaction between the gastrointestinal , urinary , gynecologic , musculoskeletal , and neurologic and endocrine systems . it is also influenced by psychological and sociocultural factors.6 several other factors may be associated with the condition , including ( 1 ) neuroplastic changes occurring in the posterior horn of the spinal cord as a consequence of electrophysiological , biochemical , and metabolic changes promoted by the initial noxious stimulus , ( 2 ) cross - sensitivity between viscera that share the same innervation , and ( 3 ) development of a visceromuscular reflex that may culminate not only in dysfunctional repercussions but also in the development of myofascial syndrome and the generation of new pain points.15 - 16 although there is a consensus that cpp has a high prevalence among women , there are a limited number of studies in our country , which can be attributed to the lack of a precise diagnosis , the heterogeneity of the group of diseases that result in cpp , and the variation of primary diseases in different populations.17 considering that knowledge about the magnitude and the demographic and socioeconomic characteristics of cpp contributes to its prevention , control , and treatment , the aim of the present study was to estimate the community prevalence of cpp in women living in ribeiro preto ( a southeastern city in the state of so paulo , brazil with a population of about 700,000 people ) , as well as to identify independent factors associated with it . the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) . women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp . the target population consisted of working - class women treated by the public health system . a total of 1,306 women over 14 years of age were contacted , but 38 of them refused to participate . the women who were contacted were selected randomly from the population using their residential address . all subjects ( or those responsible for the subjects ) gave written informed consent to participate in the study . we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions . the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ; per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification . interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study . the data collected by the interviewers were entered during the interview and sent to an electronic database . were evaluated by experts , and their diagnosis was confirmed before inclusion in the database . five percent of the subjects were selected at random and re - interviewed to check data consistency . cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . for this study , we defined cpp as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale . women who had been pregnant in the previous 12 months were excluded from the analyses . the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication . classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . for analysis , we considered only moderate and intense dysmenorrhea to be positive . dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse . the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . for analysis the clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from least possible pain ' to worst possible pain ' . the dagostino and pearson test was used to determine whether data followed a gaussian distribution . data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) . data were analyzed using the fisher exact test or chi - square test ( qualitative variable ) and the unpaired t test after confirmation of normal distribution ( quantitative variables ) . we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case . logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp . the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 . the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) . women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp . the target population consisted of working - class women treated by the public health system . a total of 1,306 women over 14 years of age were contacted , but 38 of them refused to participate . the women who were contacted were selected randomly from the population using their residential address . all subjects ( or those responsible for the subjects ) gave written informed consent to participate in the study . we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions . the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ; per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification . interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study . the data collected by the interviewers were entered during the interview and sent to an electronic database . were evaluated by experts , and their diagnosis was confirmed before inclusion in the database . five percent of the subjects were selected at random and re - interviewed to check data consistency . cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . for this study , we defined cpp as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale . women who had been pregnant in the previous 12 months were excluded from the analyses . the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication . classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . for analysis dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse . the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . for analysis the clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from least possible pain ' to worst possible pain ' . the dagostino and pearson test was used to determine whether data followed a gaussian distribution . data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) . data were analyzed using the fisher exact test or chi - square test ( qualitative variable ) and the unpaired t test after confirmation of normal distribution ( quantitative variables ) . we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case . logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp . the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 . the prevalence of cpp was 11.5% ( 147/1,278 ) . considering only women of reproductive age , the prevalence was 15.1% ( 127/841 ) . 82.4% of healthy women ( 932/1,131 ) and 90.5% of women with cpp ( 133/147 ) stated that they make regular clinic visits at basic health centers ( at least two appointments per year ) . only 4.1% of women with the disease , however , knew their diagnosis before the interview ( n = 6/147 ) . the average duration of the pain was 51.861.2 [ 6 - 360 ] months : 6 - 12 months in 34.0% of the women ( 50/147 ) , 13 - 36 months in 28.6% ( 42/147 ) , and over 36 months in 37.4% ( 55/147 ) . the mean intensity of pain obtained with a visual analogue scale ( vas ) was 58.523.4 mm . the intensity was 30 - 50 mm in 52.4% of the women ( 77/147 ) , 51 - 70 mm in 17.7% ( 26/147 ) , and 71 - 100 mm in 29.9% ( 44/147 ) . in total , 44.9% of the women reported a spontaneous onset of pain ( 66/147 ) ; in 3.4% ( 5/147 ) , the pain was related to food intake ; in 8.5% ( 9/106 excluding those who were not sexually active ) , the pain was related to intercourse ; in 21.8% ( 32/147 ) , the pain was related to physical activity ; in 30.6% ( 30/98 excluding those who were menopausal ) , the pain was related to menstruation ; in 2.0% ( 2/98 ) , the pain was related to ovulation ; in 0.7% ( 1/147 ) , the pain was related to stress ; and in 1.4% , it was related to other factors ( 2/147 ) . the frequency of self - medication was 1.4% ( 16/1131 ) and 24.5% ( 111/147 ) among healthy and cpp women , respectively . the common occupations were maidservant , hairdresser , and seamstress , with no possibility of grouping the women according to their occupations . a univariate analysis of the variables investigated is presented in table 2 . in the logistic regression , the factors independently associated with cpp were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level . in the present study , we detected a one - year prevalence of 11.5% for cpp among women from ribeiro preto and a prevalence of 15.1% in women of reproductive age . to our knowledge , this is the first time that a high prevalence of this disease has been reported in brazil . the presence of chronic pain was found to be 48.4% in women from salvador , brazil.20 although that study described pain at various sites , it made no reference to abdominal or pelvic pain . a recent review published by latthe et al.10 has shown that the worldwide prevalence of this condition ranges from 2% to 24% , which places brazil among the countries with a higher prevalence of cpp . zondervan et al . , for example , observed a community prevalence of 24.0% in women between 18 and 49 years of age.7 in that study , the questionnaire response rate was 74% . a higher response rate is natural among women with the disease , which may explain the difference in prevalence compared with our study , which had a response rate of 97.9% ( 1,278/1,306 ) . we believe that our data are representative of the entire ribeiro preto community because the social - demographic indicators of the covered area are similar to those of the general population of the city ( socioeconomic level , age distribution , sex , and ethnicity ) . a notable result was that only 4% of the women with cpp stated that they had previously received a specific diagnosis , even though 90% of them attended a basic health center . three different factors may have contributed to this finding : whether the physician is able to provide the correct diagnosis ; whether the relationship between the patient and the physician enables effective communication and explanation of the diagnosis ( which other studies have commented on21 ) ; and whether the women complain about their clinical conditions to physicians . studies have shown that gender is an important determining factor of pain complaints in women and might be the reason for diagnosis underestimation at primary care centers.22 the frequency of self - medication was also a notable finding . approximately one quarter of the women used some kind of medication ( especially analgesics and anti - inflammatory drugs ) for the relief of their symptoms in an indiscriminate way . this self - medication predisposes them to side effects and requires financial expenditures that do not guarantee clinical improvement , especially over the long term.23 we believe that this percentage may be underestimated because people responding to an interviewer may not want to report the use of their pain medication . additionally , more than one third of the women had symptoms consistent with a diagnosis of cpp for over three years , and the same proportion had symptoms that are considered severe ( vas>70 mm ) . although we have no data to support this speculation , it is plausible that the high prevalence of cpp is associated with high rates of absenteeism ( about half of these women have paid employment outside the home ) , which has a direct impact on social and economic life . we identified the following as factors independently associated with chronic pelvic pain : dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level . dyspareunia is an important element of sexual dysfunction that ranges in prevalence from 7% to 75% depending on the diagnostic criteria and the associated clinical conditions , such as cpp.24 - 29 in a recent study , we observed that dyspareunia was associated with pelvic muscle tenderness.30 although pelvic muscle tenderness may be a primary cause of cpp,31 we hypothesize that it is more often secondary to cross - talk communication between the viscera and muscles32 through neurogenic inflammation caused by the release of inflammatory mediators at the periphery in response to the antidromic stimulus over time.33 similarly , current sexual activity may be linked in some way to dyspareunia , although this relationship is still unclear . another hypothesis is that women with dysmenorrhea have a lower pain threshold , which may favor the onset of illness.34 moreover , mechanisms of viscero - visceral hyperalgesia between organs probably involve the sensitization of viscero - viscero - somatic convergent neurons.35 the most notable factor observed in the present study is the association of cpp with abdominal surgery , particularly because we observed that two - thirds of the women studied had undergone a previous abdominal surgery , and more than 40% of the women had previously undergone a cesarean section surgery . considering the high rates of cesarean section surgery in our country36 - 37 ( including this sample of our community ) , and the previous detection of an association with cpp by our group38 and by latthe et al . in a recent systematic review,14 we stress the need for detailed studies regarding this possible causal relationship between cpp and cesarean section . some studies suggest the possibility of an association between cpp and adhesions.39 however , the correlation between pelvic pain and adhesions is uncertain because adhesiolysis has not been shown to be effective in achieving pain control.40 thus , we emphasize the importance of recognizing abdominal myofascial syndrome as a differential diagnosis.41 anxiety and depression disorders are frequently concomitant with chronic pain , particularly in women , in both developed and developing countries.42 - 43 we have also observed a direct relationship between depression , anxiety , chronic pelvic pain , and quality of life.44 in addition to being a risk factor for cpp , mood disorders may make it more difficult for a women to engage in cognitively or emotionally demanding rehabilitation.45 because the individual experience of pain is personal and subjective , it is probably affected by emotional states and , therefore , by psychosocial factors . there is some evidence that it is the stress of living with chronic pain , and not personal or family predisposition , that causes depression in these patients.46 the idea that pain , particularly chronic pain , can lead to feelings of frustration , worry , anxiety , and depression seems obvious . there is also evidence , however , for reverse causality , in which negative moods and emotions can lead to or exacerbate pain.47 it therefore remains uncertain whether depression / anxiety precedes or is a consequence of chronic pain.48 low back pain is usually comorbid with cpp . this association probably reflects the same pathophysiological mechanisms,49 although each can reduce the thresholds and thus contribute to the development of the other . painful bladder syndrome and constipation are nongynecologic conditions that may cause or exacerbate cpp ( level of evidence a).2 the prevalence of constipation identified in our sample of healthy women ( 22.7% ) was similar to that observed by oliveira et al.50 because the women reported that constipation was present before cpp , we may infer that this condition may at least contribute to the development of cpp . though this was beyond the scope of our study , some studies indicate that constipation may be secondary to the dysfunction of the levator ani muscle,51 - 52 which is a common condition in cpp . several studies in the literature have shown that a lower educational level is linked to a higher prevalence of chronic pain.20,53 because cpp increases with age and most education takes place in early life , it is likely that a lower educational level increase the risk for the development of cpp , or that both are the consequences of some other undescribed factors . we have not , however , reached final conclusions about the causal relationship between these factors and cpp . a thorough investigation and other different types of studies are necessary to corroborate our results . we conclude that the prevalence of cpp in women from ribeiro preto is very high , even though only 4% sufferers are aware of their diagnosis . this confirmation is crucial for the prevention , early diagnosis , control , and resolution of cpp . the authors are grateful to cnpq ( conselho nacional de desenvolvimento cientifico e tecnologico ) for financial support .

introduction : chronic pelvic pain is a disease that directly affects the social and professional lives of women.objective:to estimate the prevalence of this clinical condition and to identify independent factors associated with it in women living in ribeiro preto , brazil.methods:a one - year cross - sectional study was conducted in a population sample of 1,278 women over the age of 14 years . the target population was predominantly composed of women who are treated by the public health system . the questionnaire was administered by interviewers who were not linked to the city health care programs . the prevalence of the morbidity was estimated . first , we identified the significant variables associated with pelvic pain ( p<0.10 ) and then we attributed values of 0 or 1 to the absence or presence of these variables . logistic regression analysis was used to identify and estimate the simultaneous impact of the independent variables . the results were expressed by odds ratio and their 95% confidence interval with p<0.05.results : the disease was found in 11.5% ( 147/1,278 ) of the sample . the independent predictors were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level.conclusion:the prevalence of chronic pelvic pain in ribeiro preto is high and is associated with conditions that can usually be prevented , controlled , or resolved by improvement of public health policies and public education .

INTRODUCTION MATERIALS AND METHODS Study design and participants Questionnaire Definitions and grouping Statistical analysis RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENT

chronic pelvic pain ( cpp ) in women is commonly described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or to lead to medical care.1 - 2 cpp may originate from one or more organ systems or pathologies and may have multiple contributing factors.3 the prevalence may vary from country to country . in primary care , the prevalence of cpp is found to be comparable to that of asthma and back pain , with values of 3.7% , 3.8% , and 4.1% , respectively.4 in a us study , the prevalence among women of reproductive age reached 24%,4 - 8 whereas in new zealand , grace , and zondervan identified a prevalence of 25.4% when considering a three - month duration of symptoms.9 this elevated prevalence was confirmed by latthe et al . in a recent review of this condition.10 the direct and indirect costs of this condition amount to over two billion dollars per year , and the condition is responsible for 10% of all gynecologic visits , 40 to 50% of all gynecologic laparoscopies and 12% of all hysterectomies.5 cpp has a direct impact on marital status as well as on the social and professional lives of women , and it thus constitutes an important public health problem . in brazil , international studies have demonstrated a high prevalence of persistent pain in women from brazil , including approximately 36% in rio de janeiro.11 according to the health ministry of brazil , in 1997 there were 1.8 million gynecologic visits and approximately 300,000 hospital admissions of women aged 15 to 69 with complaints associated with cpp.12 in a survey of diseases and/or health problems among working women in so paulo , gomes & tanaka13 reported that 13% of them mentioned abdominal and pelvic pain among their major complaints . recently , some studies have concluded that drug or alcohol abuse , abortions , inflammatory pelvic disease , cesarean sections , and psychological diseases are associated with cpp.14 the etiology of cpp in women is not clear , and cpp usually involves a complex interaction between the gastrointestinal , urinary , gynecologic , musculoskeletal , and neurologic and endocrine systems . it is also influenced by psychological and sociocultural factors.6 several other factors may be associated with the condition , including ( 1 ) neuroplastic changes occurring in the posterior horn of the spinal cord as a consequence of electrophysiological , biochemical , and metabolic changes promoted by the initial noxious stimulus , ( 2 ) cross - sensitivity between viscera that share the same innervation , and ( 3 ) development of a visceromuscular reflex that may culminate not only in dysfunctional repercussions but also in the development of myofascial syndrome and the generation of new pain points.15 - 16 although there is a consensus that cpp has a high prevalence among women , there are a limited number of studies in our country , which can be attributed to the lack of a precise diagnosis , the heterogeneity of the group of diseases that result in cpp , and the variation of primary diseases in different populations.17 considering that knowledge about the magnitude and the demographic and socioeconomic characteristics of cpp contributes to its prevention , control , and treatment , the aim of the present study was to estimate the community prevalence of cpp in women living in ribeiro preto ( a southeastern city in the state of so paulo , brazil with a population of about 700,000 people ) , as well as to identify independent factors associated with it . the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) . women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp . the target population consisted of working - class women treated by the public health system . the women who were contacted were selected randomly from the population using their residential address . we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ; per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification . interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study . were evaluated by experts , and their diagnosis was confirmed before inclusion in the database . cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) . we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case . logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp . the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 . the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) . women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp . the target population consisted of working - class women treated by the public health system . the women who were contacted were selected randomly from the population using their residential address . we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions . the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ; per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification . interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study . were evaluated by experts , and their diagnosis was confirmed before inclusion in the database . cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication . classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) . we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case . logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp . the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 . the prevalence of cpp was 11.5% ( 147/1,278 ) . the average duration of the pain was 51.861.2 [ 6 - 360 ] months : 6 - 12 months in 34.0% of the women ( 50/147 ) , 13 - 36 months in 28.6% ( 42/147 ) , and over 36 months in 37.4% ( 55/147 ) . in total , 44.9% of the women reported a spontaneous onset of pain ( 66/147 ) ; in 3.4% ( 5/147 ) , the pain was related to food intake ; in 8.5% ( 9/106 excluding those who were not sexually active ) , the pain was related to intercourse ; in 21.8% ( 32/147 ) , the pain was related to physical activity ; in 30.6% ( 30/98 excluding those who were menopausal ) , the pain was related to menstruation ; in 2.0% ( 2/98 ) , the pain was related to ovulation ; in 0.7% ( 1/147 ) , the pain was related to stress ; and in 1.4% , it was related to other factors ( 2/147 ) . the frequency of self - medication was 1.4% ( 16/1131 ) and 24.5% ( 111/147 ) among healthy and cpp women , respectively . in the logistic regression , the factors independently associated with cpp were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level . in the present study , we detected a one - year prevalence of 11.5% for cpp among women from ribeiro preto and a prevalence of 15.1% in women of reproductive age . to our knowledge , this is the first time that a high prevalence of this disease has been reported in brazil . the presence of chronic pain was found to be 48.4% in women from salvador , brazil.20 although that study described pain at various sites , it made no reference to abdominal or pelvic pain . , for example , observed a community prevalence of 24.0% in women between 18 and 49 years of age.7 in that study , the questionnaire response rate was 74% . we believe that our data are representative of the entire ribeiro preto community because the social - demographic indicators of the covered area are similar to those of the general population of the city ( socioeconomic level , age distribution , sex , and ethnicity ) . although we have no data to support this speculation , it is plausible that the high prevalence of cpp is associated with high rates of absenteeism ( about half of these women have paid employment outside the home ) , which has a direct impact on social and economic life . we identified the following as factors independently associated with chronic pelvic pain : dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level . dyspareunia is an important element of sexual dysfunction that ranges in prevalence from 7% to 75% depending on the diagnostic criteria and the associated clinical conditions , such as cpp.24 - 29 in a recent study , we observed that dyspareunia was associated with pelvic muscle tenderness.30 although pelvic muscle tenderness may be a primary cause of cpp,31 we hypothesize that it is more often secondary to cross - talk communication between the viscera and muscles32 through neurogenic inflammation caused by the release of inflammatory mediators at the periphery in response to the antidromic stimulus over time.33 similarly , current sexual activity may be linked in some way to dyspareunia , although this relationship is still unclear . another hypothesis is that women with dysmenorrhea have a lower pain threshold , which may favor the onset of illness.34 moreover , mechanisms of viscero - visceral hyperalgesia between organs probably involve the sensitization of viscero - viscero - somatic convergent neurons.35 the most notable factor observed in the present study is the association of cpp with abdominal surgery , particularly because we observed that two - thirds of the women studied had undergone a previous abdominal surgery , and more than 40% of the women had previously undergone a cesarean section surgery . some studies suggest the possibility of an association between cpp and adhesions.39 however , the correlation between pelvic pain and adhesions is uncertain because adhesiolysis has not been shown to be effective in achieving pain control.40 thus , we emphasize the importance of recognizing abdominal myofascial syndrome as a differential diagnosis.41 anxiety and depression disorders are frequently concomitant with chronic pain , particularly in women , in both developed and developing countries.42 - 43 we have also observed a direct relationship between depression , anxiety , chronic pelvic pain , and quality of life.44 in addition to being a risk factor for cpp , mood disorders may make it more difficult for a women to engage in cognitively or emotionally demanding rehabilitation.45 because the individual experience of pain is personal and subjective , it is probably affected by emotional states and , therefore , by psychosocial factors . there is some evidence that it is the stress of living with chronic pain , and not personal or family predisposition , that causes depression in these patients.46 the idea that pain , particularly chronic pain , can lead to feelings of frustration , worry , anxiety , and depression seems obvious . there is also evidence , however , for reverse causality , in which negative moods and emotions can lead to or exacerbate pain.47 it therefore remains uncertain whether depression / anxiety precedes or is a consequence of chronic pain.48 low back pain is usually comorbid with cpp . painful bladder syndrome and constipation are nongynecologic conditions that may cause or exacerbate cpp ( level of evidence a).2 the prevalence of constipation identified in our sample of healthy women ( 22.7% ) was similar to that observed by oliveira et al.50 because the women reported that constipation was present before cpp , we may infer that this condition may at least contribute to the development of cpp . though this was beyond the scope of our study , some studies indicate that constipation may be secondary to the dysfunction of the levator ani muscle,51 - 52 which is a common condition in cpp . several studies in the literature have shown that a lower educational level is linked to a higher prevalence of chronic pain.20,53 because cpp increases with age and most education takes place in early life , it is likely that a lower educational level increase the risk for the development of cpp , or that both are the consequences of some other undescribed factors . we conclude that the prevalence of cpp in women from ribeiro preto is very high , even though only 4% sufferers are aware of their diagnosis .

oligodendrocyte injury and myelin loss are principal pathological characteristics of demyelinating diseases such as multiple sclerosis ( ms ) which is a chronic degenerative central nervous system ( cns ) disease . nowadays , the relevance of primary and/or secondary alterations in glutamate signaling leading to excitotoxic oligodendrocyte damage to ms is accepted . in addition , excitotoxic injury to oligodendrocyte progenitors due to hypoxic ischemia is thought to contribute to pathogenesis of myelination disturbances in periventricular leukomalacia , which is the cerebral white matter damage in the premature infant . excitotoxicity is a phenomenon , which is characterized by overactivation of glutamate receptors ( glurs ) ; this event elicits cell death . later , olney et al . discovered that excitotoxic vulnerability existed in all central neurons that contained glurs . since then , glutamate excitotoxicity has been described in both acute injury to the cns and in chronic neurodegenerative disorders . based on earlier findings , there are two types of ionotropic glurs : n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa)/kainate ( ka ) receptors . nmda receptors have the main role in glutamate toxicity in neural cells ; in addition , ampa / kainate receptors contribute in neuronal excitotoxicity . ampa and kainate receptors are commonly present in astrocytes , oligodendrocytes and microglia . while nmda receptors are expressed in clusters on oligodendrocyte processes , ampa and kainate receptors are diffusely located on oligodendrocyte somata . ca overload , during excitotoxicity process , can trigger cell death in both neuronal and glial cells . in fact , ca overload mainly occurs through nmda receptor , but the molecular properties of ampa / kainate receptors in oligodendrocytes , ( lack of glutr2 subunit in ampa receptors , and low spread of glutr6 subunit in kainate receptors ) , result in more permeability to ca in these cells , so it makes them more susceptible to excitotoxicity insult . the oln-93 cells in their morphological features and their antigenic properties resemble 5 - 10 days old ( postnatal time ) cultured rat brain oligodendrocytes . according to the developmental stages of oligodendroglial cells , berberine has several pharmacological actions , including anti - inflammatory , antipyretic , anti - diarrheal , hypoglycemic , hypocholesterolemic , antidepressant , and alzheimer 's disease - amelioration effect . recently , it was reported that berbery extract reduced neuronal damage in the gerbil hippocampus after transient forebrain ischemia . in this study , we evaluated the effects of berberine on oln-93 oligodendrocytes during oxygen - glucose deprivation / reperfusion ( ogd / r ) , which is a reliable model of ischemia and excitotoxicity . besides , intracellular calcium levels were measured as an important factor in excitotoxic phenomenon and its involvement in ischemic induced injury . the stock solution was prepared at 1 mmol / l in deionized water and kept at 20c . dulbecco 's modified eagle 's medium ( dmem ) with glutamine , glucose / glutamine - free dmem , fetal bovine serum ( fbs ) were purchased from gibco . penicillin - streptomycin , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ( mtt ) , poly - l - lysine , dizocilpine ( mk-801 ) , nbqx ( 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ) , and fura 2-am were purchased from sigma chemical co. ( st . oln-93 cell line was purchased from pasteur institute of iran ( tehran , iran ) . the cells were grown in dmem medium supplemented with 10% fbs and 100 iu / ml penicillin and 100 g / ml streptomycin in a humidified 95% air and 5% co2 incubator at 37c . the cells were subcultured twice a week by gentle scraping and cultured on poly - l - lysine - coated 12-well plates at a density of 5 10 . culture dishes were coated with poly - l - lysine 24 h before the experiment . poly - l - lysine ( 150,000 - 300,000 mw ) was dissolved in deionized water and the plate wells were filled with this solution ( 20 g / ml ) . after 5 min standing at room temperature , the solution was aspirated and the plates were left to dry in a laminar flow hood overnight . briefly , the culture medium was replaced with glucose / glutamine - free dmem , and cells were exposed to hypoxia for 30 , 60 , and 240 min in a small anaerobic chamber previously filled with 95% ( v / v ) n2 and 5% ( v / v ) co2 at 37c . to terminate the ogd , the chamber was opened and the medium was replaced with dmem , and the cultures were then placed in an incubator with 5% co2 for 24 h. to examine the drug effects , cell cultures were treated 3 h before ogd with berberine , at concentrations of 0.5 - 4 m . these concentrations were chosen based on the results of preliminary experiments at nontoxic levels of berberine ( data not shown ) . to investigate the effects of various inhibitors on ogd - induced cell death , mk-801 , a noncompetitive antagonist of the nmda receptor or nbqx , an ampa / kainate receptor antagonist , oligodendroglial cell viability was measured using the colorimetric mtt assay , as previously described by mosmann . briefly , cells were incubated with 0.5 mg / ml mtt in dmem , at 37c under 5% co2 , for 3 h. the blue formazan reduction product , produced by the action of succinate dehydrogenase in living cells on the dye , was dissolved in 100 l dmso , and the optical density was read at 570 nm using a dynex mmx microplate reader ( dynex , richfield , mn , usa ) . data were expressed as the percentage of viable cells in ogd - exposed plates compared with control normoxic plates determined by mtt reduction . measurement of intracellular free calcium concentration was performed using a ca - sensitive indicator fura 2-am with an olympus ix-71 inverted microscope and ccd camera . fluorescence emission images of intracellular fura-2 at 510 nm , after excitation at two different 340 and 380 nm were acquired . ratiometrically analysis of image pairs was carried out using imagej from the national institute of health ( nih ) , bethesda , maryland , usa . cells were placed in a flow - chamber and washed with isotonic buffer containing ( in mm ) : 128 nacl , 2.5 kcl , 2 cacl2 , 1 mgcl2 , 10 glucose , and 10 hepes , ph 7.4 for 10 min . the flow rate was 0.5 ml / min and the temperature was kept on 37c through all the process . chemical ogd was started by flow of sodium azide ( nan3 : 20 mm ) in glucose - free isotonic buffer for 30 min . the concentration of sodium azide ( 20 mm ) was selected based on the study by marino et al . our experiments showed that this concentration was producing the acceptable elevation of intracellular calcium in oln-93 cells during 30 min - chemical ogd ( data not shown ) . images were taken for 4-min - durations along with 10 min - pauses at start , middle and the end of 30 min chemical ogd . berberine and other pharmacologic agents were dissolved in nan3 ( 20 mm ) to compare the alteration of intracellular calcium levels in presence and absence of drugs during 30 min chemical ogd . all results are expressed as means standard deviation , the significance of differences was evaluated with student 's t - test by office excel 2007 from microsoft inc . , 1 microsoft way , redmond , wa , usa . for multiple comparisons , we used one - way anova ( tukey 's post - hoc ) by spss 16 from international business machines corporation , 1 new orchard road , armonk , new york 10504 - 1722 , usa . the stock solution was prepared at 1 mmol / l in deionized water and kept at 20c . dulbecco 's modified eagle 's medium ( dmem ) with glutamine , glucose / glutamine - free dmem , fetal bovine serum ( fbs ) were purchased from gibco . penicillin - streptomycin , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ( mtt ) , poly - l - lysine , dizocilpine ( mk-801 ) , nbqx ( 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ) , and fura 2-am were purchased from sigma chemical co. ( st . oln-93 cell line was purchased from pasteur institute of iran ( tehran , iran ) . the cells were grown in dmem medium supplemented with 10% fbs and 100 iu / ml penicillin and 100 g / ml streptomycin in a humidified 95% air and 5% co2 incubator at 37c . the cells were subcultured twice a week by gentle scraping and cultured on poly - l - lysine - coated 12-well plates at a density of 5 10 . culture dishes were coated with poly - l - lysine 24 h before the experiment . poly - l - lysine ( 150,000 - 300,000 mw ) was dissolved in deionized water and the plate wells were filled with this solution ( 20 g / ml ) . after 5 min standing at room temperature , the solution was aspirated and the plates were left to dry in a laminar flow hood overnight . briefly , the culture medium was replaced with glucose / glutamine - free dmem , and cells were exposed to hypoxia for 30 , 60 , and 240 min in a small anaerobic chamber previously filled with 95% ( v / v ) n2 and 5% ( v / v ) co2 at 37c . to terminate the ogd , the chamber was opened and the medium was replaced with dmem , and the cultures were then placed in an incubator with 5% co2 for 24 h. to examine the drug effects , cell cultures were treated 3 h before ogd with berberine , at concentrations of 0.5 - 4 m . these concentrations were chosen based on the results of preliminary experiments at nontoxic levels of berberine ( data not shown ) . to investigate the effects of various inhibitors on ogd - induced cell death , mk-801 , a noncompetitive antagonist of the nmda receptor or nbqx , an ampa / kainate receptor antagonist , oligodendroglial cell viability was measured using the colorimetric mtt assay , as previously described by mosmann . briefly , cells were incubated with 0.5 mg / ml mtt in dmem , at 37c under 5% co2 , for 3 h. the blue formazan reduction product , produced by the action of succinate dehydrogenase in living cells on the dye , was dissolved in 100 l dmso , and the optical density was read at 570 nm using a dynex mmx microplate reader ( dynex , richfield , mn , usa ) . data were expressed as the percentage of viable cells in ogd - exposed plates compared with control normoxic plates determined by mtt reduction . measurement of intracellular free calcium concentration was performed using a ca - sensitive indicator fura 2-am with an olympus ix-71 inverted microscope and ccd camera . fluorescence emission images of intracellular fura-2 at 510 nm , after excitation at two different 340 and 380 nm were acquired . ratiometrically analysis of image pairs was carried out using imagej from the national institute of health ( nih ) , bethesda , maryland , usa . cells were placed in a flow - chamber and washed with isotonic buffer containing ( in mm ) : 128 nacl , 2.5 kcl , 2 cacl2 , 1 mgcl2 , 10 glucose , and 10 hepes , ph 7.4 for 10 min . the flow rate was 0.5 ml / min and the temperature was kept on 37c through all the process . chemical ogd was started by flow of sodium azide ( nan3 : 20 mm ) in glucose - free isotonic buffer for 30 min . the concentration of sodium azide ( 20 mm ) was selected based on the study by marino et al . our experiments showed that this concentration was producing the acceptable elevation of intracellular calcium in oln-93 cells during 30 min - chemical ogd ( data not shown ) . images were taken for 4-min - durations along with 10 min - pauses at start , middle and the end of 30 min chemical ogd . berberine and other pharmacologic agents were dissolved in nan3 ( 20 mm ) to compare the alteration of intracellular calcium levels in presence and absence of drugs during 30 min chemical ogd . all results are expressed as means standard deviation , the significance of differences was evaluated with student 's t - test by office excel 2007 from microsoft inc . , 1 microsoft way , redmond , wa , usa . for multiple comparisons , we used one - way anova ( tukey 's post - hoc ) by spss 16 from international business machines corporation , 1 new orchard road , armonk , new york 10504 - 1722 , usa . we examined the effect of berberine ( 0.5 - 4 m ) on 30 , 60 , and 240 min ogd followed by 24 h reperfusion in oln-93 cultures . mtt assay demonstrated that cell viability decreased to 53% , 46% , and 35% during 30 , 60 , and 240 min ogd / r , respectively . pretreatment of cultures with berberine ( 0.5 - 4 m ) , significantly attenuated ogd - induced cell injury in oln-93 cells in a concentration - dependent manner ( anova ; p < 0.05 ) . the peak of protective effect was at 2 m concentration for berberine through all 3 time - scheduled experiments . at this concentration , berberine increased cell viability to 83% , 77% , and 79% during 30 , 60 , and 240 min ogd / r , respectively ( student 's t - test , p < 0.001 ) [ figure 1a - c ] . the effects of berberine ( 0.5 - 4 m ) , and dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]quinoxaline-2,3-dione ( nbqx , 30 m ) during ( a ) 30 min , ( b ) 60 min , ( c ) 240 min oxygen - glucose deprivation/24 h reperfusion induced cell injury in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . the results are presented as mean standard deviation ( * * * , p < 0.001 ) to evaluate the effects of inhibitors of ionotropic glurs on ogd / r - induced cell death , mk-801 ( 10 m ) and nbqx ( 30 m ) were added to medium 3 h before ogd . our results demonstrated that mk-801 and nbqx could significantly protect the oln-93 cells from ogd - induced cell death during all 3 time - scheduled assessments in compare with sham ( student 's t - test , p < 0.001 ) . mk-801 increased cell viability to 78% , 69% , and 66% during 30 , 60 , and 240 min ogd / r , respectively . in addition , nbqx improved cell viability to 72% , 67% , and 57% during 30 , 60 , and 240 min ogd / r , respectively [ figure 1a - c ] . to determine the intracellular calcium levels during ischemia , the oln-93 cells were exposed to sodium azide ( nan3 : 20 mm ) , as a model of chemical ogd , for 30 min . sodium azide was able to induce 141% increase in intracellular ca concentration , which was statistically significant compared to the ca levels prior to chemical ogd treatment of oln-93 cells bathed in ca - containing medium . furthermore , the ischemia - evoked increase in intracellular ca levels was decreased to 108% by berberine ( 2 m ) , 120% by mk-801 ( 10 m ) and 124% in the presence of nbqx ( 30 m ) compared with pre ogd levels . reduction in intracellular ca levels in presence of berberine and ionotropic glurs was significant in compared with chemical ogd in absence of these pharmacologic agents [ figure 2 ] . the results based on fura 2-am fluorescence ratio ( f340 nm / f380 nm ) to assess the intracellular ca levels during 30-min chemical oxygen - glucose deprivation by sodium azide ( 20 mm ) in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . berberine ( 2 m ) , dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ( nbqx , 30 m ) reduced intracellular ca levels significantly ( p < 0.001 ) we examined the effect of berberine ( 0.5 - 4 m ) on 30 , 60 , and 240 min ogd followed by 24 h reperfusion in oln-93 cultures . mtt assay demonstrated that cell viability decreased to 53% , 46% , and 35% during 30 , 60 , and 240 min ogd / r , respectively . pretreatment of cultures with berberine ( 0.5 - 4 m ) , significantly attenuated ogd - induced cell injury in oln-93 cells in a concentration - dependent manner ( anova ; p < 0.05 ) . the peak of protective effect was at 2 m concentration for berberine through all 3 time - scheduled experiments . at this concentration , berberine increased cell viability to 83% , 77% , and 79% during 30 , 60 , and 240 min ogd / r , respectively ( student 's t - test , p < 0.001 ) [ figure 1a - c ] . the effects of berberine ( 0.5 - 4 m ) , and dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]quinoxaline-2,3-dione ( nbqx , 30 m ) during ( a ) 30 min , ( b ) 60 min , ( c ) 240 min oxygen - glucose deprivation/24 h reperfusion induced cell injury in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . the results are presented as mean standard deviation ( * * * , p < 0.001 ) to evaluate the effects of inhibitors of ionotropic glurs on ogd / r - induced cell death , mk-801 ( 10 m ) and nbqx ( 30 m ) were added to medium 3 h before ogd . our results demonstrated that mk-801 and nbqx could significantly protect the oln-93 cells from ogd - induced cell death during all 3 time - scheduled assessments in compare with sham ( student 's t - test , p < 0.001 ) . mk-801 increased cell viability to 78% , 69% , and 66% during 30 , 60 , and 240 min ogd / r , respectively . in addition , nbqx improved cell viability to 72% , 67% , and 57% during 30 , 60 , and 240 min ogd / r , respectively [ figure 1a - c ] . to determine the intracellular calcium levels during ischemia , the oln-93 cells were exposed to sodium azide ( nan3 : 20 mm ) , as a model of chemical ogd , for 30 min . sodium azide was able to induce 141% increase in intracellular ca concentration , which was statistically significant compared to the ca levels prior to chemical ogd treatment of oln-93 cells bathed in ca - containing medium . furthermore , the ischemia - evoked increase in intracellular ca levels was decreased to 108% by berberine ( 2 m ) , 120% by mk-801 ( 10 m ) and 124% in the presence of nbqx ( 30 m ) compared with pre ogd levels . reduction in intracellular ca levels in presence of berberine and ionotropic glurs was significant in compared with chemical ogd in absence of these pharmacologic agents [ figure 2 ] . the results based on fura 2-am fluorescence ratio ( f340 nm / f380 nm ) to assess the intracellular ca levels during 30-min chemical oxygen - glucose deprivation by sodium azide ( 20 mm ) in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . berberine ( 2 m ) , dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ( nbqx , 30 m ) reduced intracellular ca levels significantly ( p < 0.001 ) oligodendrocyte loss is one of the hallmarks of demyelinating diseases , including ms . in this regard , the excitotoxicity phenomenon by its subsequent intracellular calcium overload has been described as a cause of oligodendroglial death . we examined the effect of berberine ( 0.5 - 4 m ) on oln-93 oligodendrocytes during 30 , 60 , and 240 min ogd followed by 24 h reperfusion . furthermore , the alteration of intracellular calcium levels during 30 min chemical ogd by nan3 ( 20 mm ) in the absence and presence of berberine ( 2 m ) was evaluated by fura-2 am method . the results revealed that 30 , 60 , and 240 min ogd / r could induce cell death in oln-93 oligodendrocytes . the excitotoxicity pathway is initiated by ischemic glutamate release and following activation of ionotropic glurs and intracellular calcium overload . remarkable distribution of ionotropic glurs in oligodendrocyte has been revealed by the existence of ampa / kainate receptors on oligodendroglial somata and nmda receptors on the oligodendroglial processes . therefore , nbqx , the ampa / kainate receptor antagonist , and mk-801 , the selective nmda receptor antagonist , are two important pharmacologic agents to protect the oligodendrocyte against excitotoxicity - induced cell injury . based on the previous studies ; ogd - induced injury to oligodendrocyte processes and somata is ca - dependent , but the route of ca influx into the processes and somata are different . salter and fern demonstrated that using nbqx ( 30 m ) to block ca influx through ampa / kainate receptors during ogd had no effect on loss of processes but prevented the loss of oligodendrocyte somata . on the contrary , application of mk-801 ( 10 m ) could largely prevent ogd - induced injury to processes of oligodendroglial cells . the protective effect of mk-801 occurred by blocking nmda receptor , which is the route of ca influx into oligodendroglial processes . our findings revealed that blocking the ampa / kainate receptors by nbqx ( 30 m ) as well blocking nmda receptors by mk-801 ( 10 m ) could significantly protect the oln-93 oligodendrocyte from excitotoxicity - induced cell injury during 30 , 60 , and 240 min - ogd / r trials . according to the developmental stages of oligodendroglial cells , the oln-93 cell line is located between an oligodendroglial precursor cell and a late immature oligodendrocyte . thus , the position of ionotropic glurs in oln-93 follows other oligodendrocytes ; consequently , our results are in accord with salter and fern 's findings . the results demonstrated that berberine ( 0.5 - 4 m ) could significantly protect the oln-93 cells during 30 , 60 , and 240 min ogd / r in a concentration - dependent manner . this finding is consistent with the earlier experiments about the effects of berberine on neurons . by the way , zhou et al . found the neuroprotective effects of berberine on stroke models in vitro and in vivo ; furthermore , zhang et al . suggested that berberine could regulate neuronal apoptosis in cerebral ischemia , which might be dependent on the degree of cell injury . excitotoxicity insult alters ca homeostasis as the cellular ca overload is a trigger for oligodendroglial death . pervious studies have revealed that excitotoxicity in oligodendroglial lineage could be prevented when ca - free medium was used . indeed , there are the evidences that ca entry through ampa and kainate receptors alone is sufficient to trigger excitotoxicity in oligodendroglial cells , and the ca influx through nmda receptors causes injury to oligodendrocyte processes . although , the oligodendrocyte processes are too small , nmda receptors during excitotoxicity may raise intracellular ca to toxic levels within such a confined space . this indicates that sufficient ca influx occurs through the nmda receptors on processes to result in injury . because of the important role of ca in excitotoxic cell injury , we examined the intracellular calcium levels in absence and presence of berberine ( 2 m ) during 30 min - chemical ogd . sodium azide ( nan3 ) , the well - established complex iv inhibitor , was used to induce chemical hypoxia . complex iv is a part of five complexes ( complex i - complex v ) which construct the mitochondrial respiratory chain ; complex iv defects in acute ms lesions affected oligodendrocytes , astrocytes , and axons . in vitro model of brain ischemia by sodium azide increases intracellular ca in neurons . the main source of calcium increase induced by nan3 is extracellular , involving glur activation in a first step and calcium channel opening in a second step . therefore , we used sodium azide in glucose - free isotonic buffer to investigate the intracellular ca overload during chemical ogd in oln-93 oligodendrocytes . the results revealed a significant rising in intracellular ca levels in oln-93 oligodendrocyte due to 30 min - chemical ogd . furthermore , the antagonists of ionotropic glurs : mk-801 ( 10 m ) and nbqx ( 30 m ) significantly attenuated the intracellular calcium levels during chemical ogd . this finding confirmed the results of mtt assay from 30 min - ogd / r trial . as mentioned above , the elevation of intracellular ca levels is a major cause of excitotoxicity - induced cell death . therefore , attenuation of the intracellular ca overload by berbeine could be a major factor that protected the onl-93 oligodendrocytes during ogd - induced excitotoxicity insult . our results demonstrated that berberine could protect oln-93 oligodendrocyte against excitotoxicity - induced cell injury in a concentration - dependent manner during all 3 time - scheduled ogd / r ischemic models . overall , these findings advocate the results of other studies about therapeutic potential of brebrine in the treatment of neuropathological conditions associated with ischemic induced excitotoxicity . furthermore , these observations support the hypothesis that ca current has a critical role during excitotoxicity insult .

background : oligodendrocytes , the myelinating glial cells of central nervous system , are highly vulnerable to ischemic - induced excitotoxic insult , a phenomenon in which calcium overload triggers cell death . berberine is an alkaloid extracted from medicinal herbs as coptidis rhizoma with several pharmacological effects like inhibition of neuronal apoptosis in cerebral ischemia.methods:we examined the effects of berberine ( 0.5 - 4 m ) and glutamate receptors antagonists ( mk-801 [ 10 m ] and nbqx [ 30 m ] ) on oln-93 cell line ( a permanent immature rat oligodendrocyte ) during ( 30 , 60 , 240 min ) oxygen - glucose deprivation ( ogd)/24 h reperfusion . the cells were cultured in 12-well plates . the cells were exposed to glucose - free medium and hypoxia in a small anaerobic chamber . cell viability was evaluated by mtt ( 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ) assay . the intracellular calcium levels also were evaluated by ca2 + -sensitive indicator fura-2/am in presence or absence of berberine ( 2 m ) during 30 min chemical ogd by nan3 ( 20 mm ) . student 's t - test and anova were used for statistical analysis.results:berberine , mk-801and nbqx significantly increased oligodendrocyte viability in all 3 time - scheduled oxygen - glucose deprivation / reperfusion . berberine at 2 m produced peak of protection , and increased cell viability to 83% , 77% , and 79% during 30 , 60 , 240 min ischemic experiments , respectively ( p < 0.001 ) . berberine significantly attenuated intracellular ca2 + rise induced by chemical ischemia , and this effect of berberine was significantly stronger than mk-801 and nbqx ( p < 0.001).conclusions : we concluded that berberine protected oln-93 oligodendrocyte against ischemic induced excitotoxic injury . attenuation of intracellular ca2 + overload by berberine may be the key mechanism that saved oln-93 from excitotoxicity damage .

INTRODUCTION METHODS Materials Cell culture Oxygen-glucose deprivation and drug exposure Analysis of cell viability Measurement of intracellular free calcium Statistical analysis RESULTS The effects of berberine, on oxygen-glucose deprivation/reperfusion-induced cell injury in OLN-93 cells The effects of MK-801 and NBQX on oxygen-glucose deprivation-induced cell injury in OLN-93 cells The effects of berberine, MK-801 and NBQX on intracellular calcium levels during 30 min chemical oxygen-glucose deprivation in OLN-93 cells DISCUSSION CONCLUSIONS

excitotoxicity is a phenomenon , which is characterized by overactivation of glutamate receptors ( glurs ) ; this event elicits cell death . in this study , we evaluated the effects of berberine on oln-93 oligodendrocytes during oxygen - glucose deprivation / reperfusion ( ogd / r ) , which is a reliable model of ischemia and excitotoxicity . besides , intracellular calcium levels were measured as an important factor in excitotoxic phenomenon and its involvement in ischemic induced injury . penicillin - streptomycin , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ( mtt ) , poly - l - lysine , dizocilpine ( mk-801 ) , nbqx ( 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ) , and fura 2-am were purchased from sigma chemical co. ( st . the cells were grown in dmem medium supplemented with 10% fbs and 100 iu / ml penicillin and 100 g / ml streptomycin in a humidified 95% air and 5% co2 incubator at 37c . the cells were subcultured twice a week by gentle scraping and cultured on poly - l - lysine - coated 12-well plates at a density of 5 10 . briefly , the culture medium was replaced with glucose / glutamine - free dmem , and cells were exposed to hypoxia for 30 , 60 , and 240 min in a small anaerobic chamber previously filled with 95% ( v / v ) n2 and 5% ( v / v ) co2 at 37c . to terminate the ogd , the chamber was opened and the medium was replaced with dmem , and the cultures were then placed in an incubator with 5% co2 for 24 h. to examine the drug effects , cell cultures were treated 3 h before ogd with berberine , at concentrations of 0.5 - 4 m . to investigate the effects of various inhibitors on ogd - induced cell death , mk-801 , a noncompetitive antagonist of the nmda receptor or nbqx , an ampa / kainate receptor antagonist , oligodendroglial cell viability was measured using the colorimetric mtt assay , as previously described by mosmann . briefly , cells were incubated with 0.5 mg / ml mtt in dmem , at 37c under 5% co2 , for 3 h. the blue formazan reduction product , produced by the action of succinate dehydrogenase in living cells on the dye , was dissolved in 100 l dmso , and the optical density was read at 570 nm using a dynex mmx microplate reader ( dynex , richfield , mn , usa ) . cells were placed in a flow - chamber and washed with isotonic buffer containing ( in mm ) : 128 nacl , 2.5 kcl , 2 cacl2 , 1 mgcl2 , 10 glucose , and 10 hepes , ph 7.4 for 10 min . chemical ogd was started by flow of sodium azide ( nan3 : 20 mm ) in glucose - free isotonic buffer for 30 min . the concentration of sodium azide ( 20 mm ) was selected based on the study by marino et al . our experiments showed that this concentration was producing the acceptable elevation of intracellular calcium in oln-93 cells during 30 min - chemical ogd ( data not shown ) . images were taken for 4-min - durations along with 10 min - pauses at start , middle and the end of 30 min chemical ogd . berberine and other pharmacologic agents were dissolved in nan3 ( 20 mm ) to compare the alteration of intracellular calcium levels in presence and absence of drugs during 30 min chemical ogd . all results are expressed as means standard deviation , the significance of differences was evaluated with student 's t - test by office excel 2007 from microsoft inc . penicillin - streptomycin , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ( mtt ) , poly - l - lysine , dizocilpine ( mk-801 ) , nbqx ( 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ) , and fura 2-am were purchased from sigma chemical co. ( st . the cells were grown in dmem medium supplemented with 10% fbs and 100 iu / ml penicillin and 100 g / ml streptomycin in a humidified 95% air and 5% co2 incubator at 37c . the cells were subcultured twice a week by gentle scraping and cultured on poly - l - lysine - coated 12-well plates at a density of 5 10 . briefly , the culture medium was replaced with glucose / glutamine - free dmem , and cells were exposed to hypoxia for 30 , 60 , and 240 min in a small anaerobic chamber previously filled with 95% ( v / v ) n2 and 5% ( v / v ) co2 at 37c . to terminate the ogd , the chamber was opened and the medium was replaced with dmem , and the cultures were then placed in an incubator with 5% co2 for 24 h. to examine the drug effects , cell cultures were treated 3 h before ogd with berberine , at concentrations of 0.5 - 4 m . to investigate the effects of various inhibitors on ogd - induced cell death , mk-801 , a noncompetitive antagonist of the nmda receptor or nbqx , an ampa / kainate receptor antagonist , oligodendroglial cell viability was measured using the colorimetric mtt assay , as previously described by mosmann . briefly , cells were incubated with 0.5 mg / ml mtt in dmem , at 37c under 5% co2 , for 3 h. the blue formazan reduction product , produced by the action of succinate dehydrogenase in living cells on the dye , was dissolved in 100 l dmso , and the optical density was read at 570 nm using a dynex mmx microplate reader ( dynex , richfield , mn , usa ) . cells were placed in a flow - chamber and washed with isotonic buffer containing ( in mm ) : 128 nacl , 2.5 kcl , 2 cacl2 , 1 mgcl2 , 10 glucose , and 10 hepes , ph 7.4 for 10 min . chemical ogd was started by flow of sodium azide ( nan3 : 20 mm ) in glucose - free isotonic buffer for 30 min . the concentration of sodium azide ( 20 mm ) was selected based on the study by marino et al . our experiments showed that this concentration was producing the acceptable elevation of intracellular calcium in oln-93 cells during 30 min - chemical ogd ( data not shown ) . images were taken for 4-min - durations along with 10 min - pauses at start , middle and the end of 30 min chemical ogd . berberine and other pharmacologic agents were dissolved in nan3 ( 20 mm ) to compare the alteration of intracellular calcium levels in presence and absence of drugs during 30 min chemical ogd . all results are expressed as means standard deviation , the significance of differences was evaluated with student 's t - test by office excel 2007 from microsoft inc . we examined the effect of berberine ( 0.5 - 4 m ) on 30 , 60 , and 240 min ogd followed by 24 h reperfusion in oln-93 cultures . mtt assay demonstrated that cell viability decreased to 53% , 46% , and 35% during 30 , 60 , and 240 min ogd / r , respectively . pretreatment of cultures with berberine ( 0.5 - 4 m ) , significantly attenuated ogd - induced cell injury in oln-93 cells in a concentration - dependent manner ( anova ; p < 0.05 ) . the peak of protective effect was at 2 m concentration for berberine through all 3 time - scheduled experiments . at this concentration , berberine increased cell viability to 83% , 77% , and 79% during 30 , 60 , and 240 min ogd / r , respectively ( student 's t - test , p < 0.001 ) [ figure 1a - c ] . the effects of berberine ( 0.5 - 4 m ) , and dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]quinoxaline-2,3-dione ( nbqx , 30 m ) during ( a ) 30 min , ( b ) 60 min , ( c ) 240 min oxygen - glucose deprivation/24 h reperfusion induced cell injury in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . the results are presented as mean standard deviation ( * * * , p < 0.001 ) to evaluate the effects of inhibitors of ionotropic glurs on ogd / r - induced cell death , mk-801 ( 10 m ) and nbqx ( 30 m ) were added to medium 3 h before ogd . our results demonstrated that mk-801 and nbqx could significantly protect the oln-93 cells from ogd - induced cell death during all 3 time - scheduled assessments in compare with sham ( student 's t - test , p < 0.001 ) . mk-801 increased cell viability to 78% , 69% , and 66% during 30 , 60 , and 240 min ogd / r , respectively . in addition , nbqx improved cell viability to 72% , 67% , and 57% during 30 , 60 , and 240 min ogd / r , respectively [ figure 1a - c ] . to determine the intracellular calcium levels during ischemia , the oln-93 cells were exposed to sodium azide ( nan3 : 20 mm ) , as a model of chemical ogd , for 30 min . furthermore , the ischemia - evoked increase in intracellular ca levels was decreased to 108% by berberine ( 2 m ) , 120% by mk-801 ( 10 m ) and 124% in the presence of nbqx ( 30 m ) compared with pre ogd levels . reduction in intracellular ca levels in presence of berberine and ionotropic glurs was significant in compared with chemical ogd in absence of these pharmacologic agents [ figure 2 ] . the results based on fura 2-am fluorescence ratio ( f340 nm / f380 nm ) to assess the intracellular ca levels during 30-min chemical oxygen - glucose deprivation by sodium azide ( 20 mm ) in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . berberine ( 2 m ) , dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ( nbqx , 30 m ) reduced intracellular ca levels significantly ( p < 0.001 ) we examined the effect of berberine ( 0.5 - 4 m ) on 30 , 60 , and 240 min ogd followed by 24 h reperfusion in oln-93 cultures . mtt assay demonstrated that cell viability decreased to 53% , 46% , and 35% during 30 , 60 , and 240 min ogd / r , respectively . pretreatment of cultures with berberine ( 0.5 - 4 m ) , significantly attenuated ogd - induced cell injury in oln-93 cells in a concentration - dependent manner ( anova ; p < 0.05 ) . the peak of protective effect was at 2 m concentration for berberine through all 3 time - scheduled experiments . at this concentration , berberine increased cell viability to 83% , 77% , and 79% during 30 , 60 , and 240 min ogd / r , respectively ( student 's t - test , p < 0.001 ) [ figure 1a - c ] . the effects of berberine ( 0.5 - 4 m ) , and dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f]quinoxaline-2,3-dione ( nbqx , 30 m ) during ( a ) 30 min , ( b ) 60 min , ( c ) 240 min oxygen - glucose deprivation/24 h reperfusion induced cell injury in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . the results are presented as mean standard deviation ( * * * , p < 0.001 ) to evaluate the effects of inhibitors of ionotropic glurs on ogd / r - induced cell death , mk-801 ( 10 m ) and nbqx ( 30 m ) were added to medium 3 h before ogd . our results demonstrated that mk-801 and nbqx could significantly protect the oln-93 cells from ogd - induced cell death during all 3 time - scheduled assessments in compare with sham ( student 's t - test , p < 0.001 ) . mk-801 increased cell viability to 78% , 69% , and 66% during 30 , 60 , and 240 min ogd / r , respectively . in addition , nbqx improved cell viability to 72% , 67% , and 57% during 30 , 60 , and 240 min ogd / r , respectively [ figure 1a - c ] . to determine the intracellular calcium levels during ischemia , the oln-93 cells were exposed to sodium azide ( nan3 : 20 mm ) , as a model of chemical ogd , for 30 min . furthermore , the ischemia - evoked increase in intracellular ca levels was decreased to 108% by berberine ( 2 m ) , 120% by mk-801 ( 10 m ) and 124% in the presence of nbqx ( 30 m ) compared with pre ogd levels . reduction in intracellular ca levels in presence of berberine and ionotropic glurs was significant in compared with chemical ogd in absence of these pharmacologic agents [ figure 2 ] . the results based on fura 2-am fluorescence ratio ( f340 nm / f380 nm ) to assess the intracellular ca levels during 30-min chemical oxygen - glucose deprivation by sodium azide ( 20 mm ) in oln-93 cell line ( a permanent immature rat oligodendrocyte ) . berberine ( 2 m ) , dizocilpine ( mk-801 , 10 m ) and 2,3-dihydroxy-6-nitro-7-sulfamoyl - benzo[f ] quinoxaline-2,3-dione ( nbqx , 30 m ) reduced intracellular ca levels significantly ( p < 0.001 ) oligodendrocyte loss is one of the hallmarks of demyelinating diseases , including ms . in this regard , the excitotoxicity phenomenon by its subsequent intracellular calcium overload has been described as a cause of oligodendroglial death . we examined the effect of berberine ( 0.5 - 4 m ) on oln-93 oligodendrocytes during 30 , 60 , and 240 min ogd followed by 24 h reperfusion . furthermore , the alteration of intracellular calcium levels during 30 min chemical ogd by nan3 ( 20 mm ) in the absence and presence of berberine ( 2 m ) was evaluated by fura-2 am method . the results revealed that 30 , 60 , and 240 min ogd / r could induce cell death in oln-93 oligodendrocytes . therefore , nbqx , the ampa / kainate receptor antagonist , and mk-801 , the selective nmda receptor antagonist , are two important pharmacologic agents to protect the oligodendrocyte against excitotoxicity - induced cell injury . salter and fern demonstrated that using nbqx ( 30 m ) to block ca influx through ampa / kainate receptors during ogd had no effect on loss of processes but prevented the loss of oligodendrocyte somata . on the contrary , application of mk-801 ( 10 m ) could largely prevent ogd - induced injury to processes of oligodendroglial cells . our findings revealed that blocking the ampa / kainate receptors by nbqx ( 30 m ) as well blocking nmda receptors by mk-801 ( 10 m ) could significantly protect the oln-93 oligodendrocyte from excitotoxicity - induced cell injury during 30 , 60 , and 240 min - ogd / r trials . according to the developmental stages of oligodendroglial cells , the oln-93 cell line is located between an oligodendroglial precursor cell and a late immature oligodendrocyte . the results demonstrated that berberine ( 0.5 - 4 m ) could significantly protect the oln-93 cells during 30 , 60 , and 240 min ogd / r in a concentration - dependent manner . this finding is consistent with the earlier experiments about the effects of berberine on neurons . suggested that berberine could regulate neuronal apoptosis in cerebral ischemia , which might be dependent on the degree of cell injury . because of the important role of ca in excitotoxic cell injury , we examined the intracellular calcium levels in absence and presence of berberine ( 2 m ) during 30 min - chemical ogd . the main source of calcium increase induced by nan3 is extracellular , involving glur activation in a first step and calcium channel opening in a second step . therefore , we used sodium azide in glucose - free isotonic buffer to investigate the intracellular ca overload during chemical ogd in oln-93 oligodendrocytes . the results revealed a significant rising in intracellular ca levels in oln-93 oligodendrocyte due to 30 min - chemical ogd . furthermore , the antagonists of ionotropic glurs : mk-801 ( 10 m ) and nbqx ( 30 m ) significantly attenuated the intracellular calcium levels during chemical ogd . as mentioned above , the elevation of intracellular ca levels is a major cause of excitotoxicity - induced cell death . therefore , attenuation of the intracellular ca overload by berbeine could be a major factor that protected the onl-93 oligodendrocytes during ogd - induced excitotoxicity insult . our results demonstrated that berberine could protect oln-93 oligodendrocyte against excitotoxicity - induced cell injury in a concentration - dependent manner during all 3 time - scheduled ogd / r ischemic models .

lysosomal storage disorders continuous recycling of their macromolecular constituents is essential for the functional integrity of long - lived cells . this molecular rejuvenation depends not only on permanent biosynthesis but also on permanent degradation of macromolecules . to handle the potential risky turnover of macromolecular constituents , hydrolytic processes are largely contained in specific acid subcellular compartments named lysosomes ( lysein = cleave , somos = body ) by their discoverer christian de duve ( de duve et al . henri g. hers ( 1963)first realized that deficiency of a particular lysosomal enzyme ( acid alpha - glucosidase ) was the cause of glycogen storage disease type 2 , also called pompe disease . this discovery led to the concept of lysosomal storage disorders ( lsds ) , of which more than 70 have now been described . the birth prevalence of individual lsds is thought to vary between 1 in 20,000100,000 live births . collectively , the entire group of lsds affects at least 1 in 5,00010,000 live births ( meikle et al . lysosomal storage disorders may present with a broad range of phenotypes , with variable age of onset , symptom severity , and degree of central nervous system ( cns ) involvement . many lsds manifest as a spectrum encompassing more severe ( infantile ) and milder ( juvenile and adult ) forms . infantile forms often display cns involvement and are therefore called neuronopathic , in contrast to the non - neuronopathic subtypes . for instance , in gaucher disease ( beta - glucocerebrosidase deficiency ) , the most severe phenotype presents with severe neurological involvement and death soon after birth , whereas at the other end of the phenotypic spectrum , patients are found in whom diagnosis is made in late adulthood based on minimal visceral symptoms ( aerts et al . the general principle of treatment is lowering the excessive amount of stored substrate , which can be achieved either by enhanced substrate degradation or by reduced substrate production . first , hematopoietic cell transplantation ( hct ) with cells from unaffected individuals can deliver the missing enzyme to the deficient cells of the recipient . hct has been used in an attempt to treat numerous different lsds , but to now , it has only been proven effective in a small number of disorders ( see boelens et al . the second approach to enhance substrate degradation is by direct intravenous delivery of the deficient enzyme , which may partly restore the deficient enzymatic capacity . this concept , named enzyme replacement therapy ( ert ) , is based on the principle that lysosomal proteins can reach the subcellular lysosomes via endocytosis mediated by lectins on the plasma membrane , such as the mannose 6-phosphate receptor and the mannose receptor . the first enzyme preparation used to treat non - neuronopathic type 1 gaucher disease consisted of placenta - derived glucocerebrosidase with modified , mannose - terminated glycans , allowing more selective uptake by tissue macrophages that are the prominent storage cells in gaucher disease ( barton et al . this product was later replaced by recombinant enzyme ( cerezyme ; genzyme ) , which proved to be very successful in treating the visceral and bone complications of gaucher disease ( de fost et al . an alternative recombinant enzyme preparation ( velaglucerase alfa ; shire ) , produced in human cells , was registered for ert of type 1 gaucher disease ( aerts et al . an exciting new development forms the production of recombinant glucocerebrosidase by protalix in plant cells ( aviezer et al . 2009 ) . the plant - derived recombinant enzyme preparation ( named taliglucerase alfa ) is hoped to be comparatively effective and increase the choice between treatment modalities ( aviezer et al . a third and more recent development , also aimed at enhancing substrate degradation , is the use of small compounds that act as chaperones for mutant enzymes ( see smid et al . 2010 for a recent review ) . whereas the above - mentioned therapeutic approaches are based on stimulating the breakdown of the accumulated substrate , an alternative strategy is aimed at decreasing the amount of substrate by partially inhibiting its synthesis . this approach , requiring well - tolerated and specific enzymatic inhibitors , is named substrate reduction therapy ( srt ) . the most well - known and studied compound that acts by srt is a glucosylceramide synthase inhibitor , n - butyldeoxynojirimycin ( miglustat ) , which catalyses the first step in the glucosylceramide biosynthetic pathway ( platt et al . miglustat has been extensively studied for its use in gaucher disease type 1and is registered for patients with mild to moderate gaucher disease type 1 who are unable or unwilling to receive ert ( cox et al . the long - term experience with miglustat treatment in type 1 gaucher patients is satisfactory ( elstein et al . 2007 ) , and anecdotal reports suggest that combination srt and ert may be beneficial for some type 3 neuronopathic gaucher patients ( cox - brinkman et al . the development of an alternative small - molecule compound for oral substrate reduction therapy , eliglustat tartrate , by genzyme is also exciting ( mceachern et al . 2010b ) , and a 2-year follow - up of a phase ii trial indicated major improvements in hematological , visceral , and skeletal manifestations in adult patients with type 1 gaucher disease on a par with ert ( lukina et al . moreover , as ert is not able to prevent or treat neurological abnormalities in severely affected patients because the enzymes are unable to pass the blood brain barrier ( schiffmann et al . 1997 ) , such compounds could provide a much needed treatment option for these patients . another approach being studied for treating cns disease is direct introduction of the deficient enzyme into the brain ( lonser et al . alternative strategies to deliver modified enzymes from the circulation across the blood brain barrier are also being investigated ( begley 2004 ; urayama et al . numerous hurdles , such as transient expression of transgenes , immunological responses , and tumor induction , have so far discouraged the clinical application of gene therapy . however , as in recent years enormous progress has been made with respect to efficacy and safety of viral vectors , with encouraging results in application in animal models for lsds , there is still great hope for application of gene therapy ( fig . 1 ) . fig . 1gaucher cell accumulating the glycosphingolipid glucosylceramide and specifically secreting the biomarker chitotriosidase that can be detected in plasma . example of corrections in plasma chitotriosidase in gaucher disease patients receiving enzyme replacement therapy gaucher cell accumulating the glycosphingolipid glucosylceramide and specifically secreting the biomarker chitotriosidase that can be detected in plasma . example of corrections in plasma chitotriosidase in gaucher disease patients receiving enzyme replacement therapy monitoring disease manifestations and therapeutic intervention accurate quantitative monitoring of disease manifestations as well as of the therapeutic efficacy of any type of treatment is crucial for clinical management of patients with lsds . disease onset and progression can be monitored by clinical , radiological , and laboratory assessments . clinical assessments obviously differ among the various lsds , as illustrated below for gaucher disease and fabry disease . in practice , clinical therapeutic efficacy is often difficult to assess , as organ involvement can sometimes not be quantitatively assessed and is highly variable among patients . moreover , some symptoms have an irreversible nature , such as advanced renal failure in fabry disease or bone infarctions in gaucher disease . increasingly , disease - specific biochemical abnormalities are used to monitor disease progression and treatment efficacy for lsds . biomarkers are generally defined as chemical entities , ranging from simple metabolites to complex proteins , which indicate the presence of a biological process linked to the clinical manifestations and outcome of a particular disease . the value of biomarkers is appreciated by most clinicians and biomedical researchers as well as the authorities . in a recent report on biomarkers , it is expected that they will help increase the rate of success of new developments and to expedite the development of drugs . also , biomarkers are key in the shift away from the one size fits all to the right drug at the right dose in the right patient approach . hence , biomarkers play an important role for scientists and industry in drug development and for regulators in the approval process an obvious example is the measurement of blood glucose and/or glycosylated hemoglobin in diabetic individuals . the first group consists of molecules that accumulate in tissues and body fluids directly due to the enzymatic defect . the second group includes molecules produced by storage cells in a specific lsd and can be measured either in plasma , urine , or cerebrospinal fluid ( csf ) . identifying and applying such biomarkers for gaucher disease and fabry disease are discussed , and their use is illustrated . brady and co - workers first showed that the primary defect in gaucher disease is a marked deficiency in activity of the lysosomal enzyme glucocerebrosidase ( brady et al . deficiencies in glucocerebrosidase result in accumulation of its lipid substrate glucosylceramide in the lysosomal compartment of macrophages throughout the body . different phenotypes ( types 1 , 2 , and 3 ) are generally recognized and are differentiated on the basis of the presence or absence of neurological symptoms . whether a strict division in three different phenotypes is still valid has been the subject of debate , as there are an increasing number of reports on neurological manifestations in patients with type 1 gaucher disease . however , the neurological signs and symptoms in type 1 gaucher disease are of a totally different kind from and , in the majority of cases , of much less severity than , the signs and symptoms associated with types 2 and 3 disease ( biegstraaten et al . 2008 ) . it has also become apparent that complete deficiencies in glucocerebrosidase activity occur , resulting in major skin permeability abnormalities with lethal consequences either prenatally or shortly after birth ( sidransky 2004 ) . the most common gaucher phenotype is non - neuronopathic type 1 manifestation , with a highly variable age of onset and severity . even spontaneous disease regression has been observed in some relatively mildly affected type 1 gaucher patients ( boomsma et al . atypical bone pain , pathological fractures , avascular necrosis , and extremely painful bone crises may also have a great impact on quality of life . gammopathies and metabolic abnormalities such as insulin resistance and lipoprotein abnormalities are encountered in type 1 gaucher patients ( langeveld et al . 2007 , 2008b ; wennekes et al . 2009 ; de fost et al . 2009 , 2008 ) . it is prevalent among ashkenazi jews , with a carrier frequency as high as about 1 in 15 and an incidence of about 1 in 1,000 . the most common mutation in the glucocerebrosidase gene of caucasians , including ashkenazi jews , encodes the amino acid substitution n370s . the heteroallelic presence of the n370s mutation is always associated with a non - neuronopathic course ( ohashi et al . some , but not all , homozygotes for the n370s mutation develop significant clinical symptoms ( beutler et al . 2001 ; aerts et al . 2003 ) . twin studies and the poor predictive power of genotype phenotype investigations in gaucher disease have clearly pointed out that epigenetic factors also play a key role in gaucher disease manifestation ( biegstraaten et al . the diagnosis of gaucher disease can be confirmed by demonstration of reduced glucocerebrosidase activity in lysates of various cell types and even urine samples using the fluorogenic substrate 4-methylumbelliferyl beta - glucoside ( aerts et al . fluorescent analogues of the lipid glucosylceramide can be exploited to measure glucocerebrosidase activity in cultured cells ( van weely et al . highly specific monoclonal antibodies against human glucocerebrosidase have been produced and can be used in purification of the protein , western blot analysis , and microscopy techniques ( aerts et al . although glucocerebrosidase is present in the lysosomes of all cell types , patients with type 1 gaucher disease only store glucosylceramide in macrophages . it is believed that the storage material stems from the breakdown of exogenous lipids derived from the turnover of blood cells . the glucosylceramide - loaded macrophages of gaucher patients show a characteristic morphology with a wrinkled - paper appearance of their cytoplasm , which contains lysosomal inclusion bodies ; these cells are referred to as gaucher cells . in recent decades , it has become apparent that gaucher cells are not inert containers of storage material but are viable , chronically activated macrophages that contribute to the diverse clinical manifestations of gaucher disease . these mature , activated macrophages are surrounded by newly formed , highly inflammatory macrophages in tissue lesions of patients with gaucher disease ( boven et al . consistent with these observations , patients with gaucher disease show increased plasma levels of several proinflammatory and anti - inflammatory cytokines , chemokines , and hydrolases ( aerts and hollak 1997 ; aerts et al . factors released by gaucher cells and surrounding macrophages are thought to play a crucial role in the development of common abnormalities in gaucher patients , such as osteopenia , activation of coagulation , hypermetabolism , gammopathies , multiple myeloma , and hypolipoproteinemias ( aerts and hollak 1997 ) . biomarkers of gaucher cells given the prominent role of gaucher cells in the pathophysiology of the disorder , considerable attention has been focused on identifying plasma markers for such macrophages . abnormalities in levels of tartrate - resistant acid phosphatase ( trap ) , angiotensin - converting enzyme ( ace ) , hexosaminidase , and lysozyme in serum samples from gaucher patients had been documented for some time ( aerts and hollak 1997 ) . more recently , increased plasma levels of various cathepsins , including cathepsin k , were reported in patients with gaucher disease ( moran et al . all of these proteins are known to be produced by macrophages ; however , none of them appears to be a specific marker for the pathological gaucher cells , and their levels in the serum of symptomatic patients with gaucher disease may overlap with those observed in healthy individuals . the need for a very sensitive and specific marker for gaucher cells prompted a search for such a parameter . this search led to the discovery of a striking abnormality in the serum of symptomatic patients with gaucher disease . chitotriosidase serum from such individuals showed a 1,000-fold increased capacity to degrade the fluorogenic substrate 4-methylumbelliferyl chitotrioside ( hollak et al . 1994 ) . the chitotriosidase protein was subsequently purified and its complementary dna ( cdna ) cloned ( renkema et al . 1995 ; boot et al . 1995 ) . chitotriosidase was found to be the human analogue of chitinases from lower organisms ( rao et al . the enzyme hydrolyzes chitin , the natural polymer of beta-1,4 n - acetylglucosamine , is a member of the large chitinase protein family created by gene duplications ( bussink et al . the chitinase protein family includes one other active enzyme amcase ( boot et al . 2001)and several inactive lectins ( bussink et al . 2007 ; renkema et al . 1998 ) . chitotriosidase is specifically expressed by phagocytes in humans , including macrophages and neutrophils ( renkema et al . it has been shown to exert a fungistatic action of degradation of the protective chitin layer at the hyphal tip of fungi ( van eijk et al . . in situ hybridization and histochemistry of bone marrow aspirates and sections of spleens from patients with gaucher disease revealed that chitotriosidase is massively produced by storage cells . this is also supported by the close linear relationship between chitotriosidase and glucosylceramide levels in different spleen sections from patients with gaucher disease ( bussink et al . 2006 ) . in a culture model of gaucher cells , chitotriosidase accounts for almost 10% of the total secreted protein . these observations help us to understand the very specific , gross elevation of chitotriosidase levels in the blood of patients with gaucher disease . a relationship between the total body burden of storage cells in patients with gaucher disease and their plasma chitotriosidase levels has been noted . the plasma chitotriosidase level does not reflect any one particular clinical symptom of gaucher disease , suggesting , rather , that it reflects the sum of secreted enzyme by gaucher cells in various body locations . plasma chitotriosidase levels can be determined by monitoring hydrolysis of the fluorogenic substrate 4-methylumbelliferyl - chitobioside . however , the ability of chitotriosidase to transglycosylate as well as hydrolyze this substrate complicates the enzyme assay ( aguilera et al . 2003 ) . special care has to be taken to ensure that the enzyme activity is truly proportional to the amount of chitotriosidase protein . a far more convenient , sensitive , and accurate detection is feasible by measuring the activity of chitotriosidase toward the recently designed fluorogenic substrate 4-methylumbelliferyl - deoxy - chitotrioside ( aguilera et al . interpretation of plasma chitotriosidase levels is intrinsically complicated by the common occurrence of a particular 24-base - pair duplication in the chitotriosidase gene , preventing the formation of chitotriosidase protein ( aguilera et al . about one in every three individuals carries this abnormality , and about one in every 20 individuals , including patients with gaucher disease , is homozygous for this trait ( aguilera et al . it has been established that carriers of the 24-bp duplication show half the amount of plasma chitotriosidase detected in individuals with the wild - type chitotriosidase genotype ( aguilera et al . it is therefore common to correct plasma chitotriosidase by a factor of two when examining patients with gaucher disease who are carriers of the 24-bp duplication . other polymorphisms have also been detected , of which the g102s substitution is the most frequent ( allele frequency of about 0.25 ) . this polymorphism leads to an enzyme with a slightly impaired catalytic activity toward the 4-methylumbelliferyl - chitotrioside substrate compared with wild type . however , g102s enzyme activity is normal when using 4-methylumbelliferyl - deoxy - chitobioside as substrate ( bussink et al . it should be mentioned that increased plasma chitotriosidase activity is not unique for gaucher patients . plasma chitotriosidase activity is increased , albeit much more modestly , in several lysosomal and nonlysosomal diseases , such as sarcoidosis , visceral leishmaniasis , leprosy , arthritis , multiple sclerosis , thalassemia , chronic obstructive pulmonary disease ( copd ) , malaria , and atherosclerosis ( hollak et al . ; vedder et al . 2006a ; boot et al . 2010 ; iyer et al . 2009 ; boven et al . 2006 ; boot et al . parc / ccl18 the high frequency of chitotriosidase deficiency prompted us to search for an alternative marker of gaucher cells . using surface - enhanced laser distortion / ionization time of flight ( seldi - tof ) mass spectrometric analyses , we discovered massive overproduction and secretion by gaucher cells of the pulmonary and activation - regulated chemokine ( parc / ccl18 ) , of which the messenger rna ( mrna ) was previously observed to be up - regulated in the spleen of a patient with gaucher disease ( moran et al . this plasma parc / ccl18 can not be reliably quantified using seldi - tof , but reliable quantification is obtained by elisa ( van breemen et al . plasma parc / ccl18 levels are 10- to 40-fold elevated in symptomatic gaucher patients ( boot et al . due to its basic nature and small molecular mass , parc / ccl18 levels in urine are proportional to those in the circulation . therefore , urinary parc / ccl18 excretion offers insight into the body burden of gaucher cells ( boot et al . 2006 ) . measurement of plasma parc / ccl18 has been found to yield an excellent additional tool to monitor changes in body burden of gaucher cells . it is particularly useful for evaluating patients that are chitotriosidase deficient ( cox et al . mip-1 and mip-1 van breemen and co - workers reported markedly elevated levels of the chemokines macrophage inflammatory protein ( mip)-1 and mip-1 in plasma of symptomatic gaucher patients ( van breemen et al . interestingly , with immunohistochemistry , these proteins were found to be produced by surrounding inflammatory spleen macrophages and not by mature gaucher cells ( van breemen et al . the different cellular source is also reflected in the observation that plasma chitotriosidase and parc / ccl18 , both stemming from gaucher cells , respond comparably to ert . however , corrections in plasma mip-1 and mip-1 following ert are not proportional to those found with the true gaucher - cell biomarkers ( van breemen et al . a relationship was observed between plasma mip-1 and skeletal disease : stable high plasma mip-1 levels despite prolonged ert were found to correlate with ongoing skeletal disease ( van breemen et al . rigorous analysis of a large cohort of gaucher patients is required to establish the value of plasma mip-1 ( or mip-1 ) as biomarker , especially its value as prognostic marker for skeletal response to therapy . clinical applications of gaucher - cell biomarkers plasma chitotriosidase measurement is commonly employed as a first screen in the diagnosis of gaucher disease . increasing plasma levels seem to reflect gradual accumulation of storage cells in the patient s body . in an attempt to assess the utility of plasma chitotriosidase activity measurement as a biomarker for treatment efficacy , hollak and co - workers investigated the relationship between enzyme activity and clinical parameters ( hollak et al . chitotriosidase levels were usually > 20,000 nmol / ml / h and always > 15,000 nmol / ml / h , whereas patients with less severe disease tended to have lower values . during enzyme supplementation therapy , the mean decrease in 12 months was 32% ( range 082% ) , and 78% of patients had a decrease of > 15% . in six patients with a decrease in chitotriosidase activity of < 15% , the clinical response to treatment was inferior to that of other patients , with less reduction in organomegaly in four and bone problems in two . in addition , chitotriosidase response was related to disease severity ; less reduction in plasma activity was seen in more severely affected individuals . on the basis of this investigation , it has been proposed that in patients in whom the initiation of treatment is questionable based solely on clinical parameters , a chitotriosidase activity > 15,000 nmol / ml / h may serve as an indicator of a high gaucher - cell burden and an indication for treatment initiation ( hollak et al . 2001 ) . a reduction in chitotriosidase activity of < 15% after 12 months of treatment , in combination with an insufficient response of at least one clinical parameter , should be a reason to consider dose increase . furthermore , a sustained increase in chitotriosidase at any point during treatment should alert the physician to the possibility of clinical deterioration and the need for dose adjustment . a recent retrospective analysis by deegan and co - workers confirmed the value of the use of plasma chitotriosidase in gaucher disease management and presented evidence for comparable use of parc / ccl18 ( deegan et al . 2005 ) . in conclusion , next to radiological monitoring of the bone marrow and skeleton ( maas et al . 2002 ) , measurement of plasma levels of chitotriosidase and/or parc / ccl18 offers valuable information for clinical management of type 1 gaucher patients . lipid biomarkers of gaucher disease increased plasma concentrations of glucosylceramide , the primary storage lipid , have been documented for gaucher patients ( groener et al . plasma glucosylceramide is not used as a biomarker , as its increase generally is not very pronounced . moreover , the exact relation between circulating glucosylceramide and storage cells in tissues is far from clear . interestingly , besides glucosylceramide , the ganglioside gm3 is also elevated in gaucher plasma samples ( ghauharali - van der vlugt et al . elevated glycosphingolipids such as gm3 are thought to cause insulin resistance ( aerts et al . indeed , a recent study revealed that gaucher patients are insulin resistant without overt hyperglycemia ( langeveld et al . other secondary lipid abnormalities in gaucher patients have been noted by thorough investigations by fuller and co - workers , such as elevated levels of phosphatidylglycerol ( hein et al . discovery of additional protein biomarkers for gaucher disease ongoing attention is paid to the detection of useful protein biomarkers for gaucher disease by a thorough survey of protein composition of bodily fluids , or cell and tissue specimens of symptomatic gaucher patients . the latter approach has more recently become feasible due to the availability of mass spectrometric techniques that allow accurate analysis of proteins , even in complex mixtures such as plasma and urine samples . recently , label - free liquid chromatography mass spectrometry ( lc - ms ) quantification methods have been developed . these methods are typically based on determining peak area ratios of the same peptides between different conditions . using a label - free lc - ms approach ( so - called lc - ms ) , a series of plasma specimens from type 1 gaucher patients prior to and after therapy were studied ( vissers et al . 2007 ) . marked therapy - induced differences were noted in the gaucher disease protein plasma profile . comparison with the normal plasma profile revealed that many protein abnormalities in symptomatic patients were at least partially corrected by successful therapy ( vissers et al . lc - ms is used by us to study the proteome of laser - capture - dissected gaucher cells from spleens of gaucher patients . fabry disease is an x - linked lsd resulting from deficient activity of the lysosomal hydrolase -galactosidase a ( ec 3.2.1.22 ) ( desnick and ioannou 2001 ; brady et al . relatively high residual enzyme activity is often noted in patients with a milder variant of fabry disease with predominantly cardiac abnormalities , whereas there is little or no kidney dysfunction and no painful acroparesthesia ( desnick and ioannou 2001 ) . however , many female heterozygotes also display symptoms despite considerable amounts of circulating residual enzyme that varies due to random x inactivation . this sharply contrasts with the general lack of symptoms among carriers of another x - linked lysosomal hydrolase deficiency , hunter disease . deficiency of -galactosidase a results in accumulation of its glycosphingolipid substrates in lysosomes of endothelial , perithelial , and smooth - muscle cells of the vascular system , as well as renal epithelial cells , myocardial cells , and cells of the autonomic nervous system . the accumulating glycosphingolipids contain terminal -galactosyl moieties , such as globotriaosylceramide ( gb3 ; also known as ceramide trihexoside : cth ) ; galabiosylceramide ( gb2 ) ; and , to a lesser extent , blood group b , b1 , and p1 antigens ( desnick and ioannou 2001 ) . two different recombinant -galactosidase a preparations are in use for treating fabry disease ( schiffmann et al . one enzyme is produced by chinese hamster ovary ( cho ) cells with classic recombinant technology ( agalsidase , fabrazyme ) , and the other enzyme is produced by cultured human skin fibroblasts with an activated promoter of the -gal a gene ( agalsidase , replagal ) . both recombinant enzymes are quite comparable in properties and differ only slightly in glycan composition ( blom et al . the two enzyme preparations have independently been examined in clinical investigations and are both registered in europe for treating fabry patients . although both enzyme therapies were found to result in the desired clearance of globotriaosylceramide from the endothelium , the clinical effects are not as robust as anticipated . in some patients , stabilization of renal function and improvement in cardiac hypertrophy occurs upon therapy , but a considerable number experiences progressive complications ( vedder et al . numerous screening studies for fabry disease have been undertaken and are ongoing ( linthorst et al . 2010 ) . a serious complication in this connection is the difficulty of distinguishing whether some of the commonly encountered abnormalities in the gla gene are truly disease - causing mutations or polymorphisms that are not obligate disease causing ( froissart et al . other screening procedures are based on the demonstration of reduced enzymatic activity in blood cells , plasma , or dried blood spots . an associated limitation of such methods is the inability to reliably detect female carriers and some atypically affected male hemizygotes . as an alternative screening method , it is contemplated by hopwood and colleagues to use quantification of -gal a protein with specific antibodies in analogy to screening for other lysosomal enzymopathies ( tan et al . fabry biomarkers following the successful biomarker discovery for gaucher disease , attempts have been made to identify comparable metabolite and protein biomarkers for fabry disease . lack of prominent plasma protein abnormalities in symptomatic fabry patients , abnormalities are encountered that point to a low - grade inflammatory disorder ( schiffmann 2009 ) . indeed , increased circulating levels of c - reactive protein ( crp ) and the hydrolases myeloperoxidase , metalloproteinase 9 , and chitotriosidase , have been reported for symptomatic fabry hemizygotes ( vedder et al . 2006a ; kaneski et al . the abnormalities in the hydrolases , all produced by phagocytes , are not very striking and certainly not specific for fabry disease . as inflammation is not thought to be a major component of fabry disease , the value of the above - mentioned hydrolases as biomarkers seems limited . monitoring corrections induced by therapy in the levels of abnormal hydrolases may , however , be informative . for example , clear reductions were noted in elevated chitotriosidase in male fabry patients during enzyme therapy and relapses following the induction of neutralizing antibodies against the therapeutic enzyme ( vedder et al . the prevalent concept is that fabry disease is a systemic vasculopathy due to gb3 storage in endothelial cells . considerable attention has therefore been focused on identifying plasma protein abnormalities reflecting endothelial activation . known this has been further stimulated by various reports demonstrating disturbed vascular circulation and a prothrombotic state in fabry disease ( moore et al . laboratory investigations that have been performed to assess determinants of coagulation or activation of the endothelium in fabry patients are not always in accordance . in a very recent , thorough study conducted with a large cohort of fabry patients in the academic medical center in amsterdam , only minimal abnormalities in indicators of coagulation , fibrinolysis , and platelet and endothelial activation next to targeted analysis of plasma proteins already known to reflect endothelial activation , the search for protein biomarkers of fabry disease has been extended to analysis of the entire plasma proteome . moore and co - workers were the first to elegantly investigate plasma of children with fabry disease prior to and after ert using tryptic digestion of plasma protein and differentially labelling peptides with stable isotopes , such that consistent mass differences were introduced into selected amino acid residues ( moore et al . the lc - ms analysis showed only modest therapy - induced changes in a few proteins . most importantly , it stimulated the investigators to further analyze 2-antiplasmin concentrations in fabry patients using citrate - based plasma specimens and a chromogenic method . the mean level of -2-antiplasmin in 34 fabry patients aged 1055 years was 85% vs. normal laboratory mean of 105 ; range 82123% . a systematic proteomics analysis of blood specimens from fabry patients conducted at the academic medical center in amsterdam has not led to the discovery of prominent abnormalities in plasma proteins in symptomatic fabry patients ( aerts and co - workers , manuscript in preparation ) . lipid abnormalities as potential fabry biomarkers for a long time , the primary accumulating globoside gb3 has been considered as a surrogate marker for fabry disease . reduction of gb3 in tissue biopsies has actually served as the criterion for the registration of agalsidase beta . the globoside gb3 is not only elevated inside storage cells but is also present in abnormally high concentrations in bodily fluids , such as plasma and urine . it is very well documented that in symptomatic fabry hemizygotes both plasma and urinary gb3 are increased ( desnick and ioannou 2001 ) . intriguingly , in female carriers of fabry disease , urinary gb3 is generally increased , but plasma gb3 levels tend to be in the normal range ( vedder et al . mass - spectrometry - based and high - performance liquid chromatography ( hplc)-based procedures have become available , allowing accurate quantification of the globoside ( mills et al . 2004 ; fauler et al . 2005 ; fuller et al . 2005 ; auray - blais et al . 2007 ; groener et al . 2007 ) . demonstration of increased gb3 , either in plasma or urine , is of great value for diagnostic purposes , particularly for putative fabry females carrying a gla mutation with unclear consequences . it has been reported that urinary gb3 levels correlate with the predicted severity of gla mutations ( auray - blais et al . globotriaosylceramide ( gb3 ) the value of plasma or urinary gb3 as biomarker to monitor progression of fabry disease is questionable . in several investigations , plasma or urinary gb3 has been found to poorly reflect fabry disease manifestation and therapeutic outcome ( vedder et al . 2007c ; young et al . 2005 ; whitfield et al . 2005 ; bekri et al . for example , many male fabry patients lacking endogenous -galactosidase a develop neutralizing antibodies during ert ( linthorst et al . the occurrence of such antibodies is accompanied by a relapse in elevated plasma gb3 , but the clinical significance of this remains unclear ( vedder et al . the poor predictive value of plasma or urinary gb3 levels for fabry disease manifestation is not entirely surprising . prominent gb3 accumulation has been noted in placental tissue of a fabry hemizygote ( popli et al . 2006b ) , a finding illustrating that onset of clinical complications occurs only after several years of lipid deposition . gb3 accumulation apparently occurs in hemizygotes at or even before birth , long before any clinical symptoms are prominent . the same discrepancy between early storage of gb3 and clinical symptoms is also noted in fabry mice generated by disruption of the gla gene ( ohshima et al . plasma gb3 concentrations in some presymptomatic boys have been found to exceed those in symptomatic adult hemizygotes ( vedder et al . 2007c ) , the absence of infantile manifestations in fabry patients completely lacking -galactosidase a activity also indicates that gb3 accumulation does not cause immediately , and maybe even not directly , signs of disease . an investigation by barbey and co - workers provided evidence for the presence of an unidentified substance in plasma of symptomatic fabry disease patients that stimulates proliferation of vascular smooth - muscle cells and cardiomyocytes in vitro ( barbey et al . it is conceivable that this substance is a causative factor in the development of left ventricular hypertrophy and increased intima media thickness in fabry patients ( boutouyrie et al . globotriaosylsphingosine ( lysogb3 ) the puzzling findings prompted us to re - examine gb3 and its metabolites in fabry patients ( aerts et al . , it was discovered that plasma of fabry patients contains markedly increased concentrations of deacylated globotriaosylceramide , globotriaosylsphingosine ( here abbreviated as lysogb3 ) . the relative increase in plasma concentrations of the cationic amphiphilic lysogb3 spectacularly exceeds that of gb3 by more than an order of magnitude . also in the case of symptomatic female fabry patients , clearly increased levels of lysogb3 were detected , whereas concomitantly , gb3 concentrations were in the high normal range ( aerts et al . thus , measurement of plasma lysogb3 seems to offer a very useful diagnostic tool , particularly in the case of female fabry patients . meanwhile , the findings for lysogb3 in fabry disease have been confirmed by independent investigations ( togawa et al . auray - blais and co - workers demonstrated the presence of elevated lysogb3 in urine samples of fabry patients ( auray - blais et al . the relationship between plasma lysogb3 concentrations and fabry disease manifestations has been investigated ( rombach et al . it is clear that in fabry hemizygotes , both in humans and mice , plasma lysogb3 is already increased at birth and consequently does not correlate strictly with symptoms ( aerts et al . some degree of correlation of plasma lysogb3 levels and disease manifestation seems to exist for female hemizygotes . preliminary data indicate that high plasma lysogb3 correlates with increased risk for cerebrovascular disease in males ( rombach et al . of interest , life - time exposure to plasma lysogb3 is found to correlate with disease severity in male as well as female patients ( rombach et al . these observations suggest , but certainly do not prove , that lysogb3 plays a direct role in the pathogenesis of fabry disease . a role as pathogenic factor for lysogb3 is suggested by the elegant studies of ortiz and co - workers ( sanchez - nio et al . they demonstrated that exposure of cultured glomerular podocytes to lysogb3 increased the expression of tumor growth factor ( tgf)-beta-1 , extracellular matrix proteins ( fibronectin and type iv collagen ) , and cd74 . it was concluded from these findings that lysogb3 may have a role in glomerular injury in fabry disease by promoting the release of secondary mediators of glomerular injury common to diabetic nephropathy ( sanchez - nio et al . it has already been found that lysogb3 at concentrations occurring in plasma of symptomatic fabry patients is able to induce proliferation of smooth - muscle cells in vitro , possibly explaining the increased intima media thickness in fabry patients ( boutouyrie et al . the effect of ert on plasma lysogb3 has recently been reported ( van breemen et al . 2011 ) . a prominent lsd is gaucher disease ( beutler et al . 2001 ; aerts et al . brady and co - workers first showed that the primary defect in gaucher disease is a marked deficiency in activity of the lysosomal enzyme glucocerebrosidase ( brady et al . deficiencies in glucocerebrosidase result in accumulation of its lipid substrate glucosylceramide in the lysosomal compartment of macrophages throughout the body . different phenotypes ( types 1 , 2 , and 3 ) are generally recognized and are differentiated on the basis of the presence or absence of neurological symptoms . whether a strict division in three different phenotypes is still valid has been the subject of debate , as there are an increasing number of reports on neurological manifestations in patients with type 1 gaucher disease . however , the neurological signs and symptoms in type 1 gaucher disease are of a totally different kind from and , in the majority of cases , of much less severity than , the signs and symptoms associated with types 2 and 3 disease ( biegstraaten et al . 2008 ) . it has also become apparent that complete deficiencies in glucocerebrosidase activity occur , resulting in major skin permeability abnormalities with lethal consequences either prenatally or shortly after birth ( sidransky 2004 ) . the most common gaucher phenotype is non - neuronopathic type 1 manifestation , with a highly variable age of onset and severity . even spontaneous disease regression has been observed in some relatively mildly affected type 1 gaucher patients ( boomsma et al . atypical bone pain , pathological fractures , avascular necrosis , and extremely painful bone crises may also have a great impact on quality of life . gammopathies and metabolic abnormalities such as insulin resistance and lipoprotein abnormalities are encountered in type 1 gaucher patients ( langeveld et al . 2007 , 2008b ; wennekes et al . 2009 ; de fost et al . 2009 , 2008 ) . it is prevalent among ashkenazi jews , with a carrier frequency as high as about 1 in 15 and an incidence of about 1 in 1,000 . the most common mutation in the glucocerebrosidase gene of caucasians , including ashkenazi jews , encodes the amino acid substitution n370s . the heteroallelic presence of the n370s mutation is always associated with a non - neuronopathic course ( ohashi et al . 1991 ; van weely et al . some , but not all , homozygotes for the n370s mutation develop significant clinical symptoms ( beutler et al . 2001 ; aerts et al . twin studies and the poor predictive power of genotype phenotype investigations in gaucher disease have clearly pointed out that epigenetic factors also play a key role in gaucher disease manifestation ( biegstraaten et al . the diagnosis of gaucher disease can be confirmed by demonstration of reduced glucocerebrosidase activity in lysates of various cell types and even urine samples using the fluorogenic substrate 4-methylumbelliferyl beta - glucoside ( aerts et al . fluorescent analogues of the lipid glucosylceramide can be exploited to measure glucocerebrosidase activity in cultured cells ( van weely et al . highly specific monoclonal antibodies against human glucocerebrosidase have been produced and can be used in purification of the protein , western blot analysis , and microscopy techniques ( aerts et al . although glucocerebrosidase is present in the lysosomes of all cell types , patients with type 1 gaucher disease only store glucosylceramide in macrophages . it is believed that the storage material stems from the breakdown of exogenous lipids derived from the turnover of blood cells . the glucosylceramide - loaded macrophages of gaucher patients show a characteristic morphology with a wrinkled - paper appearance of their cytoplasm , which contains lysosomal inclusion bodies ; these cells are referred to as gaucher cells . in recent decades , it has become apparent that gaucher cells are not inert containers of storage material but are viable , chronically activated macrophages that contribute to the diverse clinical manifestations of gaucher disease . these mature , activated macrophages are surrounded by newly formed , highly inflammatory macrophages in tissue lesions of patients with gaucher disease ( boven et al . consistent with these observations , patients with gaucher disease show increased plasma levels of several proinflammatory and anti - inflammatory cytokines , chemokines , and hydrolases ( aerts and hollak 1997 ; aerts et al . factors released by gaucher cells and surrounding macrophages are thought to play a crucial role in the development of common abnormalities in gaucher patients , such as osteopenia , activation of coagulation , hypermetabolism , gammopathies , multiple myeloma , and hypolipoproteinemias ( aerts and hollak 1997 ) . biomarkers of gaucher cells given the prominent role of gaucher cells in the pathophysiology of the disorder , considerable attention has been focused on identifying plasma markers for such macrophages . abnormalities in levels of tartrate - resistant acid phosphatase ( trap ) , angiotensin - converting enzyme ( ace ) , hexosaminidase , and lysozyme in serum samples from gaucher patients had been documented for some time ( aerts and hollak 1997 ) . more recently , increased plasma levels of various cathepsins , including cathepsin k , were reported in patients with gaucher disease ( moran et al . all of these proteins are known to be produced by macrophages ; however , none of them appears to be a specific marker for the pathological gaucher cells , and their levels in the serum of symptomatic patients with gaucher disease may overlap with those observed in healthy individuals . the need for a very sensitive and specific marker for gaucher cells prompted a search for such a parameter . this search led to the discovery of a striking abnormality in the serum of symptomatic patients with gaucher disease . chitotriosidase serum from such individuals showed a 1,000-fold increased capacity to degrade the fluorogenic substrate 4-methylumbelliferyl chitotrioside ( hollak et al . 1994 ) . the chitotriosidase protein was subsequently purified and its complementary dna ( cdna ) cloned ( renkema et al . 1995 ; boot et al . 1995 ) . chitotriosidase was found to be the human analogue of chitinases from lower organisms ( rao et al . the enzyme hydrolyzes chitin , the natural polymer of beta-1,4 n - acetylglucosamine , is a member of the large chitinase protein family created by gene duplications ( bussink et al . the chitinase protein family includes one other active enzyme amcase ( boot et al . 2001)and several inactive lectins ( bussink et al . 2007 ; renkema et al . 1998 ) . chitotriosidase is specifically expressed by phagocytes in humans , including macrophages and neutrophils ( renkema et al . it has been shown to exert a fungistatic action of degradation of the protective chitin layer at the hyphal tip of fungi ( van eijk et al . . in situ hybridization and histochemistry of bone marrow aspirates and sections of spleens from patients with gaucher disease revealed that chitotriosidase is massively produced by storage cells . this is also supported by the close linear relationship between chitotriosidase and glucosylceramide levels in different spleen sections from patients with gaucher disease ( bussink et al . 2006 ) . in a culture model of gaucher cells , chitotriosidase accounts for almost 10% of the total secreted protein . these observations help us to understand the very specific , gross elevation of chitotriosidase levels in the blood of patients with gaucher disease . a relationship between the total body burden of storage cells in patients with gaucher disease and their plasma chitotriosidase levels has been noted . the plasma chitotriosidase level does not reflect any one particular clinical symptom of gaucher disease , suggesting , rather , that it reflects the sum of secreted enzyme by gaucher cells in various body locations . plasma chitotriosidase levels can be determined by monitoring hydrolysis of the fluorogenic substrate 4-methylumbelliferyl - chitobioside . however , the ability of chitotriosidase to transglycosylate as well as hydrolyze this substrate complicates the enzyme assay ( aguilera et al . special care has to be taken to ensure that the enzyme activity is truly proportional to the amount of chitotriosidase protein . a far more convenient , sensitive , and accurate detection is feasible by measuring the activity of chitotriosidase toward the recently designed fluorogenic substrate 4-methylumbelliferyl - deoxy - chitotrioside ( aguilera et al . interpretation of plasma chitotriosidase levels is intrinsically complicated by the common occurrence of a particular 24-base - pair duplication in the chitotriosidase gene , preventing the formation of chitotriosidase protein ( aguilera et al . about one in every three individuals carries this abnormality , and about one in every 20 individuals , including patients with gaucher disease , is homozygous for this trait ( aguilera et al . it has been established that carriers of the 24-bp duplication show half the amount of plasma chitotriosidase detected in individuals with the wild - type chitotriosidase genotype ( aguilera et al . 1998 ) . it is therefore common to correct plasma chitotriosidase by a factor of two when examining patients with gaucher disease who are carriers of the 24-bp duplication . other polymorphisms have also been detected , of which the g102s substitution is the most frequent ( allele frequency of about 0.25 ) . this polymorphism leads to an enzyme with a slightly impaired catalytic activity toward the 4-methylumbelliferyl - chitotrioside substrate compared with wild type . however , g102s enzyme activity is normal when using 4-methylumbelliferyl - deoxy - chitobioside as substrate ( bussink et al . it should be mentioned that increased plasma chitotriosidase activity is not unique for gaucher patients . plasma chitotriosidase activity is increased , albeit much more modestly , in several lysosomal and nonlysosomal diseases , such as sarcoidosis , visceral leishmaniasis , leprosy , arthritis , multiple sclerosis , thalassemia , chronic obstructive pulmonary disease ( copd ) , malaria , and atherosclerosis ( hollak et al . 1994 ; guo et al . parc / ccl18 the high frequency of chitotriosidase deficiency prompted us to search for an alternative marker of gaucher cells . using surface - enhanced laser distortion / ionization time of flight ( seldi - tof ) mass spectrometric analyses , we discovered massive overproduction and secretion by gaucher cells of the pulmonary and activation - regulated chemokine ( parc / ccl18 ) , of which the messenger rna ( mrna ) was previously observed to be up - regulated in the spleen of a patient with gaucher disease ( moran et al . this plasma parc / ccl18 can not be reliably quantified using seldi - tof , but reliable quantification is obtained by elisa ( van breemen et al . plasma parc / ccl18 levels are 10- to 40-fold elevated in symptomatic gaucher patients ( boot et al . 2005 ) . due to its basic nature and small molecular mass , parc / ccl18 levels in urine therefore , urinary parc / ccl18 excretion offers insight into the body burden of gaucher cells ( boot et al . 2006 ) . measurement of plasma parc / ccl18 has been found to yield an excellent additional tool to monitor changes in body burden of gaucher cells . it is particularly useful for evaluating patients that are chitotriosidase deficient ( cox et al . mip-1 and mip-1 van breemen and co - workers reported markedly elevated levels of the chemokines macrophage inflammatory protein ( mip)-1 and mip-1 in plasma of symptomatic gaucher patients ( van breemen et al . 2007 ) . interestingly , with immunohistochemistry , these proteins were found to be produced by surrounding inflammatory spleen macrophages and not by mature gaucher cells ( van breemen et al . the different cellular source is also reflected in the observation that plasma chitotriosidase and parc / ccl18 , both stemming from gaucher cells , respond comparably to ert . however , corrections in plasma mip-1 and mip-1 following ert are not proportional to those found with the true gaucher - cell biomarkers ( van breemen et al . 2009 ) . a relationship was observed between plasma mip-1 and skeletal disease : stable high plasma mip-1 levels despite prolonged ert were found to correlate with ongoing skeletal disease ( van breemen et al . rigorous analysis of a large cohort of gaucher patients is required to establish the value of plasma mip-1 ( or mip-1 ) as biomarker , especially its value as prognostic marker for skeletal response to therapy . clinical applications of gaucher - cell biomarkers plasma chitotriosidase measurement is commonly employed as a first screen in the diagnosis of gaucher disease . increasing plasma levels seem to reflect gradual accumulation of storage cells in the patient s body . in an attempt to assess the utility of plasma chitotriosidase activity measurement as a biomarker for treatment efficacy , hollak and co - workers investigated the relationship between enzyme activity and clinical parameters ( hollak et al . chitotriosidase levels were usually > 20,000 nmol / ml / h and always > 15,000 nmol / ml / h , whereas patients with less severe disease tended to have lower values . during enzyme supplementation therapy , the mean decrease in 12 months was 32% ( range 082% ) , and 78% of patients had a decrease of > 15% . in six patients with a decrease in chitotriosidase activity of < 15% , the clinical response to treatment was inferior to that of other patients , with less reduction in organomegaly in four and bone problems in two . in addition , chitotriosidase response was related to disease severity ; less reduction in plasma activity was seen in more severely affected individuals . on the basis of this investigation , it has been proposed that in patients in whom the initiation of treatment is questionable based solely on clinical parameters , a chitotriosidase activity > 15,000 nmol / ml / h may serve as an indicator of a high gaucher - cell burden and an indication for treatment initiation ( hollak et al . 2001 ) . a reduction in chitotriosidase activity of < 15% after 12 months of treatment , in combination with an insufficient response of at least one clinical parameter , should be a reason to consider dose increase . furthermore , a sustained increase in chitotriosidase at any point during treatment should alert the physician to the possibility of clinical deterioration and the need for dose adjustment . a recent retrospective analysis by deegan and co - workers confirmed the value of the use of plasma chitotriosidase in gaucher disease management and presented evidence for comparable use of parc / ccl18 ( deegan et al . 2005 ) . in conclusion , next to radiological monitoring of the bone marrow and skeleton ( maas et al . 2002 ) , measurement of plasma levels of chitotriosidase and/or parc / ccl18 offers valuable information for clinical management of type 1 gaucher patients . lipid biomarkers of gaucher disease increased plasma concentrations of glucosylceramide , the primary storage lipid , have been documented for gaucher patients ( groener et al . plasma glucosylceramide is not used as a biomarker , as its increase generally is not very pronounced . moreover , the exact relation between circulating glucosylceramide and storage cells in tissues is far from clear . interestingly , besides glucosylceramide , the ganglioside gm3 is also elevated in gaucher plasma samples ( ghauharali - van der vlugt et al . elevated glycosphingolipids such as gm3 are thought to cause insulin resistance ( aerts et al . indeed , a recent study revealed that gaucher patients are insulin resistant without overt hyperglycemia ( langeveld et al . other secondary lipid abnormalities in gaucher patients have been noted by thorough investigations by fuller and co - workers , such as elevated levels of phosphatidylglycerol ( hein et al . the diagnostic value and possible physiological consequences warrant further investigations . discovery of additional protein biomarkers for gaucher disease ongoing attention is paid to the detection of useful protein biomarkers for gaucher disease by a thorough survey of protein composition of bodily fluids , or cell and tissue specimens of symptomatic gaucher patients . the latter approach has more recently become feasible due to the availability of mass spectrometric techniques that allow accurate analysis of proteins , even in complex mixtures such as plasma and urine samples . recently , label - free liquid chromatography mass spectrometry ( lc - ms ) quantification methods have been developed . these methods are typically based on determining peak area ratios of the same peptides between different conditions . using a label - free lc - ms approach ( so - called lc - ms ) , a series of plasma specimens from type 1 gaucher patients prior to and after therapy were studied ( vissers et al . 2007 ) . marked therapy - induced differences were noted in the gaucher disease protein plasma profile . comparison with the normal plasma profile revealed that many protein abnormalities in symptomatic patients were at least partially corrected by successful therapy ( vissers et al . lc - ms is used by us to study the proteome of laser - capture - dissected gaucher cells from spleens of gaucher patients . fabry disease is an x - linked lsd resulting from deficient activity of the lysosomal hydrolase -galactosidase a ( ec 3.2.1.22 ) ( desnick and ioannou 2001 ; brady et al . 1967 ) . in male patients with severe forms of fabry disease , there is usually severely reduced or complete lack of enzymatic activity . relatively high residual enzyme activity is often noted in patients with a milder variant of fabry disease with predominantly cardiac abnormalities , whereas there is little or no kidney dysfunction and no painful acroparesthesia ( desnick and ioannou 2001 ) . however , many female heterozygotes also display symptoms despite considerable amounts of circulating residual enzyme that varies due to random x inactivation . this sharply contrasts with the general lack of symptoms among carriers of another x - linked lysosomal hydrolase deficiency , hunter disease . deficiency of -galactosidase a results in accumulation of its glycosphingolipid substrates in lysosomes of endothelial , perithelial , and smooth - muscle cells of the vascular system , as well as renal epithelial cells , myocardial cells , and cells of the autonomic nervous system . the accumulating glycosphingolipids contain terminal -galactosyl moieties , such as globotriaosylceramide ( gb3 ; also known as ceramide trihexoside : cth ) ; galabiosylceramide ( gb2 ) ; and , to a lesser extent , blood group b , b1 , and p1 antigens ( desnick and ioannou 2001 ) . two different recombinant -galactosidase a preparations are in use for treating fabry disease ( schiffmann et al . one enzyme is produced by chinese hamster ovary ( cho ) cells with classic recombinant technology ( agalsidase , fabrazyme ) , and the other enzyme is produced by cultured human skin fibroblasts with an activated promoter of the -gal a gene ( agalsidase , replagal ) . both recombinant enzymes are quite comparable in properties and differ only slightly in glycan composition ( blom et al . the two enzyme preparations have independently been examined in clinical investigations and are both registered in europe for treating fabry patients . although both enzyme therapies were found to result in the desired clearance of globotriaosylceramide from the endothelium , the clinical effects are not as robust as anticipated . in some patients , stabilization of renal function and improvement in cardiac hypertrophy occurs upon therapy , but a considerable number experiences progressive complications ( vedder et al . numerous screening studies for fabry disease have been undertaken and are ongoing ( linthorst et al . a serious complication in this connection is the difficulty of distinguishing whether some of the commonly encountered abnormalities in the gla gene are truly disease - causing mutations or polymorphisms that are not obligate disease causing ( froissart et al . other screening procedures are based on the demonstration of reduced enzymatic activity in blood cells , plasma , or dried blood spots . an associated limitation of such methods is the inability to reliably detect female carriers and some atypically affected male hemizygotes . as an alternative screening method , it is contemplated by hopwood and colleagues to use quantification of -gal a protein with specific antibodies in analogy to screening for other lysosomal enzymopathies ( tan et al . fabry biomarkers following the successful biomarker discovery for gaucher disease , attempts have been made to identify comparable metabolite and protein biomarkers for fabry disease . lack of prominent plasma protein abnormalities in symptomatic fabry patients , abnormalities are encountered that point to a low - grade inflammatory disorder ( schiffmann 2009 ) . indeed , increased circulating levels of c - reactive protein ( crp ) and the hydrolases myeloperoxidase , metalloproteinase 9 , and chitotriosidase , have been reported for symptomatic fabry hemizygotes ( vedder et al . 2006a ; kaneski et al . 2006 ; shah et al . 2007 ) . the abnormalities in the hydrolases , all produced by phagocytes , are not very striking and certainly not specific for fabry disease . as inflammation is not thought to be a major component of fabry disease , the value of the above - mentioned hydrolases as biomarkers seems limited . monitoring corrections induced by therapy in the levels of abnormal hydrolases may , however , be informative . for example , clear reductions were noted in elevated chitotriosidase in male fabry patients during enzyme therapy and relapses following the induction of neutralizing antibodies against the therapeutic enzyme ( vedder et al . the prevalent concept is that fabry disease is a systemic vasculopathy due to gb3 storage in endothelial cells . known plasma proteins reflecting endothelial activation have been considered as candidate biomarkers for fabry disease . this has been further stimulated by various reports demonstrating disturbed vascular circulation and a prothrombotic state in fabry disease ( moore et al . laboratory investigations that have been performed to assess determinants of coagulation or activation of the endothelium in fabry patients are not always in accordance . in a very recent , thorough study conducted with a large cohort of fabry patients in the academic medical center in amsterdam , only minimal abnormalities in indicators of coagulation , fibrinolysis , and platelet and endothelial activation next to targeted analysis of plasma proteins already known to reflect endothelial activation , the search for protein biomarkers of fabry disease has been extended to analysis of the entire plasma proteome . moore and co - workers were the first to elegantly investigate plasma of children with fabry disease prior to and after ert using tryptic digestion of plasma protein and differentially labelling peptides with stable isotopes , such that consistent mass differences were introduced into selected amino acid residues ( moore et al . the lc - ms analysis showed only modest therapy - induced changes in a few proteins . most importantly , it stimulated the investigators to further analyze 2-antiplasmin concentrations in fabry patients using citrate - based plasma specimens and a chromogenic method . the mean level of -2-antiplasmin in 34 fabry patients aged 1055 years was 85% vs. normal laboratory mean of 105 ; range 82123% . a systematic proteomics analysis of blood specimens from fabry patients conducted at the academic medical center in amsterdam has not led to the discovery of prominent abnormalities in plasma proteins in symptomatic fabry patients ( aerts and co - workers , manuscript in preparation ) . lipid abnormalities as potential fabry biomarkers for a long time , the primary accumulating globoside gb3 has been considered as a surrogate marker for fabry disease . reduction of gb3 in tissue biopsies has actually served as the criterion for the registration of agalsidase beta . the globoside gb3 is not only elevated inside storage cells but is also present in abnormally high concentrations in bodily fluids , such as plasma and urine . it is very well documented that in symptomatic fabry hemizygotes both plasma and urinary gb3 are increased ( desnick and ioannou 2001 ) . intriguingly , in female carriers of fabry disease , urinary gb3 is generally increased , but plasma gb3 levels tend to be in the normal range ( vedder et al . mass - spectrometry - based and high - performance liquid chromatography ( hplc)-based procedures have become available , allowing accurate quantification of the globoside ( mills et al . ; fuller et al . 2005 ; auray - blais et al . 2007 ; groener et al . 2007 ) . demonstration of increased gb3 , either in plasma or urine , is of great value for diagnostic purposes , particularly for putative fabry females carrying a gla mutation with unclear consequences . it has been reported that urinary gb3 levels correlate with the predicted severity of gla mutations ( auray - blais et al . globotriaosylceramide ( gb3 ) the value of plasma or urinary gb3 as biomarker to monitor progression of fabry disease is questionable . in several investigations , plasma or urinary gb3 has been found to poorly reflect fabry disease manifestation and therapeutic outcome ( vedder et al . 2007c ; young et al . 2005 ; whitfield et al . 2005 ; bekri et al . 2006 ) . for example , many male fabry patients lacking endogenous -galactosidase a develop neutralizing antibodies during ert ( linthorst et al . the occurrence of such antibodies is accompanied by a relapse in elevated plasma gb3 , but the clinical significance of this remains unclear ( vedder et al . the poor predictive value of plasma or urinary gb3 levels for fabry disease manifestation is not entirely surprising . prominent gb3 accumulation has been noted in placental tissue of a fabry hemizygote ( popli et al . 2006b ) , a finding illustrating that onset of clinical complications occurs only after several years of lipid deposition . accumulation apparently occurs in hemizygotes at or even before birth , long before any clinical symptoms are prominent . the same discrepancy between early storage of gb3 and clinical symptoms is also noted in fabry mice generated by disruption of the gla gene ( ohshima et al . plasma gb3 concentrations in some presymptomatic boys have been found to exceed those in symptomatic adult hemizygotes ( vedder et al . a activity also indicates that gb3 accumulation does not cause immediately , and maybe even not directly , signs of disease . an investigation by barbey and co - workers provided evidence for the presence of an unidentified substance in plasma of symptomatic fabry disease patients that stimulates proliferation of vascular smooth - muscle cells and cardiomyocytes in vitro ( barbey et al . it is conceivable that this substance is a causative factor in the development of left ventricular hypertrophy and increased intima media thickness in fabry patients ( boutouyrie et al . globotriaosylsphingosine ( lysogb3 ) the puzzling findings prompted us to re - examine gb3 and its metabolites in fabry patients ( aerts et al . , it was discovered that plasma of fabry patients contains markedly increased concentrations of deacylated globotriaosylceramide , globotriaosylsphingosine ( here abbreviated as lysogb3 ) . the relative increase in plasma concentrations of the cationic amphiphilic lysogb3 spectacularly exceeds that of gb3 by more than an order of magnitude . also in the case of symptomatic female fabry patients , clearly increased levels of lysogb3 were detected , whereas concomitantly , gb3 concentrations were in the high normal range ( aerts et al . thus , measurement of plasma lysogb3 seems to offer a very useful diagnostic tool , particularly in the case of female fabry patients . meanwhile , the findings for lysogb3 in fabry disease have been confirmed by independent investigations ( togawa et al . auray - blais and co - workers demonstrated the presence of elevated lysogb3 in urine samples of fabry patients ( auray - blais et al . the relationship between plasma lysogb3 concentrations and fabry disease manifestations has been investigated ( rombach et al . it is clear that in fabry hemizygotes , both in humans and mice , plasma lysogb3 is already increased at birth and consequently does not correlate strictly with symptoms ( aerts et al . some degree of correlation of plasma lysogb3 levels and disease manifestation seems to exist for female hemizygotes . preliminary data indicate that high plasma lysogb3 correlates with increased risk for cerebrovascular disease in males ( rombach et al . of interest , life - time exposure to plasma lysogb3 is found to correlate with disease severity in male as well as female patients ( rombach et al . these observations suggest , but certainly do not prove , that lysogb3 plays a direct role in the pathogenesis of fabry disease . a role as pathogenic factor for lysogb3 is suggested by the elegant studies of ortiz and co - workers ( sanchez - nio et al . they demonstrated that exposure of cultured glomerular podocytes to lysogb3 increased the expression of tumor growth factor ( tgf)-beta-1 , extracellular matrix proteins ( fibronectin and type iv collagen ) , and cd74 . it was concluded from these findings that lysogb3 may have a role in glomerular injury in fabry disease by promoting the release of secondary mediators of glomerular injury common to diabetic nephropathy ( sanchez - nio et al . it has already been found that lysogb3 at concentrations occurring in plasma of symptomatic fabry patients is able to induce proliferation of smooth - muscle cells in vitro , possibly explaining the increased intima media thickness in fabry patients ( boutouyrie et al . 2002 ; barbey et al . the effect of ert on plasma lysogb3 has recently been reported ( van breemen et al . 2011 ) . a major limitation so far in evaluating lsds has been the lack of a technology that allows selective labeling of active lysosomal hydrolases in vitro and in vivo . the availability of such a method would allow more advanced diagnostic tests and more sophisticated monitoring of tissue targeting of therapeutic proteins , and would boost fundamental research on lysosomal hydrolases . recently we conceived such a method for the lysosomal glucocerebrosidase based on activity - based covalent labeling of the enzyme s nucleophile , glutamate 340 , with fluorescent probes . the catalytic mechanism of glucocerebrosidase , a retaining beta - glucosidase , has been elucidated in detail ( liou and grabowski 2009 ; rempel and withers 2008 ) . we employed the fact that conduritol b - epoxide and cyclophellitol are potent suicide inhibitors of glucocerebrosidase ( van es et al . 1993 ) . by coupling a fluorescent boron - dipyrromethene ( bodipy ) moiety via a triazole linker to cyclophellitol , extremely potent fluorescent suicide inhibitors for glucocerebrosidase were generated ( witte et al . 2010 ) . the probes are amphiphilic and easily penetrate cells , allowing in vitro and in vivo labeling of glucocerebrosidase in cells and whole organisms . the term inhibody is coined for these types of probes , as they act as suicide inhibitors and allow direct visualization of originally active enzyme molecules with comparable methods as used for specific antibodies . the detection power of the developed inhibodies for glucocerebrosidase is amazing : as little as a few attomoles of enzyme can be visualized on slab gels by fluorescence scanning . the specificity of glucocerebrosidase labeling with the inhibody probes is also extraordinary high , even when tissues are analyzed ( atsumi et al . it has been demonstrated that the probes can be used for diagnostic purposes , allowing visualization of active glucocerebrosidase in very few cultured skin fibroblasts . the probes can also be employed for fluorescence - activated cell - sorting analysis , fluorescence microscopy , and pulse - chase experiments ( atsumi et al . 1993 ) . it is envisioned that comparable specific probes can be designed for other retaining lysosomal glycosidases by adaptations in the sugar configuration . an interesting potential application for inhibodies is to be found in subtle labeling of therapeutic recombinant protein and monitoring its tissue distribution following intravenous administration . the use of a near - infrared fluorophore to enable visualization of therapeutic glucocerebrosidase in individual patients is appealing . a similar approach was recently reported using a radio - tagged suicide inhibitor , allowing visualization of administered labeled glucocerebrosidase in mice using positron emission tomography ( phenix et al . it now becomes feasible to obtain information about the exact tissue distribution of enzymatically active glucocerebrosidase . such information may render new insights into gaucher disease and help our understanding of why carriership for the disorder constitutes a risk factor for parkinsonism ( goker - alpan et al . it is transported to lysosomes independently of the mannose-6-phophate receptor system ( aerts et al . 1991a ; rijnboutt et al . 1991b ) .. the membrane protein limp2 has been shown to act as sorting receptor in certain cell types , such as fibroblasts ( reczek et al . other cell types , including blood cells , apparently can employ an alternative sorting receptor ( balreira et al . 2008 ) . analysis of limp2-deficient mice and humans with the available inhibody probes for glucocerebrosidase may be of great value in this connection . a next challenge is designing comparable probes that recognize and label several beta - glucosidases . besides glucocerebrosidase , humans contain other beta - glucosidases , such as gba2 ( van weely et al . 2007 ; yildiz et al . 2006 ; dekker et al . 2010 ) and gba3 that might play a role in the pathogenesis of gaucher disease . in summary , the search for biomarkers of gaucher disease have been extraordinary productive . next to elevations in circulating glucosylceramide and gangliosides such as gm3 in gaucher patients , striking abnormalities in plasma concentrations of some proteins have been identified . for example , increases in chitotriosidase up to 10,000-fold above normal have been identified in some gaucher patients . for some of these proteins , i.e. , chitotriosidase and parc / ccl18 , it has been demonstrated that they stem from storage cells . the circulating levels of these proteins offer insight into the total burden of storage cells in gaucher patients . regular monitoring of chitotriosidase , or parc / ccl18 in chitotriosidase - deficient individuals , with sensitive assays is widely applied in gaucher clinics and assists in clinical decision making . accurate methods have been developed to quantify the primary storage lipid gb3 in plasma and urine specimens . however , it is generally felt that measurement of plasma and urinary gb3 is only useful for diagnostic purposes and offers no sensitive tool to monitor fabry disease progression . systematic and solid investigations with large cohorts of fabry patients are necessary to reveal the true value of lysogb3 as biomarker . in any case , the demonstration of increased plasma lysogb3 in female heterozygotes offers an important additional diagnostic tool . the availability of fluorescent probes , so - called inhibodies , which allow selective labeling ultrasensitive detection of glucocerebrosidase offers new opportunities for diagnosis of the disorder . in addition , the probes may be employed in the future in monitoring tissue distribution of recombinant therapeutic protein in individual gaucher patients . finally , inhibodies may boost fundamental research on glucocerebrosidase and the pathogenesis of gaucher disease and parkinsonism . in the case of lysosomal storage diseases , biochemical abnormalities can be identified in blood and/or urine samples as described above for gaucher and fabry disease . such terminology should be restricted to abnormalities that are disease specific and are in the context of disease manifestation , for example , circulating protein or metabolite markers stemming from storage cells . generally , these biomarkers reflect not one particular symptom but rather the total body burden of storage cells . thus , such biomarkers are not true surrogate markers of disease that accurately reflect a particular symptom in one organ . although some of the available biomarkers , such as chitotriosidase , may assist in clinical management , further studies analyzing the correlation between long - term biomarker responses and objective clinical outcome parameters following therapeutic interventions are still warranted .

a biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease . in the case of lysosomal storage disorders ( lsds ) , primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers . clinical applications of biomarkers are found in improved diagnosis , monitoring disease progression , and assessing therapeutic correction . these are illustrated by reviewing the discovery and use of biomarkers for gaucher disease and fabry disease . in addition , recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described . such probes , coined inhibodies , offer entirely new possibilities for more sophisticated molecular diagnosis , enzyme replacement therapy monitoring , and fundamental research .

Introduction Biomarkers Gaucher disease Fabry disease Inhibodies: new, versatile tools for diagnosis, monitoring therapeutic enzyme targeting and fundamental research Biomarkers, biochemical abnormalities, and surrogate markers of disease

lysosomal storage disorders continuous recycling of their macromolecular constituents is essential for the functional integrity of long - lived cells . this discovery led to the concept of lysosomal storage disorders ( lsds ) , of which more than 70 have now been described . lysosomal storage disorders may present with a broad range of phenotypes , with variable age of onset , symptom severity , and degree of central nervous system ( cns ) involvement . for instance , in gaucher disease ( beta - glucocerebrosidase deficiency ) , the most severe phenotype presents with severe neurological involvement and death soon after birth , whereas at the other end of the phenotypic spectrum , patients are found in whom diagnosis is made in late adulthood based on minimal visceral symptoms ( aerts et al . this concept , named enzyme replacement therapy ( ert ) , is based on the principle that lysosomal proteins can reach the subcellular lysosomes via endocytosis mediated by lectins on the plasma membrane , such as the mannose 6-phosphate receptor and the mannose receptor . this product was later replaced by recombinant enzyme ( cerezyme ; genzyme ) , which proved to be very successful in treating the visceral and bone complications of gaucher disease ( de fost et al . an alternative recombinant enzyme preparation ( velaglucerase alfa ; shire ) , produced in human cells , was registered for ert of type 1 gaucher disease ( aerts et al . 2007 ) , and anecdotal reports suggest that combination srt and ert may be beneficial for some type 3 neuronopathic gaucher patients ( cox - brinkman et al . 2010b ) , and a 2-year follow - up of a phase ii trial indicated major improvements in hematological , visceral , and skeletal manifestations in adult patients with type 1 gaucher disease on a par with ert ( lukina et al . numerous hurdles , such as transient expression of transgenes , immunological responses , and tumor induction , have so far discouraged the clinical application of gene therapy . example of corrections in plasma chitotriosidase in gaucher disease patients receiving enzyme replacement therapy gaucher cell accumulating the glycosphingolipid glucosylceramide and specifically secreting the biomarker chitotriosidase that can be detected in plasma . example of corrections in plasma chitotriosidase in gaucher disease patients receiving enzyme replacement therapy monitoring disease manifestations and therapeutic intervention accurate quantitative monitoring of disease manifestations as well as of the therapeutic efficacy of any type of treatment is crucial for clinical management of patients with lsds . clinical assessments obviously differ among the various lsds , as illustrated below for gaucher disease and fabry disease . moreover , some symptoms have an irreversible nature , such as advanced renal failure in fabry disease or bone infarctions in gaucher disease . biomarkers are generally defined as chemical entities , ranging from simple metabolites to complex proteins , which indicate the presence of a biological process linked to the clinical manifestations and outcome of a particular disease . also , biomarkers are key in the shift away from the one size fits all to the right drug at the right dose in the right patient approach . the second group includes molecules produced by storage cells in a specific lsd and can be measured either in plasma , urine , or cerebrospinal fluid ( csf ) . identifying and applying such biomarkers for gaucher disease and fabry disease are discussed , and their use is illustrated . different phenotypes ( types 1 , 2 , and 3 ) are generally recognized and are differentiated on the basis of the presence or absence of neurological symptoms . however , the neurological signs and symptoms in type 1 gaucher disease are of a totally different kind from and , in the majority of cases , of much less severity than , the signs and symptoms associated with types 2 and 3 disease ( biegstraaten et al . although glucocerebrosidase is present in the lysosomes of all cell types , patients with type 1 gaucher disease only store glucosylceramide in macrophages . in recent decades , it has become apparent that gaucher cells are not inert containers of storage material but are viable , chronically activated macrophages that contribute to the diverse clinical manifestations of gaucher disease . consistent with these observations , patients with gaucher disease show increased plasma levels of several proinflammatory and anti - inflammatory cytokines , chemokines , and hydrolases ( aerts and hollak 1997 ; aerts et al . factors released by gaucher cells and surrounding macrophages are thought to play a crucial role in the development of common abnormalities in gaucher patients , such as osteopenia , activation of coagulation , hypermetabolism , gammopathies , multiple myeloma , and hypolipoproteinemias ( aerts and hollak 1997 ) . all of these proteins are known to be produced by macrophages ; however , none of them appears to be a specific marker for the pathological gaucher cells , and their levels in the serum of symptomatic patients with gaucher disease may overlap with those observed in healthy individuals . this search led to the discovery of a striking abnormality in the serum of symptomatic patients with gaucher disease . in situ hybridization and histochemistry of bone marrow aspirates and sections of spleens from patients with gaucher disease revealed that chitotriosidase is massively produced by storage cells . a relationship between the total body burden of storage cells in patients with gaucher disease and their plasma chitotriosidase levels has been noted . interpretation of plasma chitotriosidase levels is intrinsically complicated by the common occurrence of a particular 24-base - pair duplication in the chitotriosidase gene , preventing the formation of chitotriosidase protein ( aguilera et al . about one in every three individuals carries this abnormality , and about one in every 20 individuals , including patients with gaucher disease , is homozygous for this trait ( aguilera et al . plasma chitotriosidase activity is increased , albeit much more modestly , in several lysosomal and nonlysosomal diseases , such as sarcoidosis , visceral leishmaniasis , leprosy , arthritis , multiple sclerosis , thalassemia , chronic obstructive pulmonary disease ( copd ) , malaria , and atherosclerosis ( hollak et al . using surface - enhanced laser distortion / ionization time of flight ( seldi - tof ) mass spectrometric analyses , we discovered massive overproduction and secretion by gaucher cells of the pulmonary and activation - regulated chemokine ( parc / ccl18 ) , of which the messenger rna ( mrna ) was previously observed to be up - regulated in the spleen of a patient with gaucher disease ( moran et al . clinical applications of gaucher - cell biomarkers plasma chitotriosidase measurement is commonly employed as a first screen in the diagnosis of gaucher disease . increasing plasma levels seem to reflect gradual accumulation of storage cells in the patient s body . during enzyme supplementation therapy , the mean decrease in 12 months was 32% ( range 082% ) , and 78% of patients had a decrease of > 15% . a recent retrospective analysis by deegan and co - workers confirmed the value of the use of plasma chitotriosidase in gaucher disease management and presented evidence for comparable use of parc / ccl18 ( deegan et al . lipid biomarkers of gaucher disease increased plasma concentrations of glucosylceramide , the primary storage lipid , have been documented for gaucher patients ( groener et al . discovery of additional protein biomarkers for gaucher disease ongoing attention is paid to the detection of useful protein biomarkers for gaucher disease by a thorough survey of protein composition of bodily fluids , or cell and tissue specimens of symptomatic gaucher patients . marked therapy - induced differences were noted in the gaucher disease protein plasma profile . fabry disease is an x - linked lsd resulting from deficient activity of the lysosomal hydrolase -galactosidase a ( ec 3.2.1.22 ) ( desnick and ioannou 2001 ; brady et al . one enzyme is produced by chinese hamster ovary ( cho ) cells with classic recombinant technology ( agalsidase , fabrazyme ) , and the other enzyme is produced by cultured human skin fibroblasts with an activated promoter of the -gal a gene ( agalsidase , replagal ) . fabry biomarkers following the successful biomarker discovery for gaucher disease , attempts have been made to identify comparable metabolite and protein biomarkers for fabry disease . the abnormalities in the hydrolases , all produced by phagocytes , are not very striking and certainly not specific for fabry disease . in a very recent , thorough study conducted with a large cohort of fabry patients in the academic medical center in amsterdam , only minimal abnormalities in indicators of coagulation , fibrinolysis , and platelet and endothelial activation next to targeted analysis of plasma proteins already known to reflect endothelial activation , the search for protein biomarkers of fabry disease has been extended to analysis of the entire plasma proteome . a systematic proteomics analysis of blood specimens from fabry patients conducted at the academic medical center in amsterdam has not led to the discovery of prominent abnormalities in plasma proteins in symptomatic fabry patients ( aerts and co - workers , manuscript in preparation ) . lipid abnormalities as potential fabry biomarkers for a long time , the primary accumulating globoside gb3 has been considered as a surrogate marker for fabry disease . 2007c ) , the absence of infantile manifestations in fabry patients completely lacking -galactosidase a activity also indicates that gb3 accumulation does not cause immediately , and maybe even not directly , signs of disease . an investigation by barbey and co - workers provided evidence for the presence of an unidentified substance in plasma of symptomatic fabry disease patients that stimulates proliferation of vascular smooth - muscle cells and cardiomyocytes in vitro ( barbey et al . also in the case of symptomatic female fabry patients , clearly increased levels of lysogb3 were detected , whereas concomitantly , gb3 concentrations were in the high normal range ( aerts et al . thus , measurement of plasma lysogb3 seems to offer a very useful diagnostic tool , particularly in the case of female fabry patients . auray - blais and co - workers demonstrated the presence of elevated lysogb3 in urine samples of fabry patients ( auray - blais et al . the relationship between plasma lysogb3 concentrations and fabry disease manifestations has been investigated ( rombach et al . however , the neurological signs and symptoms in type 1 gaucher disease are of a totally different kind from and , in the majority of cases , of much less severity than , the signs and symptoms associated with types 2 and 3 disease ( biegstraaten et al . in recent decades , it has become apparent that gaucher cells are not inert containers of storage material but are viable , chronically activated macrophages that contribute to the diverse clinical manifestations of gaucher disease . factors released by gaucher cells and surrounding macrophages are thought to play a crucial role in the development of common abnormalities in gaucher patients , such as osteopenia , activation of coagulation , hypermetabolism , gammopathies , multiple myeloma , and hypolipoproteinemias ( aerts and hollak 1997 ) . all of these proteins are known to be produced by macrophages ; however , none of them appears to be a specific marker for the pathological gaucher cells , and their levels in the serum of symptomatic patients with gaucher disease may overlap with those observed in healthy individuals . this search led to the discovery of a striking abnormality in the serum of symptomatic patients with gaucher disease . in situ hybridization and histochemistry of bone marrow aspirates and sections of spleens from patients with gaucher disease revealed that chitotriosidase is massively produced by storage cells . these observations help us to understand the very specific , gross elevation of chitotriosidase levels in the blood of patients with gaucher disease . a relationship between the total body burden of storage cells in patients with gaucher disease and their plasma chitotriosidase levels has been noted . interpretation of plasma chitotriosidase levels is intrinsically complicated by the common occurrence of a particular 24-base - pair duplication in the chitotriosidase gene , preventing the formation of chitotriosidase protein ( aguilera et al . about one in every three individuals carries this abnormality , and about one in every 20 individuals , including patients with gaucher disease , is homozygous for this trait ( aguilera et al . plasma chitotriosidase activity is increased , albeit much more modestly , in several lysosomal and nonlysosomal diseases , such as sarcoidosis , visceral leishmaniasis , leprosy , arthritis , multiple sclerosis , thalassemia , chronic obstructive pulmonary disease ( copd ) , malaria , and atherosclerosis ( hollak et al . using surface - enhanced laser distortion / ionization time of flight ( seldi - tof ) mass spectrometric analyses , we discovered massive overproduction and secretion by gaucher cells of the pulmonary and activation - regulated chemokine ( parc / ccl18 ) , of which the messenger rna ( mrna ) was previously observed to be up - regulated in the spleen of a patient with gaucher disease ( moran et al . clinical applications of gaucher - cell biomarkers plasma chitotriosidase measurement is commonly employed as a first screen in the diagnosis of gaucher disease . increasing plasma levels seem to reflect gradual accumulation of storage cells in the patient s body . during enzyme supplementation therapy , the mean decrease in 12 months was 32% ( range 082% ) , and 78% of patients had a decrease of > 15% . in addition , chitotriosidase response was related to disease severity ; less reduction in plasma activity was seen in more severely affected individuals . a recent retrospective analysis by deegan and co - workers confirmed the value of the use of plasma chitotriosidase in gaucher disease management and presented evidence for comparable use of parc / ccl18 ( deegan et al . lipid biomarkers of gaucher disease increased plasma concentrations of glucosylceramide , the primary storage lipid , have been documented for gaucher patients ( groener et al . discovery of additional protein biomarkers for gaucher disease ongoing attention is paid to the detection of useful protein biomarkers for gaucher disease by a thorough survey of protein composition of bodily fluids , or cell and tissue specimens of symptomatic gaucher patients . marked therapy - induced differences were noted in the gaucher disease protein plasma profile . although both enzyme therapies were found to result in the desired clearance of globotriaosylceramide from the endothelium , the clinical effects are not as robust as anticipated . fabry biomarkers following the successful biomarker discovery for gaucher disease , attempts have been made to identify comparable metabolite and protein biomarkers for fabry disease . the abnormalities in the hydrolases , all produced by phagocytes , are not very striking and certainly not specific for fabry disease . known plasma proteins reflecting endothelial activation have been considered as candidate biomarkers for fabry disease . in a very recent , thorough study conducted with a large cohort of fabry patients in the academic medical center in amsterdam , only minimal abnormalities in indicators of coagulation , fibrinolysis , and platelet and endothelial activation next to targeted analysis of plasma proteins already known to reflect endothelial activation , the search for protein biomarkers of fabry disease has been extended to analysis of the entire plasma proteome . a systematic proteomics analysis of blood specimens from fabry patients conducted at the academic medical center in amsterdam has not led to the discovery of prominent abnormalities in plasma proteins in symptomatic fabry patients ( aerts and co - workers , manuscript in preparation ) . lipid abnormalities as potential fabry biomarkers for a long time , the primary accumulating globoside gb3 has been considered as a surrogate marker for fabry disease . intriguingly , in female carriers of fabry disease , urinary gb3 is generally increased , but plasma gb3 levels tend to be in the normal range ( vedder et al . an investigation by barbey and co - workers provided evidence for the presence of an unidentified substance in plasma of symptomatic fabry disease patients that stimulates proliferation of vascular smooth - muscle cells and cardiomyocytes in vitro ( barbey et al . also in the case of symptomatic female fabry patients , clearly increased levels of lysogb3 were detected , whereas concomitantly , gb3 concentrations were in the high normal range ( aerts et al . thus , measurement of plasma lysogb3 seems to offer a very useful diagnostic tool , particularly in the case of female fabry patients . auray - blais and co - workers demonstrated the presence of elevated lysogb3 in urine samples of fabry patients ( auray - blais et al . the relationship between plasma lysogb3 concentrations and fabry disease manifestations has been investigated ( rombach et al . these observations suggest , but certainly do not prove , that lysogb3 plays a direct role in the pathogenesis of fabry disease . they demonstrated that exposure of cultured glomerular podocytes to lysogb3 increased the expression of tumor growth factor ( tgf)-beta-1 , extracellular matrix proteins ( fibronectin and type iv collagen ) , and cd74 . a major limitation so far in evaluating lsds has been the lack of a technology that allows selective labeling of active lysosomal hydrolases in vitro and in vivo . the availability of such a method would allow more advanced diagnostic tests and more sophisticated monitoring of tissue targeting of therapeutic proteins , and would boost fundamental research on lysosomal hydrolases . the term inhibody is coined for these types of probes , as they act as suicide inhibitors and allow direct visualization of originally active enzyme molecules with comparable methods as used for specific antibodies . the use of a near - infrared fluorophore to enable visualization of therapeutic glucocerebrosidase in individual patients is appealing . it now becomes feasible to obtain information about the exact tissue distribution of enzymatically active glucocerebrosidase . such information may render new insights into gaucher disease and help our understanding of why carriership for the disorder constitutes a risk factor for parkinsonism ( goker - alpan et al . 2010 ) and gba3 that might play a role in the pathogenesis of gaucher disease . in summary , the search for biomarkers of gaucher disease have been extraordinary productive . in addition , the probes may be employed in the future in monitoring tissue distribution of recombinant therapeutic protein in individual gaucher patients . finally , inhibodies may boost fundamental research on glucocerebrosidase and the pathogenesis of gaucher disease and parkinsonism . in the case of lysosomal storage diseases , biochemical abnormalities can be identified in blood and/or urine samples as described above for gaucher and fabry disease . thus , such biomarkers are not true surrogate markers of disease that accurately reflect a particular symptom in one organ .

the general view of the cellular plasma membrane has evolved over the last twenty years from that of a homogeneous arrangement of lipids with embedded proteins towards that of a mosaic of microdomains , each having a specific protein and lipid composition . over the last couple of decades , evidence has accumulated for organisation of the plasma membrane into lipid - based microdomains or lipid rafts . a new model of membrane architecture has been suggested in which the membrane is patchy with segregated cholesterol - rich portions , called lipid rafts . lipid rafts are envisaged as islands of highly ordered saturated lipids and cholesterol that are laterally mobile in the plane of a more fluid disordered bilayer of largely unsaturated lipids [ 3 , 4 ] . the hallmark of the lipid raft hypothesis are the spontaneous partitioning of lipids and proteins in discrete membrane domains , a behaviour based on their physicochemical characteristics and the possibility to recover these microdomains and their associated protein machinery as detergent - resistant entities using biochemical flotation experiments . microdomains appear as small dynamic structures that can aggregate into larger platforms in response to various stimuli . currently , lipid rafts are thought to allow different protein - lipid and protein - protein interactions that temporarily compartmentalise the plasma membrane . lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface . they seem to provide the necessary microenvironment in order for certain specialised signalling events to take place . recent studies have shown the importance of lipid raft formation in the acquired immune response . major histocompatibility complex- ( mhc- ) restricted t - cell activation seems to be facilitated by lipid raft formation . furthermore , we have recently found that mediators of the innate immune response also concentrate in lipid rafts in order to facilitate signal transduction [ 7 , 8 ] , thus suggesting that both the acquired and innate immune systems utilise membrane partitioning as means of activation against invading pathogens . crucial receptors for both innate and acquired immunity seem to oligomerize in nonrandom membrane structures , bringing together their signalling machinery . thus accumulation of receptors within these floating islands on the cell membrane seems to bring together intracellularly all the adaptor molecules that are necessary for signalling . in this paper , we will investigate further the mechanisms of innate immune recognition and review past and current literature that leads us to believe that membrane partitioning and lipid rafts play a central role in innate immune activation . the innate immune system constitutes the most archaic part of our immune defences and has survived through years of evolution . its function is thought to be the recognition of invading pathogens , the activation of inflammation to control the pathogen , and the subsequent activation of the acquired immune response . as part of its mechanism of activation , the innate immune system employs germ - lined encoded receptors , called pattern recognition receptors ( prrs ) in order to sense pathogens . these prrs recognise a restricted collection of microbial signatures , able to sense different types of microbial pathogens ranging from bacteria and viruses to fungi and spirochetes . lipid rafts seem to be a key feature of the innate immune response , playing a crucial role in phagocytosis , receptor - receptor as well as receptor - pathogen associations as well as signal transduction . families of prrs , such as the toll - like receptor family ( tlr ) as well as the c - type lectin family seem to localise in lipid rafts for their function thus demonstrating the importance of this membrane partitioning for the function of the innate immune response . the tlr receptor family were the first pattern recognition receptors to be identified [ 9 , 10 ] . this family of at least ten germ - line encoded receptors is able to sense microbial signatures and trigger activation leading to proinflammatory cytokine secretion . tlrs are expressed on immune cells and are able to distinguish a great variety of microbial ligands , such as cell wall components like lipopolysaccharide ( lps ) from gram - negative bacteria and lipoteichoic acid from gram - positive bacteria , bacterial flagellin , cpg dna , and viral dna or single stranded rna . all identified tlrs are type i transmembrane proteins , whose intracellular domains contain regions homologous to the intracellular domains of il-1r and are referred to as tir domains . these intracellular domains are able to trigger signalling pathways known to activate the nuclear factor kappa b ( nf-b ) [ 12 , 13 ] , which in turn leads to the secretion of proinflammatory cytokines such as tnf- , il-6 , and il-8 . tlr4 was found to recognise bacterial lipopolysaccharide ( lps ) or endotoxin [ 14 , 15 ] ; tlr2 was found to recognise lipoteichoic acid ( lta ) and peptidoglycan ; tlr3 was able to sense double - stranded viral rna ; tlr5 was found to recognise bacterial flagellin , tlr7 and tlr8 to sense single stranded viral rna , whereas tlr9 to recognise bacterial cpg dna . in addition , tlr2 was found to recognise different motifs including several components of gram - positive bacteria such as peptidoglycan , lipoteichoic acid ( lta ) , lipoarabinomannan , lipoproteins , and different lps from certain gram - negative bacteria , yeast , spirochete , and fungi [ 28 , 29 ] through its unique ability to heterodimerize with tlrs 1 and 6 . studies using diacylated and triacylated lipoproteins have revealed that diacylated lipoproteins require tlr2/6 heterodimers for activation , whereas triacylated lipoproteins induce activation of the innate immune system independently of tlr6 and mainly through tlr2/tlr1 heterodimers [ 25 , 3135 ] . the membrane distribution of tlrs as well as their intracellular trafficking has only now begun to be investigated . most tlrs ( tlr1 , tlr2 , tlr4 , tlr5 , and tlr6 ) seem to activate cells by engaging their ligands on the cell surface , whereas tlr3 , tlr7 , tlr8 , and tlr9 seem to trigger signalling intracellularly . these tlrs have been shown to reside in the er and to recognise their ligands once they have been endocytosed [ 36 , 37 ] . investigations into the innate immune recognition of bacterial endotoxin led to the discovery of the tlr family . tlr4 is the most studied tlr , mainly because of its involvement with sepsis and septic shock . sepsis is a paradoxical and complex disorder that results from an overreaction of our innate immune system to bacterial infections . the mechanisms that are designed to protect the host against infection by bacterial pathogens , either gram - negative or gram - positive , can lead to oversecretion of cytokines and fatal sepsis syndrome . it is now widely accepted that the overreaction of the host occurs at the level of the innate immune system and is directly linked to the recognition of bacterial cell wall components , such as lipopolysaccharide ( lps ) from gram - negative bacteria or lipoteichoic acid ( lta ) from gram - positive bacteria . thus the recognition of bacterial products by the innate immune system under certain conditions seems to be detrimental for the host . in the last twenty - five years , great leaps forward in our understanding of one of the seminal discoveries has been the identification of a serum protein , lipopolysaccharide - binding protein ( lbp ) , which binds lps or lta and delivers it to its cellular targets . probably the most important discovery has been that the main family of receptors employed by the innate immune system are the toll - like receptors ( tlrs ) . as far as sepsis and bacterial recognition is concerned , tlr4 seems to be the central sensor of gram - negative bacterial products [ 15 , 39 ] , whereas tlr2 seems to be the key receptor in activating the immune system against gram - positive bacteria . in addition to the involvement of tlrs , other accessory molecules seem to be involved . cd14 is believed to act as a transfer molecule for both gram - negative and gram - positive bacteria [ 40 , 41 ] . in the case of lps recognition , it has been further shown that a soluble molecule , md-2 , as well as activation clusters involving several other receptors [ 4345 ] . in the case of lta recognition , tlr2 seems to form receptor clusters as well , comprising of at least cd14 , tlr2 , tlr6 , and cd36 . thus we are moving away from the single - receptor model of activation , and a more complex picture is emerging . the mechanism that leads to activation seems to involve the careful interplay of several receptor molecules as well as serum proteins . therefore such a complex orchestration of events requires a nonrandom membrane architecture specifically geared to bring receptor molecules together and trigger activation within the lipid bilayer and lipid rafts or membrane microdomains seem to provide this platform . prrs employed by the innate immune system have been shown to have the ability to bind and recognise conserved products of pathogens that are unique to the invading microorganisms but not to the host , it is becoming increasingly apparent that the model of a single prr recognising foreign antigen is an oversimplified one . with the discovery of the toll - like receptors as the main signal transducing molecules of the innate immune system , an onslaught of research has shown that prrs are part of multicomponent sensor apparatuses . tlrs have been shown to function as homo- or heterodimers and to even form functional interactions with non - tlr molecules . many of these interactions are highly stable , whereas others are transient , forming dynamic associations in response to specific stimuli . whether homotypic , heterotypic , stable , or transient , these different protein combinations generate considerable functional diversity for the innate immune system by triggering distinct signalling cascades leading to cellular activation . there are a number of examples that suggest that tlr associations are required for cellular activation . tlr4 seems to form a complex with at least two other molecules , cd14 and md2 , in order to recognise bacterial lps . in addition , it seems to associate with a toll - like receptor homologue rp105 , which acts as a negative regulator of tlr4 responses . tlr2 has been found to heterodimerize with tlr1 or tlr6 for recognition of yeast components and to associate with tlr1 for the recognition of bacterial lipoproteins . in addition , tlr2 has been shown to also interact with scavenger receptors in order to recognise lipoproteins and most recently it was shown that tlr2 associates with cxcr4 , which acts as a negative regulator of tlr2 responses ( figure 1 ) . figure 1 depicts the possible model of tlr activation , and how tlrs and other receptors are organized in lipid rafts on the cell surface before and after stimulation . these heterodimers preexist and are not induced by the ligand ( figure 1(a ) ) . tlr2/6 heterodimers are recruited within lipid rafts and associate with lipid raft - resident proteins cd14 and cd36 upon ligand engagement . binding of appropriate microbial substances leads to energy - dependent clustering of heterotypic receptors and activation of intracellular signaling cascades that lead , for example , via nf-b to production and secretion of proinflammatory cytokines ( figure 1(b ) ) . functional associations of tlrs with non - tlr molecules have also been demonstrated , for example , tlr2 association with dectin-1 is required for macrophage and dendritic cell activation by -glucan - containing particles . more recently , functional interactions of tlr2 and cd36 have been shown to be involved in the recognition of diacylglycerides . tlr4 seems to be the best example of tlrs associating with non - tlr molecules . as it has already been mentioned , tlr4 has been shown to form at least a trimolecular complex with cd14 and md2 in order to recognise bacterial lps . the possibility that additional receptor components such as heat shock proteins [ 43 , 50 ] , cxcr4 , or cd55 have been suggested to be part of this activation cluster , possibly acting as additional lps transfer molecules . shapes of lps induce the formation of different activation clusters , involving the association of tlr4 with a variety of molecules mentioned above , which seems to determine lps responses . recent structural studies have shed some light onto tlr associations , supporting the hypothesis of cluster formation , since all tlrs that have been crystallised have been found to be in a dimer formation , thus the hypothesis has been put forward that dimerisation or clustering might be a common feature of the tlrs and might be essential for signalling . structural studies of tlrs have been an attractive area of research since structural information is crucial in understanding receptor function . in 2005 , it was surprising , that although the structure did not have a ligand , tlr3 was crystallised as a dimer . in 2007 and 2008 , three structures of tlr - ligand complexes were revealed , tlr1-tlr2-lipoprotein , tlr4-md-2-eritoran , and tlr3- dsrna [ 5355 ] . prior to the publication of the crystal structures , gayand gangloff suggested a possible model of activation , where dimerization was ligand induced . these observations have suggested the hypothesis that dimerization of the ectodomains forces the intracellular tir domains to dimerize , and this initiates signalling by recruiting the intracellular adaptor molecules , such as myd88 , mal , trif , and tram in order to initiate signalling . the structures of the tir domains of tlr1 , tlr2 , and tlr10 have been revealed . interestingly , the tir domain of tlr10 was shown to be involved in a homodimeric interaction . however , it is not certain whether the structure seen in the crystal corresponds to a physiologically relevant dimer of tlr10 tir domains because they have been found to exist as monomers in solution . interestingly it has been recently suggested that myd88 interacts with irak4 in an 8 : 4 ratio in solution , suggesting that maybe there is higher oligomer formation . in order for such higher oligomers to be formed and in order to have such a well - orchestrated accumulation of receptors and signalling machinery membrane partitioning seems to be crucial for the formation of these tlr multicomponent sensor apparatuses . tlr4 was the first one to be shown to be recruited to lipid rafts upon stimulation by bacterial lps . within these membrane microdomains it was shown that tlr4 formed clusters with non - tlr molecules that tailored the immune response against the particular pathogen [ 43 , 5962 ] . it was subsequently shown that this accumulation in lipid rafts also influenced its internalization and targeting . tlr4 was found to accumulate in lipid rafts , to internalize in a lipid - raft - dependent manner and to be targeted to the golgi apparatus . this intracellular targeting was shown to be independent of signalling , thus suggesting that accumulation in lipid rafts only facilitated ligand recognition and signalling that was initiated at the cell surface and not in the intracellular compartments where tlr4 was targeted to . more recently it had been proposed that the molecular mechanism for signalling by the tlrs must involve a series of protein conformational changes initiated by dimerization of their extracellular domains . it was suggested that this receptor - receptor association of the extracellular domains forced the association of the cytoplasmic domains as well . recently proved this experimentally , demonstrating that the death domains of human myd88 , one of the adaptor proteins used by all but one of the tlrs , and irak4 assemble into closed complexes with stoichiometries of 7 : 4 and 8 : 4 , which they called the myddosome . the ability to form 7 : 4 or even 8 : 4 stoichiometries suggests a mechanism by which clusters of activated receptors concentrate in lipid rafts and their intracellular machinery clusters as well , forming a signalling platform that seems to be crucial for tlr activation . cell membranes display a tremendous complexity of lipid and proteins designed to perform the functions cells require . lipid rafts were originally proposed as an explanation for a nonrandom membrane architecture and their function was originally thought to be linked with membrane trafficking . however , rafts proved to be able to influence organization of membrane receptors and bioactivity as well as membrane trafficking . it is now emerging that this membrane partitioning might play a major role in protein uptake and intracellular routing . it is becoming more apparent that this differential sorting on the cell surface might pre - dispose the intracellular fate of a given molecule . since the discovery of clathrin - coated pits by roth and porter in 1964 , as specialised sites for the selective recruitment of specialised molecules that are internalised into eukaryotic cells , clathrin - independent endocytic pathways have now emerged . endocytic pathways that do not rely on the formation of clathrin coated pits include the earliest identified pathways such as phagocytosis , macropinosis , and caveolae . , it has been shown that lipid rafts play a crucial role in the phagocytic uptake of latex microspheres , suggesting that these specialised microdomains on the plasma membrane are necessary for endocytosis and phagocytosis . furthermore , caveolae which is defined as small , uncoated invaginations in the plasma membrane containing the plasma protein caveolin 1 has been shown to be able to bind cholesterol and to be resistant to detergent extraction , and this has led to the suggestion that caveolae might constitute a type of lipid raft . lipid rafts are increasingly becoming linked with clathrin - independent endocytosis , since nearly all molecules that are known to be internalised independently of clathrin are found in biochemically defined rafts . it has been suggested that raft components might be taken up preferentially by clathrin - independent endocytosis . the extent to which these different pathways require lipid rafts to operate or are somehow selective for lipid rafts is currently the subject of intensive investigation . recently , nichols et al . have described a rapid lipid - raft - dependent targeting from the cell surface to the golgi apparatus . in addition , a new clathrin - independent mechanism has been described that can lead to delivery of receptor molecules from the plasma membrane to caveolin-1-containing endosomes , termed the idea that different types of endocytosis have markedly different functions is beginning to become apparent . ultimately we have to speculate that sorting at the plasma membrane might predispose the intracellular route that a molecule might take . if that is the case , then where are the raft - associated molecules , such as tlr4 , targeted to ? and most importantly why ? tlr2 has also been found to reside in lipid rafts after stimulation by gram - positive bacterial products and to be similarly targeted to the golgi apparatus ( figure 1 ) . the question that remains is whether lipid raft association is common for all tlrs expressed at the cell surface ? if this is the case , do they all follow the same intracellular route ? do different signalling cascades require differential targeting of tlrs and their ligands ? in the case of the er - resident tlrs , very little evidence of their trafficking upon stimulation exists . to date only tlr9 has been found to translocate from the er to lysosomes in response to its ligand , cpg dna . based on the findings for tlr9 , a hypothesis has been put forward that er - resident tlrs might become accessible to endosomal and lysosomal compartments after the er fuses with sites of microbial entry . if this is the case , then it would seem that er membrane fusion might be critical for microbial recognition by er - resident tlrs . the regulation of endosome dynamics is crucial for fundamental cellular functions , such as nutrient intake / digestion , membrane receptor recycling / degradation , antigen presentation , cell migration , and intracellular signaling [ 7476 ] . the system is also utilised by various pathogens to bud in and from the cell . in addition to the function as a distribution centre , it has been proposed that the endosome system serves as an intracellular signalling station . in the case of innate immunity this is certainly the case , since tlr3 , tlr7 , tlr8 , and tlr9 all reside within the endosome waiting to capture incoming pamps and trigger signalling . endosomes are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance . the question that remains is whether in addition to these morphologically distinguishable regions , endosomal membranes are further subcompartmentalized into membrane lipid rafts or microdomains . lipid rafts have mostly been studied at the plasma membrane , mainly due to accessibility for microscopy and biophysical methods . characterisation of lipid rafts has also been extensively based on their resistance to detergent solubilisation , although this has inherent limitations , as well as fluorescent microscopy . although most studies have focused on the existence of lipid rafts on plasma membranes , many intracellular organelles appear to contain raft - like domains [ 8184 ] . due to its low cholesterol content , the endoplasmic reticulum was originally thought not to contain cholesterol - dependent microdomains . however , recently several studies have reported their existence [ 85 , 86 ] . raft - like domains have been described in the golgi and trans - golgi network [ 87 , 88 ] , along the endocytic pathway as well as in the endosomes [ 8991 ] . although potential roles of lipid rafts for the outer membrane have been demonstrated , including endocytosis , exocytosis , vesicle formation , and signalling , the functions of lipid rafts in the processes of endosomal membrane dynamics are currently unknown . it has been suggested that protein and lipid sorting into and out of the endosomes may be controlled by endosomal membrane partitioning , but whether these microdomains control signalling and in particular tlr signalling has not been investigated . since most extracellular tlrs have been shown to be recruited to lipid rafts upon ligand activation , it is safe to assume that endosomal tlrs act in a similar manner , especially since the existence of cholesterol - dependent microdomains on the endosomal membranes has been proven ( figure 2 ) . thus it is safe to assume that membrane partitioning control both extracellular and intracellular tlr - dependent signalling . we are proposing a model for endosomal tlr activation , where ligation of endosomal tlrs by their respective ligands can lead to clustering within lipid rafts at the endosomal membrane and activation of intracellular signaling cascades that lead via nf-b to production and secretion of proinflammatory cytokines ( figure 2(b ) ) . c - type lectin receptors ( clrs ) , such as dectin 1 , are a family of pattern recognition receptors which bind -glucan found in the cell walls of pathogenic fungi such as candida albicans . in particular dectin 1 has been shown to mediate the phagocytosis of yeast and yeast - derived particles , such as zymosan , activating the production of inflammatory cytokines [ 9294 ] . interestingly , dectin-1 possesses an immunotyrosine - activated motif ( itam ) in its cytoplasmic tail , suggesting that it is capable of mediating signalling analogous to the bcr and tcr . since both the bcr and tcr have been found to reside in lipid rafts it was suggested that dectin-1 might also be recruited there upon activation . a recent study has revealed that dectin-1 and possibly other clrs are recruited to lipid rafts upon activation and raft integrity is important for signalling . thus suggesting that recruitment to lipid rafts is a common feature for most prrs , including tlrs and clrs . cell membranes are complicated in composition but precise in purpose : to selectively compartmentalize its constituents in order to coordinate cellular functions . in this way , the membrane is able to compartmentalize , segregate receptors as well as their signalling machinery and create oligomeric signalling platforms in order to transduce signals . once the required function has subsided , these segregated islands are involved in internalization and membrane trafficking , thus bringing the whole function to a close . the innate and acquired immune systems seem to utilise this membrane partitioning for their functions . in this paper , we have extensively looked at the use of this membrane partitioning by the innate immune system and most particular by the tlrs . the molecular mechanism involved in lps recognition and tlr signalling in general , the plasma membrane seems to be heterogeneous and to coalesce to more stable membrane - ordered assemblies upon activation by ligands . this partitioning of the membrane and the assembly of more stable raft platforms in the functionalized state must be initiated by raft - resident proteins , which form protein - lipid as well as protein - protein interactions . the tlrs and other prrs associate with the raft - resident proteins and are recruited to these floating islands forming higher oligomers , both extracellularly as well as inside the cell , concentrating their signalling machinery which finally leads to a functional , focused , and coordinated activation of the innate immune system . the lifetime of these domains and the length of the response will depend on their size and factors that may stabilise or destabilise them . these factors will include not only lipid - lipid , lipid - protein and protein - protein interactions both in the plane of the membrane but also elements of the cytoskeleton , pericellular matrix adjacent to the membrane as well as transmembrane and cytoplasmic domains of the receptors involved . in the case of tlrs , the association of the tir domains intracellularly would stabilise the ectodomains extracellularly and provide the molecular scaffold that will recruit the adaptor molecules that contribute to signalling . the challenge for the future will be to visualise the assembly and stoichiometry of these large and transient oligomeric complexes in vivo . thus refining existing biophysical methods , such as single particle tracking ( spt ) , fluorescence correlation spectroscopy ( fcs ) , and fluorescence resonance energy transfer ( fret ) will help us reveal these dynamic nanoassemblies of sterol , sphingolipid , and proteins in living cell and provide us with the first dynamic picture of the innate immune response .

in the last twenty years , the general view of the plasma membrane has changed from a homogeneous arrangement of lipids to a mosaic of microdomains . it is currently thought that islands of highly ordered saturated lipids and cholesterol , which are laterally mobile , exist in the plane of the plasma membrane . lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface . they seem to provide the necessary microenvironment in order for certain specialised signalling events to take place , such as the innate immune recognition . the innate immune system seems to employ germ - lined encoded receptors , called pattern recognition receptors ( prrs ) , in order to detect pathogens . one family of such receptors are the toll - like receptors ( tlrs ) , which are the central sensing apparatus of the innate immune system . in recent years , it has become apparent that tlrs are recruited into membrane microdomains in response to ligands . these nanoscale assemblies of sphingolipid , cholesterol , and tlrs stabilize and coalesce , forming signalling platforms , which transduce signals that lead to innate immune activation . in the current paper , we will investigate all past and current literature concerning recruitment of extracellular and intracellular tlrs into lipid rafts and how this membrane organization modulates innate immune responses .

1. Introduction 2. The Innate Immune System

the general view of the cellular plasma membrane has evolved over the last twenty years from that of a homogeneous arrangement of lipids with embedded proteins towards that of a mosaic of microdomains , each having a specific protein and lipid composition . over the last couple of decades , evidence has accumulated for organisation of the plasma membrane into lipid - based microdomains or lipid rafts . a new model of membrane architecture has been suggested in which the membrane is patchy with segregated cholesterol - rich portions , called lipid rafts . lipid rafts are envisaged as islands of highly ordered saturated lipids and cholesterol that are laterally mobile in the plane of a more fluid disordered bilayer of largely unsaturated lipids [ 3 , 4 ] . the hallmark of the lipid raft hypothesis are the spontaneous partitioning of lipids and proteins in discrete membrane domains , a behaviour based on their physicochemical characteristics and the possibility to recover these microdomains and their associated protein machinery as detergent - resistant entities using biochemical flotation experiments . microdomains appear as small dynamic structures that can aggregate into larger platforms in response to various stimuli . currently , lipid rafts are thought to allow different protein - lipid and protein - protein interactions that temporarily compartmentalise the plasma membrane . lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface . they seem to provide the necessary microenvironment in order for certain specialised signalling events to take place . furthermore , we have recently found that mediators of the innate immune response also concentrate in lipid rafts in order to facilitate signal transduction [ 7 , 8 ] , thus suggesting that both the acquired and innate immune systems utilise membrane partitioning as means of activation against invading pathogens . thus accumulation of receptors within these floating islands on the cell membrane seems to bring together intracellularly all the adaptor molecules that are necessary for signalling . in this paper , we will investigate further the mechanisms of innate immune recognition and review past and current literature that leads us to believe that membrane partitioning and lipid rafts play a central role in innate immune activation . the innate immune system constitutes the most archaic part of our immune defences and has survived through years of evolution . its function is thought to be the recognition of invading pathogens , the activation of inflammation to control the pathogen , and the subsequent activation of the acquired immune response . as part of its mechanism of activation , the innate immune system employs germ - lined encoded receptors , called pattern recognition receptors ( prrs ) in order to sense pathogens . lipid rafts seem to be a key feature of the innate immune response , playing a crucial role in phagocytosis , receptor - receptor as well as receptor - pathogen associations as well as signal transduction . families of prrs , such as the toll - like receptor family ( tlr ) as well as the c - type lectin family seem to localise in lipid rafts for their function thus demonstrating the importance of this membrane partitioning for the function of the innate immune response . the tlr receptor family were the first pattern recognition receptors to be identified [ 9 , 10 ] . this family of at least ten germ - line encoded receptors is able to sense microbial signatures and trigger activation leading to proinflammatory cytokine secretion . tlrs are expressed on immune cells and are able to distinguish a great variety of microbial ligands , such as cell wall components like lipopolysaccharide ( lps ) from gram - negative bacteria and lipoteichoic acid from gram - positive bacteria , bacterial flagellin , cpg dna , and viral dna or single stranded rna . these intracellular domains are able to trigger signalling pathways known to activate the nuclear factor kappa b ( nf-b ) [ 12 , 13 ] , which in turn leads to the secretion of proinflammatory cytokines such as tnf- , il-6 , and il-8 . in addition , tlr2 was found to recognise different motifs including several components of gram - positive bacteria such as peptidoglycan , lipoteichoic acid ( lta ) , lipoarabinomannan , lipoproteins , and different lps from certain gram - negative bacteria , yeast , spirochete , and fungi [ 28 , 29 ] through its unique ability to heterodimerize with tlrs 1 and 6 . studies using diacylated and triacylated lipoproteins have revealed that diacylated lipoproteins require tlr2/6 heterodimers for activation , whereas triacylated lipoproteins induce activation of the innate immune system independently of tlr6 and mainly through tlr2/tlr1 heterodimers [ 25 , 3135 ] . most tlrs ( tlr1 , tlr2 , tlr4 , tlr5 , and tlr6 ) seem to activate cells by engaging their ligands on the cell surface , whereas tlr3 , tlr7 , tlr8 , and tlr9 seem to trigger signalling intracellularly . investigations into the innate immune recognition of bacterial endotoxin led to the discovery of the tlr family . sepsis is a paradoxical and complex disorder that results from an overreaction of our innate immune system to bacterial infections . it is now widely accepted that the overreaction of the host occurs at the level of the innate immune system and is directly linked to the recognition of bacterial cell wall components , such as lipopolysaccharide ( lps ) from gram - negative bacteria or lipoteichoic acid ( lta ) from gram - positive bacteria . thus the recognition of bacterial products by the innate immune system under certain conditions seems to be detrimental for the host . in the last twenty - five years , great leaps forward in our understanding of one of the seminal discoveries has been the identification of a serum protein , lipopolysaccharide - binding protein ( lbp ) , which binds lps or lta and delivers it to its cellular targets . probably the most important discovery has been that the main family of receptors employed by the innate immune system are the toll - like receptors ( tlrs ) . as far as sepsis and bacterial recognition is concerned , tlr4 seems to be the central sensor of gram - negative bacterial products [ 15 , 39 ] , whereas tlr2 seems to be the key receptor in activating the immune system against gram - positive bacteria . in the case of lps recognition , it has been further shown that a soluble molecule , md-2 , as well as activation clusters involving several other receptors [ 4345 ] . in the case of lta recognition , tlr2 seems to form receptor clusters as well , comprising of at least cd14 , tlr2 , tlr6 , and cd36 . thus we are moving away from the single - receptor model of activation , and a more complex picture is emerging . therefore such a complex orchestration of events requires a nonrandom membrane architecture specifically geared to bring receptor molecules together and trigger activation within the lipid bilayer and lipid rafts or membrane microdomains seem to provide this platform . prrs employed by the innate immune system have been shown to have the ability to bind and recognise conserved products of pathogens that are unique to the invading microorganisms but not to the host , it is becoming increasingly apparent that the model of a single prr recognising foreign antigen is an oversimplified one . with the discovery of the toll - like receptors as the main signal transducing molecules of the innate immune system , an onslaught of research has shown that prrs are part of multicomponent sensor apparatuses . many of these interactions are highly stable , whereas others are transient , forming dynamic associations in response to specific stimuli . whether homotypic , heterotypic , stable , or transient , these different protein combinations generate considerable functional diversity for the innate immune system by triggering distinct signalling cascades leading to cellular activation . tlr4 seems to form a complex with at least two other molecules , cd14 and md2 , in order to recognise bacterial lps . in addition , it seems to associate with a toll - like receptor homologue rp105 , which acts as a negative regulator of tlr4 responses . in addition , tlr2 has been shown to also interact with scavenger receptors in order to recognise lipoproteins and most recently it was shown that tlr2 associates with cxcr4 , which acts as a negative regulator of tlr2 responses ( figure 1 ) . figure 1 depicts the possible model of tlr activation , and how tlrs and other receptors are organized in lipid rafts on the cell surface before and after stimulation . tlr2/6 heterodimers are recruited within lipid rafts and associate with lipid raft - resident proteins cd14 and cd36 upon ligand engagement . more recently , functional interactions of tlr2 and cd36 have been shown to be involved in the recognition of diacylglycerides . as it has already been mentioned , tlr4 has been shown to form at least a trimolecular complex with cd14 and md2 in order to recognise bacterial lps . shapes of lps induce the formation of different activation clusters , involving the association of tlr4 with a variety of molecules mentioned above , which seems to determine lps responses . in 2005 , it was surprising , that although the structure did not have a ligand , tlr3 was crystallised as a dimer . in 2007 and 2008 , three structures of tlr - ligand complexes were revealed , tlr1-tlr2-lipoprotein , tlr4-md-2-eritoran , and tlr3- dsrna [ 5355 ] . these observations have suggested the hypothesis that dimerization of the ectodomains forces the intracellular tir domains to dimerize , and this initiates signalling by recruiting the intracellular adaptor molecules , such as myd88 , mal , trif , and tram in order to initiate signalling . the structures of the tir domains of tlr1 , tlr2 , and tlr10 have been revealed . however , it is not certain whether the structure seen in the crystal corresponds to a physiologically relevant dimer of tlr10 tir domains because they have been found to exist as monomers in solution . in order for such higher oligomers to be formed and in order to have such a well - orchestrated accumulation of receptors and signalling machinery membrane partitioning seems to be crucial for the formation of these tlr multicomponent sensor apparatuses . within these membrane microdomains it was shown that tlr4 formed clusters with non - tlr molecules that tailored the immune response against the particular pathogen [ 43 , 5962 ] . this intracellular targeting was shown to be independent of signalling , thus suggesting that accumulation in lipid rafts only facilitated ligand recognition and signalling that was initiated at the cell surface and not in the intracellular compartments where tlr4 was targeted to . recently proved this experimentally , demonstrating that the death domains of human myd88 , one of the adaptor proteins used by all but one of the tlrs , and irak4 assemble into closed complexes with stoichiometries of 7 : 4 and 8 : 4 , which they called the myddosome . the ability to form 7 : 4 or even 8 : 4 stoichiometries suggests a mechanism by which clusters of activated receptors concentrate in lipid rafts and their intracellular machinery clusters as well , forming a signalling platform that seems to be crucial for tlr activation . lipid rafts were originally proposed as an explanation for a nonrandom membrane architecture and their function was originally thought to be linked with membrane trafficking . it is now emerging that this membrane partitioning might play a major role in protein uptake and intracellular routing . it is becoming more apparent that this differential sorting on the cell surface might pre - dispose the intracellular fate of a given molecule . since the discovery of clathrin - coated pits by roth and porter in 1964 , as specialised sites for the selective recruitment of specialised molecules that are internalised into eukaryotic cells , clathrin - independent endocytic pathways have now emerged . endocytic pathways that do not rely on the formation of clathrin coated pits include the earliest identified pathways such as phagocytosis , macropinosis , and caveolae . , it has been shown that lipid rafts play a crucial role in the phagocytic uptake of latex microspheres , suggesting that these specialised microdomains on the plasma membrane are necessary for endocytosis and phagocytosis . furthermore , caveolae which is defined as small , uncoated invaginations in the plasma membrane containing the plasma protein caveolin 1 has been shown to be able to bind cholesterol and to be resistant to detergent extraction , and this has led to the suggestion that caveolae might constitute a type of lipid raft . lipid rafts are increasingly becoming linked with clathrin - independent endocytosis , since nearly all molecules that are known to be internalised independently of clathrin are found in biochemically defined rafts . the extent to which these different pathways require lipid rafts to operate or are somehow selective for lipid rafts is currently the subject of intensive investigation . have described a rapid lipid - raft - dependent targeting from the cell surface to the golgi apparatus . in addition , a new clathrin - independent mechanism has been described that can lead to delivery of receptor molecules from the plasma membrane to caveolin-1-containing endosomes , termed the idea that different types of endocytosis have markedly different functions is beginning to become apparent . ultimately we have to speculate that sorting at the plasma membrane might predispose the intracellular route that a molecule might take . if that is the case , then where are the raft - associated molecules , such as tlr4 , targeted to ? the question that remains is whether lipid raft association is common for all tlrs expressed at the cell surface ? in the case of the er - resident tlrs , very little evidence of their trafficking upon stimulation exists . to date only tlr9 has been found to translocate from the er to lysosomes in response to its ligand , cpg dna . the regulation of endosome dynamics is crucial for fundamental cellular functions , such as nutrient intake / digestion , membrane receptor recycling / degradation , antigen presentation , cell migration , and intracellular signaling [ 7476 ] . the system is also utilised by various pathogens to bud in and from the cell . in addition to the function as a distribution centre , it has been proposed that the endosome system serves as an intracellular signalling station . in the case of innate immunity this is certainly the case , since tlr3 , tlr7 , tlr8 , and tlr9 all reside within the endosome waiting to capture incoming pamps and trigger signalling . the question that remains is whether in addition to these morphologically distinguishable regions , endosomal membranes are further subcompartmentalized into membrane lipid rafts or microdomains . lipid rafts have mostly been studied at the plasma membrane , mainly due to accessibility for microscopy and biophysical methods . characterisation of lipid rafts has also been extensively based on their resistance to detergent solubilisation , although this has inherent limitations , as well as fluorescent microscopy . although most studies have focused on the existence of lipid rafts on plasma membranes , many intracellular organelles appear to contain raft - like domains [ 8184 ] . raft - like domains have been described in the golgi and trans - golgi network [ 87 , 88 ] , along the endocytic pathway as well as in the endosomes [ 8991 ] . although potential roles of lipid rafts for the outer membrane have been demonstrated , including endocytosis , exocytosis , vesicle formation , and signalling , the functions of lipid rafts in the processes of endosomal membrane dynamics are currently unknown . it has been suggested that protein and lipid sorting into and out of the endosomes may be controlled by endosomal membrane partitioning , but whether these microdomains control signalling and in particular tlr signalling has not been investigated . since most extracellular tlrs have been shown to be recruited to lipid rafts upon ligand activation , it is safe to assume that endosomal tlrs act in a similar manner , especially since the existence of cholesterol - dependent microdomains on the endosomal membranes has been proven ( figure 2 ) . thus it is safe to assume that membrane partitioning control both extracellular and intracellular tlr - dependent signalling . we are proposing a model for endosomal tlr activation , where ligation of endosomal tlrs by their respective ligands can lead to clustering within lipid rafts at the endosomal membrane and activation of intracellular signaling cascades that lead via nf-b to production and secretion of proinflammatory cytokines ( figure 2(b ) ) . c - type lectin receptors ( clrs ) , such as dectin 1 , are a family of pattern recognition receptors which bind -glucan found in the cell walls of pathogenic fungi such as candida albicans . in particular dectin 1 has been shown to mediate the phagocytosis of yeast and yeast - derived particles , such as zymosan , activating the production of inflammatory cytokines [ 9294 ] . a recent study has revealed that dectin-1 and possibly other clrs are recruited to lipid rafts upon activation and raft integrity is important for signalling . cell membranes are complicated in composition but precise in purpose : to selectively compartmentalize its constituents in order to coordinate cellular functions . in this way , the membrane is able to compartmentalize , segregate receptors as well as their signalling machinery and create oligomeric signalling platforms in order to transduce signals . the innate and acquired immune systems seem to utilise this membrane partitioning for their functions . in this paper , we have extensively looked at the use of this membrane partitioning by the innate immune system and most particular by the tlrs . the molecular mechanism involved in lps recognition and tlr signalling in general , the plasma membrane seems to be heterogeneous and to coalesce to more stable membrane - ordered assemblies upon activation by ligands . this partitioning of the membrane and the assembly of more stable raft platforms in the functionalized state must be initiated by raft - resident proteins , which form protein - lipid as well as protein - protein interactions . the tlrs and other prrs associate with the raft - resident proteins and are recruited to these floating islands forming higher oligomers , both extracellularly as well as inside the cell , concentrating their signalling machinery which finally leads to a functional , focused , and coordinated activation of the innate immune system . these factors will include not only lipid - lipid , lipid - protein and protein - protein interactions both in the plane of the membrane but also elements of the cytoskeleton , pericellular matrix adjacent to the membrane as well as transmembrane and cytoplasmic domains of the receptors involved . in the case of tlrs , the association of the tir domains intracellularly would stabilise the ectodomains extracellularly and provide the molecular scaffold that will recruit the adaptor molecules that contribute to signalling . thus refining existing biophysical methods , such as single particle tracking ( spt ) , fluorescence correlation spectroscopy ( fcs ) , and fluorescence resonance energy transfer ( fret ) will help us reveal these dynamic nanoassemblies of sterol , sphingolipid , and proteins in living cell and provide us with the first dynamic picture of the innate immune response .

twenty - five adults with type 1 diabetes for > 15 years were recruited from the university of minnesota diabetes clinic and were matched by sex and age with 25 healthy volunteers . the exclusion criteria included history of or current substance abuse disorder other than tobacco and/or caffeine dependence , severe psychiatric disorder , history of seizure disorder ( not related to hypoglycemia ) , transient ischemic attack / stroke , head injury , or other disease of the central nervous system , or standard contraindication to magnetic resonance imaging ( mri ) . a previous publication reported measurements of fractional anisotropy and cognitive function in this population ( 4 ) . as noted in that publication , three subjects with diabetes reported a history of retinopathy , two reported a history of gastroparesis , and three reported histories of both retinopathy and neuropathy . fifteen of the 25 subjects with diabetes reported a history of severe hypoglycemia , defined as having seizures , loss of consciousness , or needing another person 's help to treat the symptoms of low blood glucose . there were no differences between the subjects with diabetes and the control subjects on the performance on the wechsler abbreviated scale of intelligence ( wasi ) . ten of the 25 subjects with diabetes and 3 of the 25 control subjects were taking antihypertensive medications at the time of study . twelve of the 25 subjects with diabetes and 2 of the control subjects were taking drugs to lower cholesterol . prior to undergoing the imaging protocol , blood glucose values were measured in subjects with type 1 diabetes using a reflectance meter to ensure values were between 100 and 250 mg / dl . if the blood glucose level was outside this range , it was corrected or the study was scheduled for another day . table 1 details clinical information about these subjects . demographic information of subjects data are means sd unless otherwise indicated . imaging data were acquired on a siemens trio 3 tesla scanner ( erlangen , germany ) at the university of minnesota 's center for magnetic resonance research . axial diffusion tensor imaging ( dti ) was performed using the following parameters : the field of view was positioned to cover the entire cerebrum . whole brain dti was performed using a dual spin echo , single shot , echo planar , diffusion - weighted sequence with these parameters : repetition time ( tr ) = 8,000 ms echo time ( te ) = 83 ms , 128 128 matrix , 32 cm field of view , 2-mm thick slices skip 0 , 64 slices , and b value = 1,000 . diffusion was measured along 12 noncollinear directions . a 3d t1 magnetization - prepared rapid acquisition of gradient echo ( mprage ) sequence was acquired with the following parameters : tr = 2530ms , te = 3.63 ms , imaging time ( ti ) = 1,100 ms , 1 mm isotropic , field of view = 256 mm , and 256 256 matrix . no grossly abnormal findings that would have required exclusion were observed in structural mri scans . in our previous report on this subject group ( 4 ) , individual white matter tracts were reconstructed using diffusion - weighted images . this procedure is discussed in depth in the supplementary data section in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0598/dc1 . briefly , for each subject , bilateral forceps minor , cingulum bundle , medial corona radiata , superior longitudinal fasciculus , and optic radiation regions of interest were generated in addition to six corpus callosum regions of interest : genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium using tract - based spatial statistics ( 14 ) and custom software . figure 1 demonstrates these segmented white matter tracts on a composite t1 structural brain image . groupwise differences in fractional anisotropy , a commonly used metric of white matter microstructural integrity , were calculated . those tracts that were found to have significantly different fractional anisotropy between the diabetic and control subject groups were then used for starting points for the fiber tract projection analysis described below . automated white matter image analysis technique resulting in tract - specific regions of interest : corona radiata ( red ) , superior longitudinal fasiciculus ( yellow ) , cungulum bundle ( green ) ( left ) , six corpus callosum subdivisions ( middle ) , and the optic radiations ( right ) . ( a high - quality digital representation of this figure is available in the online issue . ) the connectivity of these tracts , which included the optic radiations , posterior corona radiata , and splenium tracts , was determined using probtrack and bedpost software ( 15 ) . the tract - based spatial statistics generated skeletonized tracts described above were used as region - of - interest starting masks , and a connectivity distribution for each tract was calculated using the fsl - fdt software package ( 16 ) . this distribution represents the degree of connectivity from each starting voxel in the region of interest to every other voxel on the basis of the diffusion data and relies on a probabilistic white matter tractography approach . voxels with high measured connectivity to the starting voxel tend to be connected by white matter tracts with consistent diffusion directions . this technique relied on a probabilistic markov chain monte carlo sampling approach to determine which voxels throughout the brain are most closely linked to these starting points on the basis of the diffusion - weighted imaging data . this connectivity distribution was thresholded in the same way for each starting mask using a sum of the distribution 's mean and sd thus removing all insignificant connectivity results from the distribution . the average cortical thickness of the gray matter regions that intersected with each of these connectivity distributions was then calculated as is discussed below . t1 images from each subject were registered to a common template based on the interface between gray and white matter using a standard talairach atlas . cortical thickness ( the distance between the pial surface and white matter ) was calculated for each point throughout the cortex as the distance between the pial and white matter boundaries . the gyral surface for each subject was labeled using a probabilistic algorithm , and the cortical thickness was calculated for these named gyri and sulci ( 17 ) . the cortical segmentation and labeling method generates results that have similar reliability to manual methods ( 18 ) . imaging data were acquired on a siemens trio 3 tesla scanner ( erlangen , germany ) at the university of minnesota 's center for magnetic resonance research . axial diffusion tensor imaging ( dti ) was performed using the following parameters : the field of view was positioned to cover the entire cerebrum . whole brain dti was performed using a dual spin echo , single shot , echo planar , diffusion - weighted sequence with these parameters : repetition time ( tr ) = 8,000 ms echo time ( te ) = 83 ms , 128 128 matrix , 32 cm field of view , 2-mm thick slices skip 0 , 64 slices , and b value = 1,000 . a 3d t1 magnetization - prepared rapid acquisition of gradient echo ( mprage ) sequence was acquired with the following parameters : tr = 2530ms , te = 3.63 ms , imaging time ( ti ) = 1,100 ms , 1 mm isotropic , field of view = 256 mm , and 256 256 matrix . no grossly abnormal findings that would have required exclusion were observed in structural mri scans . in our previous report on this subject group ( 4 ) , individual white matter tracts were reconstructed using diffusion - weighted images . this procedure is discussed in depth in the supplementary data section in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0598/dc1 . briefly , for each subject , bilateral forceps minor , cingulum bundle , medial corona radiata , superior longitudinal fasciculus , and optic radiation regions of interest were generated in addition to six corpus callosum regions of interest : genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium using tract - based spatial statistics ( 14 ) and custom software . figure 1 demonstrates these segmented white matter tracts on a composite t1 structural brain image . groupwise differences in fractional anisotropy , a commonly used metric of white matter microstructural integrity , were calculated . those tracts that were found to have significantly different fractional anisotropy between the diabetic and control subject groups were then used for starting points for the fiber tract projection analysis described below . automated white matter image analysis technique resulting in tract - specific regions of interest : corona radiata ( red ) , superior longitudinal fasiciculus ( yellow ) , cungulum bundle ( green ) ( left ) , six corpus callosum subdivisions ( middle ) , and the optic radiations ( right ) . ( a high - quality digital representation of this figure is available in the online issue . ) the connectivity of these tracts , which included the optic radiations , posterior corona radiata , and splenium tracts , was determined using probtrack and bedpost software ( 15 ) . the tract - based spatial statistics generated skeletonized tracts described above were used as region - of - interest starting masks , and a connectivity distribution for each tract was calculated using the fsl - fdt software package ( 16 ) . this distribution represents the degree of connectivity from each starting voxel in the region of interest to every other voxel on the basis of the diffusion data and relies on a probabilistic white matter tractography approach . voxels with high measured connectivity to the starting voxel tend to be connected by white matter tracts with consistent diffusion directions . this technique relied on a probabilistic markov chain monte carlo sampling approach to determine which voxels throughout the brain are most closely linked to these starting points on the basis of the diffusion - weighted imaging data . this connectivity distribution was thresholded in the same way for each starting mask using a sum of the distribution 's mean and sd thus removing all insignificant connectivity results from the distribution . the average cortical thickness of the gray matter regions that intersected with each of these connectivity distributions was then calculated as is discussed below . t1 images from each subject were registered to a common template based on the interface between gray and white matter using a standard talairach atlas . cortical thickness ( the distance between the pial surface and white matter ) was calculated for each point throughout the cortex as the distance between the pial and white matter boundaries . the gyral surface for each subject was labeled using a probabilistic algorithm , and the cortical thickness was calculated for these named gyri and sulci ( 17 ) . the cortical segmentation and labeling method generates results that have similar reliability to manual methods ( 18 ) . using this dataset , we have previously reported that the subjects with type 1 diabetes had significantly lower measures of fractional anisotropy than the control subjects in the optic radiations , posterior corona radiata , and splenium . in the current analysis , the white matter tract regions of interest ( in red ) are used as starting seeds for connectivity mapping , and voxels found to have high connectivity on the basis of probabilistic tractography with these starting seeds are marked in yellow . subsequent analysis determined which gray matter regions had high connectivity with these white matter seeds . figure 2b demonstrates in a representative subject this method of using the overlap of the connectivity map with cortical boundaries to determine the connectivity of the original white matter region of interest to specific cortical regions . the upper image shows the lateral occipital gyral cortical boundary in blue , and the overlap with the optic radiations connectivity map in yellow is shown in the lower image . in this case , there are a number of voxels that overlap , and connectivity is established between the optic radiations region of interest and the lateral occipital cortical region for further statistical analysis of the thickness in this cortical region . the overlap analysis was performed in each subject 's native imaging space instead of warping to a common template in order to increase connectivity mapping accuracy , and the images presented in fig . the white matter tract regions of interest in red are used as starting seeds for connectivity mapping , and diffusion mri data are used to generate connectivity maps . the connectivity maps and starting seeds are shown for the optic radiations white matter tract ( top left ) , posterior corona radiata ( top right ) , and splenium of corpus callosum ( bottom ) . a representative axial ( on the left ) and coronal view ( on the right ) b : the overlap between the connectivity map and cortical regions is used to determine which cortical regions have high connectivity with the original white matter regions of interest . in this representative subject , the lateral occipital gyral cortical boundary is shown in blue in the upper image , and overlap with the optic radiations connectivity map is shown in yellow in the lower image . ( a high - quality digital representation of this figure is available in the online issue . ) the connectivity analysis was undertaken for each white matter region of interest and all cortical regions for each control and type 1 diabetic subject . table 2 lists the cortical regions that had a cortical - connectivity map overlap of at least one voxel on average across all subjects for each of the three white matter seeds . white matter connectivity with gyral interface masks significantly lower average cortical thickness was found in the subjects with type 1 diabetes for cortical regions with high connectivity to the optic radiations ( p = 0.012 , d = 0.86 ) and posterior corona radiata tracts ( p = 0.031 , d = 0.74 ) . figure 3 shows the relationship between average fractional anisotropy in the white matter seed regions of interest and the average cortical thickness in cortical regions found to have high connectivity with these seeds . these plots show that subjects with type 1 diabetes cluster with lower fractional anisotropy and lower cortical thickness . a pearson correlation analysis performed across all subjects between fractional anisotropy for the posterior corona radiata and connected cortex resulted in r = 0.427 ( p = 0.002 ) , and for fractional anisotropy in the optic radiations and connected cortex data r = 0.503 ( p < 0.001 ) . the average fractional anisotropy for the posterior corona radiata white matter region of interest ( left ) and optic radiations ( right ) is plotted against average cortical thickness for cortical regions found to have high connectivity to each of these seed regions . diabetic subjects are denoted by ; control subjects are denoted by . average cortical thickness in regions without high connectivity ( the remainder ) was not significantly lower than in control subjects ( p > 0.45 ) . overall brain size was not significantly different between groups . of note , in the subjects with diabetes a trend toward reduced cortical thickness was observed in the cortical regions found to have high connectivity to the splenium , but this did not reach statistical significance ( p = 0.11 ) . subject age and thickness in the cortex with high connectivity to the posterior corona radiata were found to be significantly related ( spearman correlation = 0.597 , p = 0.002 after bonferroni correction ) . diabetes duration , a1c at the time of the testing , and bmi were not significantly related to gray matter cortical thickness . performance on the grooved peg board with the dominant hand was the only neuropsychometric measure that correlated with gray matter thickness in regions with high connectivity to the posterior corona radiata ( spearman correlation = 0.51 , p = < 0.0001 after bonferroni correction ) or the optic radiations ( spearman correlation = 0.41 , p = < 0.0001 after bonferroni correction ) . using this dataset , we have previously reported that the subjects with type 1 diabetes had significantly lower measures of fractional anisotropy than the control subjects in the optic radiations , posterior corona radiata , and splenium . in the current analysis , the white matter tract regions of interest ( in red ) are used as starting seeds for connectivity mapping , and voxels found to have high connectivity on the basis of probabilistic tractography with these starting seeds are marked in yellow . subsequent analysis determined which gray matter regions had high connectivity with these white matter seeds . figure 2b demonstrates in a representative subject this method of using the overlap of the connectivity map with cortical boundaries to determine the connectivity of the original white matter region of interest to specific cortical regions . the upper image shows the lateral occipital gyral cortical boundary in blue , and the overlap with the optic radiations connectivity map in yellow is shown in the lower image . in this case , there are a number of voxels that overlap , and connectivity is established between the optic radiations region of interest and the lateral occipital cortical region for further statistical analysis of the thickness in this cortical region . the overlap analysis was performed in each subject 's native imaging space instead of warping to a common template in order to increase connectivity mapping accuracy , and the images presented in fig . the white matter tract regions of interest in red are used as starting seeds for connectivity mapping , and diffusion mri data are used to generate connectivity maps . the connectivity maps and starting seeds are shown for the optic radiations white matter tract ( top left ) , posterior corona radiata ( top right ) , and splenium of corpus callosum ( bottom ) . a representative axial ( on the left ) and coronal view ( on the right ) b : the overlap between the connectivity map and cortical regions is used to determine which cortical regions have high connectivity with the original white matter regions of interest . in this representative subject , the lateral occipital gyral cortical boundary is shown in blue in the upper image , and overlap with the optic radiations connectivity map is shown in yellow in the lower image . ( a high - quality digital representation of this figure is available in the online issue . ) the connectivity analysis was undertaken for each white matter region of interest and all cortical regions for each control and type 1 diabetic subject . table 2 lists the cortical regions that had a cortical - connectivity map overlap of at least one voxel on average across all subjects for each of the three white matter seeds . significantly lower average cortical thickness was found in the subjects with type 1 diabetes for cortical regions with high connectivity to the optic radiations ( p = 0.012 , d = 0.86 ) and posterior corona radiata tracts ( p = 0.031 , d = 0.74 ) . figure 3 shows the relationship between average fractional anisotropy in the white matter seed regions of interest and the average cortical thickness in cortical regions found to have high connectivity with these seeds . these plots show that subjects with type 1 diabetes cluster with lower fractional anisotropy and lower cortical thickness . a pearson correlation analysis performed across all subjects between fractional anisotropy for the posterior corona radiata and connected cortex resulted in r = 0.427 ( p = 0.002 ) , and for fractional anisotropy in the optic radiations and connected cortex data r = 0.503 ( p < 0.001 ) . the average fractional anisotropy for the posterior corona radiata white matter region of interest ( left ) and optic radiations ( right ) is plotted against average cortical thickness for cortical regions found to have high connectivity to each of these seed regions . diabetic subjects are denoted by ; control subjects are denoted by . average cortical thickness in regions without high connectivity ( the remainder ) was not significantly lower than in control subjects ( p > 0.45 ) . , in the subjects with diabetes a trend toward reduced cortical thickness was observed in the cortical regions found to have high connectivity to the splenium , but this did not reach statistical significance ( p = 0.11 ) . subject age and thickness in the cortex with high connectivity to the posterior corona radiata were found to be significantly related ( spearman correlation = 0.597 , p = 0.002 after bonferroni correction ) . diabetes duration , a1c at the time of the testing , and bmi were not significantly related to gray matter cortical thickness . performance on the grooved peg board with the dominant hand was the only neuropsychometric measure that correlated with gray matter thickness in regions with high connectivity to the posterior corona radiata ( spearman correlation = 0.51 , p = < 0.0001 after bonferroni correction ) or the optic radiations ( spearman correlation = 0.41 , p = < 0.0001 after bonferroni correction ) . in this study , we determined that there was linkage between the white matter tracts we had previously found to have reduced fractional anisotropy ( 4 ) and regions with reduced cortical thickness . together these findings suggest that long - standing type 1 diabetes may cause widespread microstructural alterations in the posterior cerebrum . although a number of neurological and neuropsychiatric diseases have been shown to alter gray matter morphometric measurements based on structural mr , diabetes has not been consistently associated with gray matter pathology . ( 7 ) , using a voxelwise analysis , found that relative to control subjects , subjects with type 1 diabetes had asymmetric reductions in gray matter density in the frontal and parietal lobes . on the other hand , ( 8) identified an asymmetric reduction in gray matter density in the occipital and frontal lobes in subjects with diabetic retinopathy . the voxelwise comparisons used in these investigations warped images onto each other and relied on blurring to overcome the problems with anatomic variation that occur when gyri are aligned among subjects . as a result , voxel - based morphological techniques are exquisitely sensitive to alignment errors , and discrepant conclusions between studies are the frequent result ( 19 ) . in our study , we overcame the limitation of voxel - based analyses by using techniques that measured the thickness of the cortex at the individual sulcus and gyrus level based on t1 mri images ( 13 ) . it is interesting that we found a predominantly posterior pattern of microstructual alterations in patients with type 1 diabetes because other diseases such as adrenoleukodystrophy , posterior reversible encephalopathy syndrome ( pres ) , and posterior cortical atrophy are known to display a similar pattern of change . the progressive dysmyelination present in adrenoleukodystropy , which results from accumulation of very - long - chain fatty acids , classically initiates in posterior white matter ( 20 ) before gray matter is affected . the reason for this predilection is not known and may be the result of local immunological or biochemical sensitivities . the posterior pres presents with posterior white matter edema and often occurs transiently in the face of hypertension , immunosuppressive therapy , or renal disease . the etiology of pres is unknown , although it is speculated that endothelial dysfunction ( 21 ) or cerebral autoregulation ( 22 ) may be involved . whether common mechanisms are responsible for the posterior changes seen in these diseases is unknown but should be the topic of subsequent research . we observed there to be connectivity between the gray matter regions with a reduction in cortical thickness and the white matter regions with a reduced fractional anisotropy in the subjects with type 1 diabetes . although it is intuitive to expect that cortical thickness and the neuronal process that caused this thinning would result in pathology in the axons that lead to and from these areas of cortex , there have been few human studies that have addressed this issue . in patients with posterior cortical atrophy , reduced fractional anisotropy in the splenium of the corpus callosum was found to be correlated with volume loss in the occipital cortex in patients ( 23 ) . similarly , white matter hyperintensities located in frontal or parieto - occipital white matter were found to correlate with reduced hippocampal volume in patients with alzheimer 's disease ( 24 ) . the mechanism for the concurrent white and gray matter structural findings in our study is not known , but it could involve a causal event starting in either tissue type resulting in pathology in the other or else pathology occurring in both white and gray matter simultaneously . it has been proposed that white matter may be more susceptible to microvascular disease than cortical gray matter ( 25 ) , and it is plausible that diabetic microvascular disease may be a causative factor leading to first to white and then to gray matter structural deficits . in our previous study , we found a significant correlation between reduced fractional anisotropy and reduced performance on the copy portion of the rey - osterreith complex figure drawing test and the grooved peg board test ( 4 ) . both tests are believed to assess white matter function , but our finding of a significant relationship between cortical thickness in posterior gray matter regions and performance on the grooved peg board tests suggests this region may also participate in performing the tasks of executive function and psychomotor speed . age was the only subject characteristic that related significantly to cortical thickness in areas with high connectivity to the posterior corona radiata and the optic radiations . a prospective evaluation of a larger sample size will be necessary to determine if diabetes - related characteristics such as glycemic control have an impact on gray matter cortical thickness . one limitation of our study is that we can not control for the effect of diabetic retinopathy and other related ophthalmological complications on the posterior regions in which we identified microstructural changes . however , as reported previously ( 4 ) , only two members of this group had received photocoagulation therapy for diabetic retinopathy in the years prior to the imaging study . none of our subjects reported vision loss , but it is not known if subtle retinopathy results in general in structural brain changes . in conclusion , we found that patients with long - standing type 1 diabetes have concurrent structural deficits in both the gray and the white matter located in the posterior region of the brain . future investigation will be necessary to identify both the cause and the long - term effects of these findings .

objectiveprevious studies have observed disruptions in brain white and gray matter structure in individuals with type 1 diabetes , and these structural differences have been associated with neurocognitive testing deficiencies . this study investigated the relationship between cerebral cortical thickness reductions and white matter microstructural integrity loss in a group of patients with type 1 diabetes and in healthy control subjects using diffusion tensor imaging ( dti).research design and methodstwenty - five subjects with type 1 diabetes for at least 15 years and 25 age- and sex - matched control subjects underwent structural t1 and proton - density and dti on a 3.0 tesla scanner . fractional anisotropy measurements were made on major cerebral white matter tracts , and dti tractography was performed to identify cortical regions with high connectivity to these tracts.resultsposterior white matter tracts with reduced fractional anisotropy ( optic radiations , posterior corona radiata , and the splenium region of the corpus callosum ) were found to have high connectivity with a number of posterior cortical regions , including the cuneus , precuneus , fusiform , and posterior parietal cortical regions . a significant reduction in cortical thickness in the diabetic group was observed in the regions with high connectivity to the optic radiations and posterior corona radiata tracts ( p < 0.05).conclusionsthe direct relationship between white and gray matter structural pathology has not been previously demonstrated in subjects with long - standing type 1 diabetes . the relationship between posterior white matter microstructural integrity disruption and lower cortical thickness demonstrated using a novel dti connectivity technique suggests a common or interrelated pathophysiological mechanism in type 1 diabetes .

RESEARCH DESIGN AND METHODS MRI data acquisition. DTI data processing. Fiber tract projection analysis. Cortical thickness MRI analyses. RESULTS Connectivity analysis. Relationship between gray matter cortical thickness and water matter regions with reduced fractional anisotropy. Relationships between gray matter cortical thickness and subject characteristics. DISCUSSION Supplementary Material

twenty - five adults with type 1 diabetes for > 15 years were recruited from the university of minnesota diabetes clinic and were matched by sex and age with 25 healthy volunteers . there were no differences between the subjects with diabetes and the control subjects on the performance on the wechsler abbreviated scale of intelligence ( wasi ) . ten of the 25 subjects with diabetes and 3 of the 25 control subjects were taking antihypertensive medications at the time of study . twelve of the 25 subjects with diabetes and 2 of the control subjects were taking drugs to lower cholesterol . prior to undergoing the imaging protocol , blood glucose values were measured in subjects with type 1 diabetes using a reflectance meter to ensure values were between 100 and 250 mg / dl . axial diffusion tensor imaging ( dti ) was performed using the following parameters : the field of view was positioned to cover the entire cerebrum . whole brain dti was performed using a dual spin echo , single shot , echo planar , diffusion - weighted sequence with these parameters : repetition time ( tr ) = 8,000 ms echo time ( te ) = 83 ms , 128 128 matrix , 32 cm field of view , 2-mm thick slices skip 0 , 64 slices , and b value = 1,000 . in our previous report on this subject group ( 4 ) , individual white matter tracts were reconstructed using diffusion - weighted images . briefly , for each subject , bilateral forceps minor , cingulum bundle , medial corona radiata , superior longitudinal fasciculus , and optic radiation regions of interest were generated in addition to six corpus callosum regions of interest : genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium using tract - based spatial statistics ( 14 ) and custom software . figure 1 demonstrates these segmented white matter tracts on a composite t1 structural brain image . groupwise differences in fractional anisotropy , a commonly used metric of white matter microstructural integrity , were calculated . those tracts that were found to have significantly different fractional anisotropy between the diabetic and control subject groups were then used for starting points for the fiber tract projection analysis described below . automated white matter image analysis technique resulting in tract - specific regions of interest : corona radiata ( red ) , superior longitudinal fasiciculus ( yellow ) , cungulum bundle ( green ) ( left ) , six corpus callosum subdivisions ( middle ) , and the optic radiations ( right ) . the connectivity of these tracts , which included the optic radiations , posterior corona radiata , and splenium tracts , was determined using probtrack and bedpost software ( 15 ) . this distribution represents the degree of connectivity from each starting voxel in the region of interest to every other voxel on the basis of the diffusion data and relies on a probabilistic white matter tractography approach . voxels with high measured connectivity to the starting voxel tend to be connected by white matter tracts with consistent diffusion directions . this technique relied on a probabilistic markov chain monte carlo sampling approach to determine which voxels throughout the brain are most closely linked to these starting points on the basis of the diffusion - weighted imaging data . this connectivity distribution was thresholded in the same way for each starting mask using a sum of the distribution 's mean and sd thus removing all insignificant connectivity results from the distribution . the average cortical thickness of the gray matter regions that intersected with each of these connectivity distributions was then calculated as is discussed below . t1 images from each subject were registered to a common template based on the interface between gray and white matter using a standard talairach atlas . cortical thickness ( the distance between the pial surface and white matter ) was calculated for each point throughout the cortex as the distance between the pial and white matter boundaries . the gyral surface for each subject was labeled using a probabilistic algorithm , and the cortical thickness was calculated for these named gyri and sulci ( 17 ) . axial diffusion tensor imaging ( dti ) was performed using the following parameters : the field of view was positioned to cover the entire cerebrum . whole brain dti was performed using a dual spin echo , single shot , echo planar , diffusion - weighted sequence with these parameters : repetition time ( tr ) = 8,000 ms echo time ( te ) = 83 ms , 128 128 matrix , 32 cm field of view , 2-mm thick slices skip 0 , 64 slices , and b value = 1,000 . in our previous report on this subject group ( 4 ) , individual white matter tracts were reconstructed using diffusion - weighted images . briefly , for each subject , bilateral forceps minor , cingulum bundle , medial corona radiata , superior longitudinal fasciculus , and optic radiation regions of interest were generated in addition to six corpus callosum regions of interest : genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium using tract - based spatial statistics ( 14 ) and custom software . figure 1 demonstrates these segmented white matter tracts on a composite t1 structural brain image . groupwise differences in fractional anisotropy , a commonly used metric of white matter microstructural integrity , were calculated . those tracts that were found to have significantly different fractional anisotropy between the diabetic and control subject groups were then used for starting points for the fiber tract projection analysis described below . automated white matter image analysis technique resulting in tract - specific regions of interest : corona radiata ( red ) , superior longitudinal fasiciculus ( yellow ) , cungulum bundle ( green ) ( left ) , six corpus callosum subdivisions ( middle ) , and the optic radiations ( right ) . the connectivity of these tracts , which included the optic radiations , posterior corona radiata , and splenium tracts , was determined using probtrack and bedpost software ( 15 ) . this distribution represents the degree of connectivity from each starting voxel in the region of interest to every other voxel on the basis of the diffusion data and relies on a probabilistic white matter tractography approach . voxels with high measured connectivity to the starting voxel tend to be connected by white matter tracts with consistent diffusion directions . this technique relied on a probabilistic markov chain monte carlo sampling approach to determine which voxels throughout the brain are most closely linked to these starting points on the basis of the diffusion - weighted imaging data . this connectivity distribution was thresholded in the same way for each starting mask using a sum of the distribution 's mean and sd thus removing all insignificant connectivity results from the distribution . the average cortical thickness of the gray matter regions that intersected with each of these connectivity distributions was then calculated as is discussed below . t1 images from each subject were registered to a common template based on the interface between gray and white matter using a standard talairach atlas . cortical thickness ( the distance between the pial surface and white matter ) was calculated for each point throughout the cortex as the distance between the pial and white matter boundaries . the gyral surface for each subject was labeled using a probabilistic algorithm , and the cortical thickness was calculated for these named gyri and sulci ( 17 ) . using this dataset , we have previously reported that the subjects with type 1 diabetes had significantly lower measures of fractional anisotropy than the control subjects in the optic radiations , posterior corona radiata , and splenium . in the current analysis , the white matter tract regions of interest ( in red ) are used as starting seeds for connectivity mapping , and voxels found to have high connectivity on the basis of probabilistic tractography with these starting seeds are marked in yellow . subsequent analysis determined which gray matter regions had high connectivity with these white matter seeds . figure 2b demonstrates in a representative subject this method of using the overlap of the connectivity map with cortical boundaries to determine the connectivity of the original white matter region of interest to specific cortical regions . the upper image shows the lateral occipital gyral cortical boundary in blue , and the overlap with the optic radiations connectivity map in yellow is shown in the lower image . in this case , there are a number of voxels that overlap , and connectivity is established between the optic radiations region of interest and the lateral occipital cortical region for further statistical analysis of the thickness in this cortical region . the overlap analysis was performed in each subject 's native imaging space instead of warping to a common template in order to increase connectivity mapping accuracy , and the images presented in fig . the connectivity maps and starting seeds are shown for the optic radiations white matter tract ( top left ) , posterior corona radiata ( top right ) , and splenium of corpus callosum ( bottom ) . a representative axial ( on the left ) and coronal view ( on the right ) b : the overlap between the connectivity map and cortical regions is used to determine which cortical regions have high connectivity with the original white matter regions of interest . in this representative subject , the lateral occipital gyral cortical boundary is shown in blue in the upper image , and overlap with the optic radiations connectivity map is shown in yellow in the lower image . the connectivity analysis was undertaken for each white matter region of interest and all cortical regions for each control and type 1 diabetic subject . table 2 lists the cortical regions that had a cortical - connectivity map overlap of at least one voxel on average across all subjects for each of the three white matter seeds . white matter connectivity with gyral interface masks significantly lower average cortical thickness was found in the subjects with type 1 diabetes for cortical regions with high connectivity to the optic radiations ( p = 0.012 , d = 0.86 ) and posterior corona radiata tracts ( p = 0.031 , d = 0.74 ) . figure 3 shows the relationship between average fractional anisotropy in the white matter seed regions of interest and the average cortical thickness in cortical regions found to have high connectivity with these seeds . these plots show that subjects with type 1 diabetes cluster with lower fractional anisotropy and lower cortical thickness . a pearson correlation analysis performed across all subjects between fractional anisotropy for the posterior corona radiata and connected cortex resulted in r = 0.427 ( p = 0.002 ) , and for fractional anisotropy in the optic radiations and connected cortex data r = 0.503 ( p < 0.001 ) . the average fractional anisotropy for the posterior corona radiata white matter region of interest ( left ) and optic radiations ( right ) is plotted against average cortical thickness for cortical regions found to have high connectivity to each of these seed regions . average cortical thickness in regions without high connectivity ( the remainder ) was not significantly lower than in control subjects ( p > 0.45 ) . of note , in the subjects with diabetes a trend toward reduced cortical thickness was observed in the cortical regions found to have high connectivity to the splenium , but this did not reach statistical significance ( p = 0.11 ) . subject age and thickness in the cortex with high connectivity to the posterior corona radiata were found to be significantly related ( spearman correlation = 0.597 , p = 0.002 after bonferroni correction ) . diabetes duration , a1c at the time of the testing , and bmi were not significantly related to gray matter cortical thickness . performance on the grooved peg board with the dominant hand was the only neuropsychometric measure that correlated with gray matter thickness in regions with high connectivity to the posterior corona radiata ( spearman correlation = 0.51 , p = < 0.0001 after bonferroni correction ) or the optic radiations ( spearman correlation = 0.41 , p = < 0.0001 after bonferroni correction ) . using this dataset , we have previously reported that the subjects with type 1 diabetes had significantly lower measures of fractional anisotropy than the control subjects in the optic radiations , posterior corona radiata , and splenium . in the current analysis , the white matter tract regions of interest ( in red ) are used as starting seeds for connectivity mapping , and voxels found to have high connectivity on the basis of probabilistic tractography with these starting seeds are marked in yellow . subsequent analysis determined which gray matter regions had high connectivity with these white matter seeds . figure 2b demonstrates in a representative subject this method of using the overlap of the connectivity map with cortical boundaries to determine the connectivity of the original white matter region of interest to specific cortical regions . the upper image shows the lateral occipital gyral cortical boundary in blue , and the overlap with the optic radiations connectivity map in yellow is shown in the lower image . in this case , there are a number of voxels that overlap , and connectivity is established between the optic radiations region of interest and the lateral occipital cortical region for further statistical analysis of the thickness in this cortical region . the overlap analysis was performed in each subject 's native imaging space instead of warping to a common template in order to increase connectivity mapping accuracy , and the images presented in fig . the connectivity maps and starting seeds are shown for the optic radiations white matter tract ( top left ) , posterior corona radiata ( top right ) , and splenium of corpus callosum ( bottom ) . a representative axial ( on the left ) and coronal view ( on the right ) b : the overlap between the connectivity map and cortical regions is used to determine which cortical regions have high connectivity with the original white matter regions of interest . in this representative subject , the lateral occipital gyral cortical boundary is shown in blue in the upper image , and overlap with the optic radiations connectivity map is shown in yellow in the lower image . the connectivity analysis was undertaken for each white matter region of interest and all cortical regions for each control and type 1 diabetic subject . table 2 lists the cortical regions that had a cortical - connectivity map overlap of at least one voxel on average across all subjects for each of the three white matter seeds . significantly lower average cortical thickness was found in the subjects with type 1 diabetes for cortical regions with high connectivity to the optic radiations ( p = 0.012 , d = 0.86 ) and posterior corona radiata tracts ( p = 0.031 , d = 0.74 ) . figure 3 shows the relationship between average fractional anisotropy in the white matter seed regions of interest and the average cortical thickness in cortical regions found to have high connectivity with these seeds . these plots show that subjects with type 1 diabetes cluster with lower fractional anisotropy and lower cortical thickness . a pearson correlation analysis performed across all subjects between fractional anisotropy for the posterior corona radiata and connected cortex resulted in r = 0.427 ( p = 0.002 ) , and for fractional anisotropy in the optic radiations and connected cortex data r = 0.503 ( p < 0.001 ) . the average fractional anisotropy for the posterior corona radiata white matter region of interest ( left ) and optic radiations ( right ) is plotted against average cortical thickness for cortical regions found to have high connectivity to each of these seed regions . average cortical thickness in regions without high connectivity ( the remainder ) was not significantly lower than in control subjects ( p > 0.45 ) . , in the subjects with diabetes a trend toward reduced cortical thickness was observed in the cortical regions found to have high connectivity to the splenium , but this did not reach statistical significance ( p = 0.11 ) . subject age and thickness in the cortex with high connectivity to the posterior corona radiata were found to be significantly related ( spearman correlation = 0.597 , p = 0.002 after bonferroni correction ) . diabetes duration , a1c at the time of the testing , and bmi were not significantly related to gray matter cortical thickness . performance on the grooved peg board with the dominant hand was the only neuropsychometric measure that correlated with gray matter thickness in regions with high connectivity to the posterior corona radiata ( spearman correlation = 0.51 , p = < 0.0001 after bonferroni correction ) or the optic radiations ( spearman correlation = 0.41 , p = < 0.0001 after bonferroni correction ) . in this study , we determined that there was linkage between the white matter tracts we had previously found to have reduced fractional anisotropy ( 4 ) and regions with reduced cortical thickness . together these findings suggest that long - standing type 1 diabetes may cause widespread microstructural alterations in the posterior cerebrum . although a number of neurological and neuropsychiatric diseases have been shown to alter gray matter morphometric measurements based on structural mr , diabetes has not been consistently associated with gray matter pathology . ( 7 ) , using a voxelwise analysis , found that relative to control subjects , subjects with type 1 diabetes had asymmetric reductions in gray matter density in the frontal and parietal lobes . on the other hand , ( 8) identified an asymmetric reduction in gray matter density in the occipital and frontal lobes in subjects with diabetic retinopathy . it is interesting that we found a predominantly posterior pattern of microstructual alterations in patients with type 1 diabetes because other diseases such as adrenoleukodystrophy , posterior reversible encephalopathy syndrome ( pres ) , and posterior cortical atrophy are known to display a similar pattern of change . the progressive dysmyelination present in adrenoleukodystropy , which results from accumulation of very - long - chain fatty acids , classically initiates in posterior white matter ( 20 ) before gray matter is affected . the posterior pres presents with posterior white matter edema and often occurs transiently in the face of hypertension , immunosuppressive therapy , or renal disease . we observed there to be connectivity between the gray matter regions with a reduction in cortical thickness and the white matter regions with a reduced fractional anisotropy in the subjects with type 1 diabetes . although it is intuitive to expect that cortical thickness and the neuronal process that caused this thinning would result in pathology in the axons that lead to and from these areas of cortex , there have been few human studies that have addressed this issue . in patients with posterior cortical atrophy , reduced fractional anisotropy in the splenium of the corpus callosum was found to be correlated with volume loss in the occipital cortex in patients ( 23 ) . similarly , white matter hyperintensities located in frontal or parieto - occipital white matter were found to correlate with reduced hippocampal volume in patients with alzheimer 's disease ( 24 ) . the mechanism for the concurrent white and gray matter structural findings in our study is not known , but it could involve a causal event starting in either tissue type resulting in pathology in the other or else pathology occurring in both white and gray matter simultaneously . it has been proposed that white matter may be more susceptible to microvascular disease than cortical gray matter ( 25 ) , and it is plausible that diabetic microvascular disease may be a causative factor leading to first to white and then to gray matter structural deficits . in our previous study , we found a significant correlation between reduced fractional anisotropy and reduced performance on the copy portion of the rey - osterreith complex figure drawing test and the grooved peg board test ( 4 ) . both tests are believed to assess white matter function , but our finding of a significant relationship between cortical thickness in posterior gray matter regions and performance on the grooved peg board tests suggests this region may also participate in performing the tasks of executive function and psychomotor speed . age was the only subject characteristic that related significantly to cortical thickness in areas with high connectivity to the posterior corona radiata and the optic radiations . in conclusion , we found that patients with long - standing type 1 diabetes have concurrent structural deficits in both the gray and the white matter located in the posterior region of the brain . future investigation will be necessary to identify both the cause and the long - term effects of these findings .

chondrocytes are responsible for the biosynthesis and maintenance of articular cartilage constituents , i.e. collagen and proteoglycans ( pgs ) [ 13 ] . during osteoarthritis ( oa ) , the balance of these constituents is disturbed due to changes of the anabolic and catabolic activities within chondrocytes . chondrocyte strains have been suggested to be related to the cartilage biosynthesis [ 1 , 2 , 5 ] . hence , it is important to understand the influence of the mechanical environment of chondrocytes on the mechanical signals perceived by cells . the pericellular matrix ( pcm , fig . 1 ) , which includes a shell of collagen fibres around the cells [ 68 ] , as well as relatively high pg content [ 6 , 9 , 10 ] , can modify the deformation behaviour of chondrocytes [ 1113 ] . the importance of the mechanical properties of the pcm for the cell deformations has been studied in a few studies [ 1113 ] . however , the influence of the pcm composition ( collagen , pgs and interstitial water ) on the deformation of chondrocytes is currently poorly understood.fig . 1schematic 2d presentation of articular cartilage with collagen network and chondrocytes in xy - plane . one chondron with the pericellular collagen has been zoomed on the left . on the right , a top view ( xz - plane ) of an articular cartilage sample simulated quadrant of the sample is indicated schematic 2d presentation of articular cartilage with collagen network and chondrocytes in xy - plane . one chondron with the pericellular collagen has been zoomed on the left . on the right , a top view ( xz - plane ) of an articular cartilage sample simulated quadrant of the sample is indicated the three - dimensional ( 3d ) collagen network of articular cartilage is highly organized . the collagen network architecture has been implemented in macroscopic models of cartilage [ 1520 ] . in the first theoretical studies on chondrocyte mechanics , a 3d collagen structure of the cartilage tissue our recent studies [ 13 , 26 ] showed that the arcade - like fibril network as well as the collagen fibrils in the pcm significantly influenced chondrocyte deformation in a depth - dependent manner . however , the models of those studies were not able to estimate tissue function based on composition , and they neglected split - lines in the extracellular matrix ( ecm ) . based on a recently presented macroscopic finite element ( fe ) model , which is based on a composition of biphasic structures , a microscopic model including the chondrocytes and pcms was created in 3d including collagen split - lines in the ecm . this is the first attempt to combine a 3d macroscopic tissue - level model with a cellular - level model . with this model , the effects of collagen network , fixed charge density ( fcd ) and interstitial fluid of the pcm , as well as the effect of split - lines of the ecm on static deformations and stress strain environment of the chondrocytes we hypothesized that the collagen fibrils and fcd of the pcm modulate cell deformations in an opposite manner , and that cells are deformed primarily along the collagen split - line direction of the ecm . a 3d mesh of a cartilage plug ( height 0.75 mm , diameter 3 mm ) was constructed for unconfined compression geometry ( fig . 2 ) . the mesh included three chondrocytes with the pcms in different cartilage zones , located at the axis of symmetry . the chondrocytes were initially considered round with diameters of 10 , 14.5 and 12 m in the superficial , middle and deep zones , respectively . pcm thickness was 2.5 , 3.625 and 3 m in the superficial , middle and deep zones , respectively [ 26 , 28 , 29 ] . a recently developed material with realistic depth - dependent structure and composition of articular cartilage ( non - fibrillar part , fibrillar part , fluid ) cells were modelled in a similar manner as the pcms and ecm , except that they included no fibrillar part . the information of structural , compositional and mechanical properties of the cells , pcms and ecm were obtained from the literature [ 6 , 13 , 20 , 26 , 2833].fig . one quadrant of the sample was modelled by applying symmetry conditions to both splitting ( xy- and zy - plane ) edges . fluid was allowed to flow through the cartilage edge ( and top during the swelling ) . below the mesh , the deformation of the middle zone chondron is presented after each simulation step finite element mesh for 3d cartilage sample . one quadrant of the sample was modelled by applying symmetry conditions to both splitting ( xy- and zy - plane ) edges . fluid was allowed to flow through the cartilage edge ( and top during the swelling ) . below the mesh , the deformation of the middle zone chondron is presented after each simulation step the model is based on a standard biphasic theory:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - pi + \sigma_{s } = - n_{\text{s } } pi - n_{\text{f } } pi + \sigma_{s } , $ $ \end{document}where p is the hydrostatic pressure , s is the effective solid stress tensor , i is unit tensor , and nf and ns are the fluid and solid volume fractions , respectively . differing from the original biphasic theory , the influence of changes in the solid volume fraction is accounted for in the model . then , the total stress becomes:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - pi + n_{\text{s } } \sigma_{s } = - pi + { \frac{{n_{\text{s,0 } } } } { j}}\sigma_{rs } = - pi + n_{\text{s,0 } } \sigma_{rs , j } ( j ) , $ $ \end{document}where ns,0 and ns are the initial and current volume fractions of the solid matrix , j is the volumetric deformation and rs is the real solid stress tensor . after including the osmotic swelling , eq . 2 becomes:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - \mu^{\text{f } } i - \updelta \pi i + n_{\text{s,0 } } \sigma_{rs , j } , $ $ \end{document}where is the water chemical potential and is the osmotic pressure difference ( p = + ) . by separating the fibrillar and non - fibrillar part , eq . 3 becomes:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - \mu^{\text{f } } i + n_{{{\text{s}},0 } } \left ( { \left ( { 1 - \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } } } \right)\sigma_{nf } + \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } \sigma_{f}^{i } } } \right ) - \updelta \pi i , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho_{\text{c}}^{i } $ $ \end{document } is the volume fraction of the collagen fibrils in i - th fibril with respect to the total volume of the solid matrix , nf is the stress tensor of the non - fibrillar matrix , f is the fibril stress tensor . the stress of the non - fibrillar solid matrix can be described as:5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } { \varvec{\sigma}_{{nf } } } = { \frac{2}{j}}{\mathbf{f}}{\frac{{\partial w_{\text{nf } } } } { \partial { \mathbf{c}}}}f^{t } \hfill \\ \ , = - { \frac{1}{6}}{\frac{\ln ( j)}{j}}g_{\text{m } } i\left [ { - 1 + { \frac{{3(j + n_{\text{s,0 } } ) } } { { ( - j + n_{\text{s,0 } } ) } } } + { \frac{{3\ln ( j ) + jn_{\text{s,0 } } } } { { ( - j + n_{\text{s,0 } } ) ^{2 } } } } } \right ] + { \frac{{g_{\text{m } } } } { j}}(f \cdot f^{t } - j^{2/3 } i ) , \hfill \\ \end{gathered } $ $ \end{document}where nf is the cauchy stress tensor of the non - fibrillar matrix , gm is the shear modulus , c is the cauchy - green deformation tensor , wnf is the energy function of the non - fibrillar matrix , and j is the determinant of the deformation tensor f . the shear modulus of the solid matrix in the pcm was chosen to be 10 times lower ( 90 kpa ) , than that of the ecm ( 900 kpa ) [ 27 , 35 ] . the solid matrix shear modulus of the cells was 17 kpa , which was assumed to account for all solid constituents of the cells as has also been assumed earlier . effective shear moduli depend on the water fraction of the matrix in the cells , pcms and ecm . the fibril stress tensor was given as follows : 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{f } = { \frac{\lambda } { j}}p_{\text{f } } \vec{e}_{\text{f } } \vec{e}_{\text{f } } , $ $ \end{document}where is the elongation of the fibril , pf the 1st piola kirchhoff fibril stress , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vec{e}_{\text{f } } $ $ \end{document } the current fibril direction . mechanical behaviour of the collagen fibrils depends on the total fibril strain ( f ) , strain of the nonlinear spring ( e ) and the values of e1 , k1 , e2 , k2 and n0 ( fig . material properties of the collagen fibril are described with the total fibril strain ( f ) , the strain of nonlinear spring ( e ) and the values of model parameters e1 , k1 , e2 , k2 and n0 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{1 } = e_{1 } ( e^{{k_{1 } \varepsilon_{\text{f } } - 1 } } ) $ $ \end{document}8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{2 } = e_{2 } ( e^{{k_{2 } \varepsilon_{\text{e } } - 1 } } ) $ $ \end{document}9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{\text{f } } = p_{1 } + p_{2 } , $ $ \end{document } schematic spring / dashpot model for a viscoelastic collagen fibril . material properties of the collagen fibril are described with the total fibril strain ( f ) , the strain of nonlinear spring ( e ) and the values of model parameters e1 , k1 , e2 , k2 and n0 values for e1 and k1 ( describing the behaviour of the linear part of the collagen stiffness ) were set to 4.316 mpa and 16.85 , respectively . values for e2 , k2 and n0 ( describing the behaviour of the nonlinear part of the collagen stiffness ) for ecm were previously determined to be 19.97 mpa , 41.49 and 1.424 10 mpa s , respectively . for pcm ( predominantly type vi ) collagen , 10% stiffness of the ecm ( mostly type ii ) collagen stiffness was assumed in the reference configuration . this was justified by the geometrical differences in the collagen types , type ii collagen being wider in diameter than type vi collagen . the density of each collagen fibril was presented as a function of the total collagen density:10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } \rho_{c } = \rho_{{c,{\text{tot } } } } { \frac{c}{2c + 7}}{\text { for primary fibrils , } } \hfill \\ \rho_{c } = \rho_{{c,{\text{tot } } } } { \frac{1}{2c + 7}}{\text { for secondary fibrils , } } \hfill \\ \end{gathered } $ $ \end{document}where c was 3.009 . in the ecm , the collagen fibrils ran parallel to the articular surface in the superficial zone , bending in the middle zone towards perpendicular orientation in the deep zone . thickness of superficial and middle zones were set to 10 and 40% of the total cartilage thickness , respectively . collagen fibrils were implemented in parallel planes in xy direction representing the direction of split - lines in the ecm ( fig . 1 ) . the collagen fibrils in the pcms were orientated parallel to the cell surfaces ( fig . 1 ) [ 7 , 8 ] , and the cells included no collagen fibrils . : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \updelta \pi = \varphi_{\text{int } } rt\sqrt { c_{\text{f}}^{2 } + 4{\frac{{\gamma_{\text{ext}}^ { { \pm^{2 } } } } } { { \gamma_{\text{int}}^ { { \pm^{2 } } } } } } c_{\text{ext}}^{2 } } - 2\varphi_{\text{ext } } rtc_{\text{ext } } , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varphi_{\text{int } } $ $ \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}$$ \varphi_{\text{ext } } $ $ \end{document } are osmotic coefficients , int and ext are activity coefficients , and cext is the external salt concentration ( 0.15 m ) , and cf is the fcd . r and t are the gas constant ( 8.3145 j / mol k ) and absolute temperature ( 293 k ) , respectively . the effective fcd , cf , eff , takes the extra- and intra - fibrillar water into account:12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{\text{f , eff } } = { \frac{{n_{\text{f } } c_{\text{f } } } } { { n_{\text{ext } } } } } , $ $ \end{document}where next is the volume fraction of the extra - fibrillar water as a function of the extra - fibrillar water mass fraction ( next , m):13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{ext } } = { \frac{{\rho_{\text{s } } n_{\text{ext , m } } } } { { 1 - n_{\text{ext , m } } + \rho_{\text{s } } n_{\text{ext , m } } } } } , $ $ \end{document}where s is the solid matrix mass density , which was set to 1.4338 [ 39 , 40 ] . : 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k = \alpha ( 1 - n_{\text{ext } } ) ^ { - m } , $ $ \end{document}where and m are positive material constants with the values of 1.767 10m / n s and 1.339 in the ecm , respectively . in accordance with the literature , the permeability of the pcm was chosen to be the same as that of the ecm , while the permeability of the cells was fixed at 2.6 10m / n s . a more thorough implementation of the constitutive laws into the fe - model was presented in a recent study by wilson et al . . depth - dependent composition ( distribution of fcd , collagen mass fraction and fluid fraction ) of the ecm and pcms were obtained from the literature [ 3133 , 41 ] . the fcd was distributed as follows [ 20 , 3032]:15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{\text{f } } = - 0.1z^{2 } + 0.24z + 0.035 , $ $ \end{document}where z is the normalised depth of cartilage ( 0 in top , 1 in bottom ) . the collagen mass density and fluid fraction were [ 20 , 33 , 40 , 41]:16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho_{\text{c } } = 1.4z^{2 } - 1.1z + 0.59 , $ $ \end{document}17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{f } } = - 0.2z + 0.9 . $ $ \end{document } the fcd and fluid fraction of chondrocytes are presented in table 1 , and they were consistent with the previous studies [ 23 , 29 , 42 , 43].table 1values of the compositional properties for the 3d cellsfixed charge density ( meq / ml)fluid fraction ( % ) superficial zone0.1090middle zone0.1387deep zone0.1784 values of the compositional properties for the 3d cells a total of 8,502 8-node poroelastic continuum elements in one quadrant of the sample were used in the simulations . a total of 360 elements for each pcm and 528 elements for each chondrocyte were used . chondrocytes were placed in three cartilage zones ; superficial zone , middle zone and deep zone . they were assumed to be adhesively attached to the pcm and were therefore fixed to pcm in the geometry . consequently , continuous displacements and fluid flow in the cell - pcm and pcm - ecm interfaces were assumed [ 22 , 23 , 26 ] . the loading protocol included a free swelling step , a 15% mechanical compression , and a full relaxation . boundary conditions were set as follows : ( 1 ) movement of the nodes at the bottom of the mesh was prevented in y - direction ( fig . 2 ) , ( 2 ) symmetry conditions were set on the xy - plane and yz - plane of the simulated quadrant , ( 3 ) free fluid flow was allowed at the edge of the sample , and ( 4 ) compression of the sample was applied axially for the top nodes of the mesh . the contacts between the compressing platens and cartilage specimen were assumed frictionless . the collagen mass fraction , fluid fraction and fcd in the pcm were varied relatively to the composition of the ecm in order to determine their influence on the deformation of cells in different cartilage zones . the finite element simulations were performed using abaqus 6.7 ( dassault systmes , providence , ri ) . a 3d mesh of a cartilage plug ( height 0.75 mm , diameter 3 mm ) was constructed for unconfined compression geometry ( fig . 2 ) . the mesh included three chondrocytes with the pcms in different cartilage zones , located at the axis of symmetry . the chondrocytes were initially considered round with diameters of 10 , 14.5 and 12 m in the superficial , middle and deep zones , respectively . pcm thickness was 2.5 , 3.625 and 3 m in the superficial , middle and deep zones , respectively [ 26 , 28 , 29 ] . a recently developed material with realistic depth - dependent structure and composition of articular cartilage ( non - fibrillar part , fibrillar part , fluid ) cells were modelled in a similar manner as the pcms and ecm , except that they included no fibrillar part . the information of structural , compositional and mechanical properties of the cells , pcms and ecm were obtained from the literature [ 6 , 13 , 20 , 26 , 2833].fig . one quadrant of the sample was modelled by applying symmetry conditions to both splitting ( xy- and zy - plane ) edges . fluid was allowed to flow through the cartilage edge ( and top during the swelling ) . below the mesh , the deformation of the middle zone chondron is presented after each simulation step finite element mesh for 3d cartilage sample . one quadrant of the sample was modelled by applying symmetry conditions to both splitting ( xy- and zy - plane ) edges . fluid was allowed to flow through the cartilage edge ( and top during the swelling ) . below the mesh , the deformation of the middle zone chondron is presented after each simulation step the model is based on a standard biphasic theory:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - pi + \sigma_{s } = - n_{\text{s } } pi - n_{\text{f } } pi + \sigma_{s } , $ $ \end{document}where p is the hydrostatic pressure , s is the effective solid stress tensor , i is unit tensor , and nf and ns are the fluid and solid volume fractions , respectively . differing from the original biphasic theory , the influence of changes in the solid volume fraction is accounted for in the model . then , the total stress becomes:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - pi + n_{\text{s } } \sigma_{s } = - pi + { \frac{{n_{\text{s,0 } } } } { j}}\sigma_{rs } = - pi + n_{\text{s,0 } } \sigma_{rs , j } ( j ) , $ $ \end{document}where ns,0 and ns are the initial and current volume fractions of the solid matrix , j is the volumetric deformation and rs is the real solid stress tensor . after including the osmotic swelling , eq . 2 becomes:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - \mu^{\text{f } } i - \updelta \pi i + n_{\text{s,0 } } \sigma_{rs , j } , $ $ \end{document}where is the water chemical potential and is the osmotic pressure difference ( p = + ) . by separating the fibrillar and non - fibrillar part , eq . 3 becomes:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - \mu^{\text{f } } i + n_{{{\text{s}},0 } } \left ( { \left ( { 1 - \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } } } \right)\sigma_{nf } + \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } \sigma_{f}^{i } } } \right ) - \updelta \pi i , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho_{\text{c}}^{i } $ $ \end{document } is the volume fraction of the collagen fibrils in i - th fibril with respect to the total volume of the solid matrix , nf is the stress tensor of the non - fibrillar matrix , f is the fibril stress tensor . the stress of the non - fibrillar solid matrix can be described as:5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } { \varvec{\sigma}_{{nf } } } = { \frac{2}{j}}{\mathbf{f}}{\frac{{\partial w_{\text{nf } } } } { \partial { \mathbf{c}}}}f^{t } \hfill \\ \ , = - { \frac{1}{6}}{\frac{\ln ( j)}{j}}g_{\text{m } } i\left [ { - 1 + { \frac{{3(j + n_{\text{s,0 } } ) } } { { ( - j + n_{\text{s,0 } } ) } } } + { \frac{{3\ln ( j ) + jn_{\text{s,0 } } } } { { ( - j + n_{\text{s,0 } } ) ^{2 } } } } } \right ] + { \frac{{g_{\text{m } } } } { j}}(f \cdot f^{t } - j^{2/3 } i ) , \hfill \\ \end{gathered } $ $ \end{document}where nf is the cauchy stress tensor of the non - fibrillar matrix , gm is the shear modulus , c is the cauchy - green deformation tensor , wnf is the energy function of the non - fibrillar matrix , and j is the determinant of the deformation tensor f . the shear modulus of the solid matrix in the pcm was chosen to be 10 times lower ( 90 kpa ) , than that of the ecm ( 900 kpa ) [ 27 , 35 ] . the solid matrix shear modulus of the cells was 17 kpa , which was assumed to account for all solid constituents of the cells as has also been assumed earlier . effective shear moduli depend on the water fraction of the matrix in the cells , pcms and ecm . the fibril stress tensor was given as follows : 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{f } = { \frac{\lambda } { j}}p_{\text{f } } \vec{e}_{\text{f } } \vec{e}_{\text{f } } , $ $ \end{document}where is the elongation of the fibril , pf the 1st piola kirchhoff fibril stress , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \vec{e}_{\text{f } } $ $ \end{document } the current fibril direction . mechanical behaviour of the collagen fibrils depends on the total fibril strain ( f ) , strain of the nonlinear spring ( e ) and the values of e1 , k1 , e2 , k2 and n0 ( fig . material properties of the collagen fibril are described with the total fibril strain ( f ) , the strain of nonlinear spring ( e ) and the values of model parameters e1 , k1 , e2 , k2 and n0 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{1 } = e_{1 } ( e^{{k_{1 } \varepsilon_{\text{f } } - 1 } } ) $ $ \end{document}8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{2 } = e_{2 } ( e^{{k_{2 } \varepsilon_{\text{e } } - 1 } } ) $ $ \end{document}9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{\text{f } } = p_{1 } + p_{2 } , $ $ \end{document } schematic spring / dashpot model for a viscoelastic collagen fibril . material properties of the collagen fibril are described with the total fibril strain ( f ) , the strain of nonlinear spring ( e ) and the values of model parameters e1 , k1 , e2 , k2 and n0 values for e1 and k1 ( describing the behaviour of the linear part of the collagen stiffness ) were set to 4.316 mpa and 16.85 , respectively . values for e2 , k2 and n0 ( describing the behaviour of the nonlinear part of the collagen stiffness ) for ecm were previously determined to be 19.97 mpa , 41.49 and 1.424 10 mpa s , respectively . for pcm ( predominantly type vi ) collagen , 10% stiffness of the ecm ( mostly type ii ) collagen stiffness was assumed in the reference configuration . this was justified by the geometrical differences in the collagen types , type ii collagen being wider in diameter than type vi collagen . the density of each collagen fibril was presented as a function of the total collagen density:10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } \rho_{c } = \rho_{{c,{\text{tot } } } } { \frac{c}{2c + 7}}{\text { for primary fibrils , } } \hfill \\ \rho_{c } = \rho_{{c,{\text{tot } } } } { \frac{1}{2c + 7}}{\text { for secondary fibrils , } } \hfill \\ \end{gathered } $ $ \end{document}where c was 3.009 . in the ecm , the collagen fibrils ran parallel to the articular surface in the superficial zone , bending in the middle zone towards perpendicular orientation in the deep zone . thickness of superficial and middle zones were set to 10 and 40% of the total cartilage thickness , respectively . collagen fibrils were implemented in parallel planes in xy direction representing the direction of split - lines in the ecm ( fig . 1 ) . the collagen fibrils in the pcms were orientated parallel to the cell surfaces ( fig . 1 ) [ 7 , 8 ] , and the cells included no collagen fibrils . : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \updelta \pi = \varphi_{\text{int } } rt\sqrt { c_{\text{f}}^{2 } + 4{\frac{{\gamma_{\text{ext}}^ { { \pm^{2 } } } } } { { \gamma_{\text{int}}^ { { \pm^{2 } } } } } } c_{\text{ext}}^{2 } } - 2\varphi_{\text{ext } } rtc_{\text{ext } } , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varphi_{\text{int } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varphi_{\text{ext } } $ $ \end{document } are osmotic coefficients , int and ext are activity coefficients , and cext is the external salt concentration ( 0.15 m ) , and cf is the fcd . r and t are the gas constant ( 8.3145 j / mol k ) and absolute temperature ( 293 k ) , respectively . the effective fcd , cf , eff , takes the extra- and intra - fibrillar water into account:12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{\text{f , eff } } = { \frac{{n_{\text{f } } c_{\text{f } } } } { { n_{\text{ext } } } } } , $ $ \end{document}where next is the volume fraction of the extra - fibrillar water as a function of the extra - fibrillar water mass fraction ( next , m):13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{ext } } = { \frac{{\rho_{\text{s } } n_{\text{ext , m } } } } { { 1 - n_{\text{ext , m } } + \rho_{\text{s } } n_{\text{ext , m } } } } } , $ $ \end{document}where s is the solid matrix mass density , which was set to 1.4338 [ 39 , 40 ] . : 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k = \alpha ( 1 - n_{\text{ext } } ) ^ { - m } , $ $ \end{document}where and m are positive material constants with the values of 1.767 10m / n s and 1.339 in the ecm , respectively . in accordance with the literature , the permeability of the pcm was chosen to be the same as that of the ecm , while the permeability of the cells was fixed at 2.6 10m / n s . a more thorough implementation of the constitutive laws into the fe - model was presented in a recent study by wilson et al . . depth - dependent composition ( distribution of fcd , collagen mass fraction and fluid fraction ) of the ecm and pcms were obtained from the literature [ 3133 , 41 ] . the fcd was distributed as follows [ 20 , 3032]:15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{\text{f } } = - 0.1z^{2 } + 0.24z + 0.035 , $ $ \end{document}where z is the normalised depth of cartilage ( 0 in top , 1 in bottom ) . the collagen mass density and fluid fraction were [ 20 , 33 , 40 , 41]:16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho_{\text{c } } = 1.4z^{2 } - 1.1z + 0.59 , $ $ \end{document}17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{f } } = - 0.2z + 0.9 . $ $ \end{document } the fcd and fluid fraction of chondrocytes are presented in table 1 , and they were consistent with the previous studies [ 23 , 29 , 42 , 43].table 1values of the compositional properties for the 3d cellsfixed charge density ( meq / ml)fluid fraction ( % ) superficial zone0.1090middle zone0.1387deep zone0.1784 values of the compositional properties for the 3d cells a total of 8,502 8-node poroelastic continuum elements in one quadrant of the sample were used in the simulations . a total of 360 elements for each pcm and 528 elements for each chondrocyte were used . chondrocytes were placed in three cartilage zones ; superficial zone , middle zone and deep zone . they were assumed to be adhesively attached to the pcm and were therefore fixed to pcm in the geometry . consequently , continuous displacements and fluid flow in the cell - pcm and pcm - ecm interfaces were assumed [ 22 , 23 , 26 ] . the loading protocol included a free swelling step , a 15% mechanical compression , and a full relaxation . boundary conditions were set as follows : ( 1 ) movement of the nodes at the bottom of the mesh was prevented in y - direction ( fig . 2 ) , ( 2 ) symmetry conditions were set on the xy - plane and yz - plane of the simulated quadrant , ( 3 ) free fluid flow was allowed at the edge of the sample , and ( 4 ) compression of the sample was applied axially for the top nodes of the mesh . the contacts between the compressing platens and cartilage specimen were assumed frictionless . the collagen mass fraction , fluid fraction and fcd in the pcm were varied relatively to the composition of the ecm in order to determine their influence on the deformation of cells in different cartilage zones . the finite element simulations were performed using abaqus 6.7 ( dassault systmes , providence , ri ) . in the reference configuration , chondrocyte strains and volumes were highly depth - dependent ( fig . under 15% static surface - to - surface compression , axial strains of the cells were 11% ( deep zone)49% ( superficial zone ) , whereas lateral strains were 2% ( deep zone)9% ( superficial zone ) . strains of the superficial , middle and deep zones of the ecm were also highly depth - dependent , but 1.18 times lower than those of the chondrocytes . there were virtually no differences in the cell and ecm strains parallel and perpendicular to the direction of split - lines of the ecm collagen fibrils . volumes of the superficial , middle and deep zone chondrocytes were 41 , 25 and 5% lower than those before compression , respectively.fig . 4a reference axial and lateral ( parallel and perpendicular to the split - lines ) nominal strains and volume changes ( v / v0 ) for the cells after an application of 15% surface - to - surface compression of the tissue . b corresponding axial and lateral strains of the ecm within the proximity of each chondron a reference axial and lateral ( parallel and perpendicular to the split - lines ) nominal strains and volume changes ( v / v0 ) for the cells after an application of 15% surface - to - surface compression of the tissue . b corresponding axial and lateral strains of the ecm within the proximity of each chondron collagen fibril strain distribution ( f ) in the pcm exhibited depth - dependent characteristics ( fig . 5 ) . lateral edge of the pcm was compressed more in the superficial and middle zone than in the deep zone , therefore , exhibiting low tensile fibril strains , while the fibrils especially in the superficial zone pcm exhibited larger tensile strains above and below the cell . fibril strains of the ecm in the proximity of the pcm were lower parallel than perpendicular to the split - lines . von mises stresses in the vicinity of the simulated chondrocytes exhibited depth - dependent characteristics , greatest stresses being in the superficial zone , and were slightly different parallel and perpendicular to the split - lines ( fig . 5distribution of the logarithmic tensile fibril strain of primary collagen fibrils in the proximity of the simulated cells in the reference configuration . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presentedfig . 6von mises stress distribution in the proximity of the simulated cells in the reference configuration . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presented distribution of the logarithmic tensile fibril strain of primary collagen fibrils in the proximity of the simulated cells in the reference configuration . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presented von mises stress distribution in the proximity of the simulated cells in the reference configuration . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presented the collagen mass fraction , fcd and fluid fraction of the pcms affected static cell deformation ( table 2 ) after application of 15% axial strain for the tissue . an increase in the collagen content of the pcms was followed by an increase in lateral strains of the superficial and middle zone chondrocytes , whereas the deep zone cell experienced a decrease in lateral strains ( table 2 ) . in the superficial and middle zones , lateral cell strains perpendicular to the split - line direction were affected more than those parallel to the split - lines after the change in the collagen mass fraction of the pcms . axial cell strains were reduced slightly after an increase in the collagen content of the pcms.table 2cell strains parallel ( x ) and perpendicular ( z ) to the split - line direction as well as in axial direction ( y ) in the superficial , middle and deep zoneszonecollagen mass fractionfixed charge densityfluid fraction0.1 ecm1.5 ecm0.5 ecm1.3 ecm0.75 ecm1.05 ecmsuperficialx ( % ) 7.58.39.07.28.69.8y ( % ) 50.048.848.750.147.948.7z ( % ) 7.18.98.87.68.99.7middlex ( % ) 6.36.58.16.96.77.5y ( % ) 34.934.334.234.330.134.7z ( % ) 7.07.78.48.27.68.3deepx ( % ) 3.73.15.33.22.53.4y ( % ) 10.88.59.28.66.210.1z ( % ) 2.72.34.42.31.82.5strains were calculated with different values of pcm composition cell strains parallel ( x ) and perpendicular ( z ) to the split - line direction as well as in axial direction ( y ) in the superficial , middle and deep zones strains were calculated with different values of pcm composition an increase in the pericellular fcd reduced lateral strains of each chondrocyte ( table 2 ) . in the superficial and middle zones , lateral cell strains were reduced more parallel than perpendicular to the split - lines as the pericellular fcd was increased . the pcm fluid fraction induced a decrease in lateral strains of the chondrocytes ( table 2 ) . lateral cell strains changed similarly parallel and perpendicular to the split - lines of the ecm after the change in the pericellular fluid fraction . a 3d finite element model with depth - dependent cartilage properties , including chondrocytes with pcms in the superficial , middle and deep zones was constructed in order to study the effect of the pcm composition and the split - lines of the ecm on the cell deformation . cell deformation was dependent on the collagen and pg content , as well as on the fluid fraction of the pcm . the current model supports earlier found important role of the pcm in modulating the biomechanical signals perceived by the cells [ 11 , 29 , 44 ] , and highlights for the first time the effect of the pcm composition on the mechanical behaviour of chondrocytes . however , one must be careful in comparison between this study and the other studies , as the composition and mechanical properties of the ecm and pcm can be different in these studies . axial cell strains were higher in the superficial and middle zone , and lower in the deep zone , as compared to the work by guilak et al . . on the other hand , lateral cell strains were systematically higher in the superficial and middle zones than those examined by guilak et al . thereby , also the changes in cell volumes in these studies after static tissue compression were different ; 1622% in the study by guilak et al . one reason for these differences may be that , due to quite similar axial cell strains and local tissue strains in different cartilage zones , the specimens used by guilak et al . may have exhibited more homogenous tissue structure and composition than those assumed in our simulations . on the other hand , fluid may have had difficulties to penetrate cell membranes in experimental tests , preventing the decrease in cell volumes , whereas we assumed continuous fluid flow through the pcm - cell interface in the simulations . this was a justified assumption because fluid flow should have a negligible effect under equilibrium loading . it is also not certain if 20 min relaxation period was enough for full equilibrium in the experiments by guilak et al . . at least , a theoretical study by ateshian et al . showed that it might take much longer time for chondrocytes to reach steady state . in our model , minimal and large changes in the volumes of the superficial and deep zone chondrocytes , respectively , are directly related to the assumed composition and structure of the ecm , obtained from the literature . obviously , changes in the implemented ecm properties would have affected the deformation behaviour of chondrocytes . however , as the composition , structure and mechanical properties of the simulated samples were unknown , we did not pursue to optimize the correspondence between the experiments and simulations . thus , the differences between this study and the study by guilak et al . were expected . by optimizing the model parameters , the correspondence would have likely been better , but the optimizing solution would not have been unique due to too many optimized parameters with respect to the experimental data . recently , in situ indentation studies were conducted to investigate the deformation behaviour of superficial zone chondrocytes of rabbit articular cartilage . han and herzog suggested that the superficial zone chondrocytes experience greater strains along the split - line direction than perpendicular to the split - lines . this behaviour was also observed with some material parameter choices of the present model ( e.g. with low collagen content and fcd ) . however , the comparison between these studies may be meaningless since the measurement geometries were different ( unconfined versus indentation ) . moreover , similarly as in the comparison of this study and guilak et al . , tissue composition and structure may have been significantly different in han and herzog and this study , affecting significantly the cell deformations . in the presented model , we assumed that the cells were initially round in each zone of the cartilage . however , it is known that chondrocytes are ellipsoidal in the superficial zone of cartilage [ 6 , 48 ] . our previous study showed that initially round cells became ellipsoidal in the superficial zone after the free swelling step ( 0.15 m , physiological saline solution ) prior to compression . initially spherical cell shape has also been assumed in several earlier cell level models of articular cartilage [ 26 , 42 , 4951 ] . further , it has earlier been suggested that the modelling cells initially ellipsoidal instead of spherical could slightly increase cell strains , indicating that our choice for the cell shape resulted in a better correspondence between the model and experiments than that would have been with initially ellipsoidal superficial zone cell . an increase in the collagen content of the pcms enhanced lateral cell strain in the superficial zone cell , while the lateral cell strain of the deep zone cell decreased as the collagen content increased . consistent with our previous study , these differences were due to the benninghoff - type collagen orientation . the increase in the collagen content increased the stiffness of the collagen , which induced increased cell strain in the superficial zone where the fibrils were oriented parallel to the cartilage surface . in contrast , the vertically oriented fibrils in the deep zone restricted the cell expansion more effectively as the collagen content increased . this was due to softening of the cell surroundings , allowing greater local deformation for the pcm . moreover , stresses of the collagen fibrils were reduced as a result of the loss in the fcd , amplifying local pcm strains especially in the direction parallel to the split - lines . the decrease in the fluid fraction of the pcms , which increased the pcm stiffness , induced similar changes in the cell strains as the increase in the fcd . it was suggested earlier that the fluid fraction of chondrocytes may be much lower than what was assumed in this study [ 34 , 50 ] . therefore , we performed an additional parametric study to test the effect of cell properties , i.e. , solid matrix shear modulus , water fraction and fcd , on cell deformations . decreasing the water fraction by 30% increased lateral strains by 13% ( deep to superficial zone ) and decreased axial strains by 06% ( deep to superficial zone ) . decreasing the fcd by 30% increased lateral strains by 13% ( deep to superficial zone ) and increased axial strains by 27% ( deep to superficial zone ) . the effect of the shear modulus on the deformation behaviour of the cells was much smaller than that of the water fraction or fcd . it is likely that the ratio between the ecm and pcm stiffness is depth - dependent . this is supported by the depth - dependent stiffness of the ecm and depth - independent stiffness of the pcm [ 35 , 53 ] . further , the collagen arrangement in the pcm remains rather similar through tissue depth , while the ecm has a unique depth - wise collagen organization . the collagen network of the pcm and ecm together with the depth - dependent pg molecules were accounted for in the present model . thus , the ecm / pcm stiffness ratio of the model varied thought the tissue depth . moreover , the parametric analysis of the effect of different collagen content and fcd of the pcm induced several different ecm / pcm stiffness ratios . changes in the ecm composition or stiffness , and consequently ecm / pcm stiffness ratio , would of course have had an impact on the outcome of the simulations . however , it was already a subject of our earlier study . this study was is the first study to incorporate a 3d fibril - reinforced model with swelling properties for the ecm and pcm together with chondrocytes embedded into the model . applying the composition - based model allowed the estimation of the effects of the collagen content , fcd and fluid fraction of the pcm on the deformation behaviour of cells in articular cartilage . on the contrary , dynamic loading has been shown to stimulate proteoglycan synthesis . in order to clarify the impact of cell mechanics on cartilage biosynthesis , the relationships between mechanical and biological responses of cells should be investigated in the future in static and dynamic loading configuration . for that , the number of optimized model parameters should be reduced , i.e. , as many compositional , structural and mechanical parameters of the ecm , pcm and cells as possible should be measured experimentally ( e.g. [ 17 , 56 , 57 ] ) and implemented in the model . following that , cell deformations should be compared with experimental measurements of cells in situ under mechanical and/or osmotic loading , and the rest of the parameters in the pcm and cells could be possibly optimized . this could lead to a sample - specific estimation of stress and strain environment of chondrocytes . in conclusion , based on a microscopic 3d model of articular cartilage , collagen and pgs of the pcm influence the mechanical signals detected by the chondrocytes differently . hence , their effect may be different for the cartilage biosynthesis . the pericellular fcd reduced lateral cell strains which might highlight the protective role of the pcm . furthermore , the collagen content affected lateral cell strains in the superficial and middle zone more perpendicular than parallel to split - line direction , while with fcd , the trend was opposite . however , cartilage is a very complex tissue and the interplay between collagen , pgs and fluid ultimately modulate the mechanical environment of chondrocytes . the present model may improve the understanding of the significance of the chondrocyte environment on the degeneration , adaptation and remodelling of articular cartilage . we believe that this information can be used to estimate the impact of the structural and compositional changes in pcms ( e.g. , during osteoarthrosis ) on the maintenance of articular cartilage integrity .

the aim was to assess the role of the composition changes in the pericellular matrix ( pcm ) for the chondrocyte deformation . for that , a three - dimensional finite element model with depth - dependent collagen density , fluid fraction , fixed charge density and collagen architecture , including parallel planes representing the split - lines , was created to model the extracellular matrix ( ecm ) . the pcm was constructed similarly as the ecm , but the collagen fibrils were oriented parallel to the chondrocyte surfaces . the chondrocytes were modelled as poroelastic with swelling properties . deformation behaviour of the cells was studied under 15% static compression . due to the depth - dependent structure and composition of cartilage , axial cell strains were highly depth - dependent . an increase in the collagen content and fluid fraction in the pcms increased the lateral cell strains , while an increase in the fixed charge density induced an inverse behaviour . axial cell strains were only slightly affected by the changes in pcm composition . we conclude that the pcm composition plays a significant role in the deformation behaviour of chondrocytes , possibly modulating cartilage development , adaptation and degeneration . the development of cartilage repair materials could benefit from this information .

Introduction Materials and methods Fibril-reinforced biphasic swelling model Simulations Results Discussion Conclusions

the pericellular matrix ( pcm , fig . 1 ) , which includes a shell of collagen fibres around the cells [ 68 ] , as well as relatively high pg content [ 6 , 9 , 10 ] , can modify the deformation behaviour of chondrocytes [ 1113 ] . however , the influence of the pcm composition ( collagen , pgs and interstitial water ) on the deformation of chondrocytes is currently poorly understood.fig . on the right , a top view ( xz - plane ) of an articular cartilage sample simulated quadrant of the sample is indicated the three - dimensional ( 3d ) collagen network of articular cartilage is highly organized . in the first theoretical studies on chondrocyte mechanics , a 3d collagen structure of the cartilage tissue our recent studies [ 13 , 26 ] showed that the arcade - like fibril network as well as the collagen fibrils in the pcm significantly influenced chondrocyte deformation in a depth - dependent manner . however , the models of those studies were not able to estimate tissue function based on composition , and they neglected split - lines in the extracellular matrix ( ecm ) . based on a recently presented macroscopic finite element ( fe ) model , which is based on a composition of biphasic structures , a microscopic model including the chondrocytes and pcms was created in 3d including collagen split - lines in the ecm . with this model , the effects of collagen network , fixed charge density ( fcd ) and interstitial fluid of the pcm , as well as the effect of split - lines of the ecm on static deformations and stress strain environment of the chondrocytes we hypothesized that the collagen fibrils and fcd of the pcm modulate cell deformations in an opposite manner , and that cells are deformed primarily along the collagen split - line direction of the ecm . a recently developed material with realistic depth - dependent structure and composition of articular cartilage ( non - fibrillar part , fibrillar part , fluid ) cells were modelled in a similar manner as the pcms and ecm , except that they included no fibrillar part . 3 becomes:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - \mu^{\text{f } } i + n_{{{\text{s}},0 } } \left ( { \left ( { 1 - \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } } } \right)\sigma_{nf } + \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } \sigma_{f}^{i } } } \right ) - \updelta \pi i , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho_{\text{c}}^{i } $ $ \end{document } is the volume fraction of the collagen fibrils in i - th fibril with respect to the total volume of the solid matrix , nf is the stress tensor of the non - fibrillar matrix , f is the fibril stress tensor . the shear modulus of the solid matrix in the pcm was chosen to be 10 times lower ( 90 kpa ) , than that of the ecm ( 900 kpa ) [ 27 , 35 ] . mechanical behaviour of the collagen fibrils depends on the total fibril strain ( f ) , strain of the nonlinear spring ( e ) and the values of e1 , k1 , e2 , k2 and n0 ( fig . in the ecm , the collagen fibrils ran parallel to the articular surface in the superficial zone , bending in the middle zone towards perpendicular orientation in the deep zone . collagen fibrils were implemented in parallel planes in xy direction representing the direction of split - lines in the ecm ( fig . the collagen fibrils in the pcms were orientated parallel to the cell surfaces ( fig . in accordance with the literature , the permeability of the pcm was chosen to be the same as that of the ecm , while the permeability of the cells was fixed at 2.6 10m / n s . depth - dependent composition ( distribution of fcd , collagen mass fraction and fluid fraction ) of the ecm and pcms were obtained from the literature [ 3133 , 41 ] . $ $ \end{document } the fcd and fluid fraction of chondrocytes are presented in table 1 , and they were consistent with the previous studies [ 23 , 29 , 42 , 43].table 1values of the compositional properties for the 3d cellsfixed charge density ( meq / ml)fluid fraction ( % ) superficial zone0.1090middle zone0.1387deep zone0.1784 values of the compositional properties for the 3d cells a total of 8,502 8-node poroelastic continuum elements in one quadrant of the sample were used in the simulations . the collagen mass fraction , fluid fraction and fcd in the pcm were varied relatively to the composition of the ecm in order to determine their influence on the deformation of cells in different cartilage zones . a recently developed material with realistic depth - dependent structure and composition of articular cartilage ( non - fibrillar part , fibrillar part , fluid ) cells were modelled in a similar manner as the pcms and ecm , except that they included no fibrillar part . 3 becomes:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{tot } = - \mu^{\text{f } } i + n_{{{\text{s}},0 } } \left ( { \left ( { 1 - \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } } } \right)\sigma_{nf } + \sum\limits_{i = 1}^{\text{totf } } { \rho_{c}^{i } \sigma_{f}^{i } } } \right ) - \updelta \pi i , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho_{\text{c}}^{i } $ $ \end{document } is the volume fraction of the collagen fibrils in i - th fibril with respect to the total volume of the solid matrix , nf is the stress tensor of the non - fibrillar matrix , f is the fibril stress tensor . the shear modulus of the solid matrix in the pcm was chosen to be 10 times lower ( 90 kpa ) , than that of the ecm ( 900 kpa ) [ 27 , 35 ] . mechanical behaviour of the collagen fibrils depends on the total fibril strain ( f ) , strain of the nonlinear spring ( e ) and the values of e1 , k1 , e2 , k2 and n0 ( fig . values for e2 , k2 and n0 ( describing the behaviour of the nonlinear part of the collagen stiffness ) for ecm were previously determined to be 19.97 mpa , 41.49 and 1.424 10 mpa s , respectively . in the ecm , the collagen fibrils ran parallel to the articular surface in the superficial zone , bending in the middle zone towards perpendicular orientation in the deep zone . collagen fibrils were implemented in parallel planes in xy direction representing the direction of split - lines in the ecm ( fig . the collagen fibrils in the pcms were orientated parallel to the cell surfaces ( fig . in accordance with the literature , the permeability of the pcm was chosen to be the same as that of the ecm , while the permeability of the cells was fixed at 2.6 10m / n s . depth - dependent composition ( distribution of fcd , collagen mass fraction and fluid fraction ) of the ecm and pcms were obtained from the literature [ 3133 , 41 ] . $ $ \end{document } the fcd and fluid fraction of chondrocytes are presented in table 1 , and they were consistent with the previous studies [ 23 , 29 , 42 , 43].table 1values of the compositional properties for the 3d cellsfixed charge density ( meq / ml)fluid fraction ( % ) superficial zone0.1090middle zone0.1387deep zone0.1784 values of the compositional properties for the 3d cells a total of 8,502 8-node poroelastic continuum elements in one quadrant of the sample were used in the simulations . the collagen mass fraction , fluid fraction and fcd in the pcm were varied relatively to the composition of the ecm in order to determine their influence on the deformation of cells in different cartilage zones . in the reference configuration , chondrocyte strains and volumes were highly depth - dependent ( fig . under 15% static surface - to - surface compression , axial strains of the cells were 11% ( deep zone)49% ( superficial zone ) , whereas lateral strains were 2% ( deep zone)9% ( superficial zone ) . strains of the superficial , middle and deep zones of the ecm were also highly depth - dependent , but 1.18 times lower than those of the chondrocytes . there were virtually no differences in the cell and ecm strains parallel and perpendicular to the direction of split - lines of the ecm collagen fibrils . 4a reference axial and lateral ( parallel and perpendicular to the split - lines ) nominal strains and volume changes ( v / v0 ) for the cells after an application of 15% surface - to - surface compression of the tissue . b corresponding axial and lateral strains of the ecm within the proximity of each chondron a reference axial and lateral ( parallel and perpendicular to the split - lines ) nominal strains and volume changes ( v / v0 ) for the cells after an application of 15% surface - to - surface compression of the tissue . b corresponding axial and lateral strains of the ecm within the proximity of each chondron collagen fibril strain distribution ( f ) in the pcm exhibited depth - dependent characteristics ( fig . lateral edge of the pcm was compressed more in the superficial and middle zone than in the deep zone , therefore , exhibiting low tensile fibril strains , while the fibrils especially in the superficial zone pcm exhibited larger tensile strains above and below the cell . fibril strains of the ecm in the proximity of the pcm were lower parallel than perpendicular to the split - lines . von mises stresses in the vicinity of the simulated chondrocytes exhibited depth - dependent characteristics , greatest stresses being in the superficial zone , and were slightly different parallel and perpendicular to the split - lines ( fig . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presentedfig . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presented distribution of the logarithmic tensile fibril strain of primary collagen fibrils in the proximity of the simulated cells in the reference configuration . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presented von mises stress distribution in the proximity of the simulated cells in the reference configuration . projections in the xy- ( parallel to split - lines ) and zy - planes ( perpendicular to split - lines ) are presented the collagen mass fraction , fcd and fluid fraction of the pcms affected static cell deformation ( table 2 ) after application of 15% axial strain for the tissue . an increase in the collagen content of the pcms was followed by an increase in lateral strains of the superficial and middle zone chondrocytes , whereas the deep zone cell experienced a decrease in lateral strains ( table 2 ) . in the superficial and middle zones , lateral cell strains perpendicular to the split - line direction were affected more than those parallel to the split - lines after the change in the collagen mass fraction of the pcms . axial cell strains were reduced slightly after an increase in the collagen content of the pcms.table 2cell strains parallel ( x ) and perpendicular ( z ) to the split - line direction as well as in axial direction ( y ) in the superficial , middle and deep zoneszonecollagen mass fractionfixed charge densityfluid fraction0.1 ecm1.5 ecm0.5 ecm1.3 ecm0.75 ecm1.05 ecmsuperficialx ( % ) 7.58.39.07.28.69.8y ( % ) 50.048.848.750.147.948.7z ( % ) 7.18.98.87.68.99.7middlex ( % ) 6.36.58.16.96.77.5y ( % ) 34.934.334.234.330.134.7z ( % ) 7.07.78.48.27.68.3deepx ( % ) 3.73.15.33.22.53.4y ( % ) 10.88.59.28.66.210.1z ( % ) 2.72.34.42.31.82.5strains were calculated with different values of pcm composition cell strains parallel ( x ) and perpendicular ( z ) to the split - line direction as well as in axial direction ( y ) in the superficial , middle and deep zones strains were calculated with different values of pcm composition an increase in the pericellular fcd reduced lateral strains of each chondrocyte ( table 2 ) . in the superficial and middle zones , lateral cell strains were reduced more parallel than perpendicular to the split - lines as the pericellular fcd was increased . the pcm fluid fraction induced a decrease in lateral strains of the chondrocytes ( table 2 ) . lateral cell strains changed similarly parallel and perpendicular to the split - lines of the ecm after the change in the pericellular fluid fraction . a 3d finite element model with depth - dependent cartilage properties , including chondrocytes with pcms in the superficial , middle and deep zones was constructed in order to study the effect of the pcm composition and the split - lines of the ecm on the cell deformation . cell deformation was dependent on the collagen and pg content , as well as on the fluid fraction of the pcm . the current model supports earlier found important role of the pcm in modulating the biomechanical signals perceived by the cells [ 11 , 29 , 44 ] , and highlights for the first time the effect of the pcm composition on the mechanical behaviour of chondrocytes . however , one must be careful in comparison between this study and the other studies , as the composition and mechanical properties of the ecm and pcm can be different in these studies . axial cell strains were higher in the superficial and middle zone , and lower in the deep zone , as compared to the work by guilak et al . in our model , minimal and large changes in the volumes of the superficial and deep zone chondrocytes , respectively , are directly related to the assumed composition and structure of the ecm , obtained from the literature . obviously , changes in the implemented ecm properties would have affected the deformation behaviour of chondrocytes . by optimizing the model parameters , the correspondence would have likely been better , but the optimizing solution would not have been unique due to too many optimized parameters with respect to the experimental data . han and herzog suggested that the superficial zone chondrocytes experience greater strains along the split - line direction than perpendicular to the split - lines . further , it has earlier been suggested that the modelling cells initially ellipsoidal instead of spherical could slightly increase cell strains , indicating that our choice for the cell shape resulted in a better correspondence between the model and experiments than that would have been with initially ellipsoidal superficial zone cell . an increase in the collagen content of the pcms enhanced lateral cell strain in the superficial zone cell , while the lateral cell strain of the deep zone cell decreased as the collagen content increased . the increase in the collagen content increased the stiffness of the collagen , which induced increased cell strain in the superficial zone where the fibrils were oriented parallel to the cartilage surface . in contrast , the vertically oriented fibrils in the deep zone restricted the cell expansion more effectively as the collagen content increased . this was due to softening of the cell surroundings , allowing greater local deformation for the pcm . moreover , stresses of the collagen fibrils were reduced as a result of the loss in the fcd , amplifying local pcm strains especially in the direction parallel to the split - lines . the decrease in the fluid fraction of the pcms , which increased the pcm stiffness , induced similar changes in the cell strains as the increase in the fcd . the effect of the shear modulus on the deformation behaviour of the cells was much smaller than that of the water fraction or fcd . this is supported by the depth - dependent stiffness of the ecm and depth - independent stiffness of the pcm [ 35 , 53 ] . further , the collagen arrangement in the pcm remains rather similar through tissue depth , while the ecm has a unique depth - wise collagen organization . the collagen network of the pcm and ecm together with the depth - dependent pg molecules were accounted for in the present model . moreover , the parametric analysis of the effect of different collagen content and fcd of the pcm induced several different ecm / pcm stiffness ratios . changes in the ecm composition or stiffness , and consequently ecm / pcm stiffness ratio , would of course have had an impact on the outcome of the simulations . this study was is the first study to incorporate a 3d fibril - reinforced model with swelling properties for the ecm and pcm together with chondrocytes embedded into the model . applying the composition - based model allowed the estimation of the effects of the collagen content , fcd and fluid fraction of the pcm on the deformation behaviour of cells in articular cartilage . following that , cell deformations should be compared with experimental measurements of cells in situ under mechanical and/or osmotic loading , and the rest of the parameters in the pcm and cells could be possibly optimized . in conclusion , based on a microscopic 3d model of articular cartilage , collagen and pgs of the pcm influence the mechanical signals detected by the chondrocytes differently . the pericellular fcd reduced lateral cell strains which might highlight the protective role of the pcm . furthermore , the collagen content affected lateral cell strains in the superficial and middle zone more perpendicular than parallel to split - line direction , while with fcd , the trend was opposite . the present model may improve the understanding of the significance of the chondrocyte environment on the degeneration , adaptation and remodelling of articular cartilage .

the arab gulf countries , namely , bahrain , kuwait , oman , qatar , saudi arabia , and united arab emirates have undergone a rapid change in their socio - economic situation , food consumption patterns , and lifestyle and health status during the past four decades . this was mainly caused by the sharp increase in income due to accumulated oil revenues . communicable diseases have almost diminished and diet - related chronic diseases have become the main health problems the concept of food - based dietary guidelines ( fbdg ) has been promoted by the food and agriculture organization ( fao ) and the world health organization ( who ) of the united nations since the 1992 international conference on nutrition in rome , italy . one of the recommendations of this conference was that individual countries should develop simple dietary guidelines that are based on their specific public health concerns and relevant to people of different ages , lifestyles , and cultures . the action needed to promote healthy nutrition and a healthy lifestyle has been emphasized at several meetings in arab gulf counties [ 2 , 3 ] . in 1997 , the first workshop on diet , nutrition and a healthy lifestyle in the arab gulf countries was held in manama , bahrain . two main recommendations were proposed : establishing food - based dietary guidelines ( fbdg ) for the arab gulf countries , and promoting a healthy lifestyle in these countries . a simplified arabic version of the fbdg for the arab gulf countries was published in a booklet to be used by the public . it was distributed to many health authorities in the region , and it is also available in the website of the arab center of nutrition for more dissemination . therefore an attempt was done to release the second version , including all the comments received from the public , nutritionists , and professionals . the main objectives of this paper , therefore , are to summarize the steps taken to develope the food - based dietary guidelines for the arab gulf countries , and to explore the scientific bases of these guidelines . developing food - based dietary guidelines ( fbdg ) for the arab gulf countries step 1 ( determine the purpose and goals for establishing fbdg)the arab gulf countries are facing great challenges to prevent and control several nutritional problems and diet - related chronic diseases . two types of nutritional and health problems occur : those associated with change in lifestyle , such as obesity , cardiovascular disease , diabetes , hypertension , cancer , dental caries , and osteoporosis and those associated with nutrient deficiencies , such as iron deficiency anemia , undernutrition among preschool children , and deficiencies of vitamin d and calcium . in addition , food - borne diseases are a problem of concern in these countries . therefore , the need for simple dietary guidelines to address the burden of these diseases is urgent , especially as some of these diseases contribute to more than 50% of total mortality in this region .there are no specific food - based dietary guidelines used by arab countries in general . nutritionists , dietitians and other health workers in the arabian gulf region have been relying on fbdg developed for other countries , such as usa , canada , and uk , to convey nutrition messages to the public.in oman , an attempt was made to establish guidelines to healthy eating . however , these guidelines lack some issues related to the prevention and control of diet - related chronic disease such as reduced intake of salt and added sugar in foods , weight maintenance , and avoiding alcoholic drinks and smoking . encouraging drinking of water and other liquids were given very little attention ; it was mentioned in conjunction with physical activity , without any further advices . in general , these guidelines were prepared for the people who are responsible to educate the public , but not directed to the public . in general our work is different than omani guidelines in two main issues : ( 1 ) the omani guidelines were prepared for the health workers and not for the public , and ( 2 ) the omani guidelines were focused in portion size of the food groups while the current guidelines focused on food - based advices.an attempt , therefore , has been made to develop fbdg specifically for arab gulf countries to provide simple and practical dietary guidelines to help prevent and control the main nutrition - related disorders and that take into account the socio - cultural , lifestyle , and dietary habits of the people in these countries . step 2 ( characteristics of fbdg)to successfully promote healthy eating habits , a number of specific characteristics of fbdg were identified based on current knowledge and available information on food , nutrition , and health status in the countries of the region . these characteristics can be summarized as follows [ 2 , 6].the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries.they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods.they should consider the local dietary habits and food consumption patterns in the arab gulf countries.they should take into account food safety situation . they should consider the cultural and religious background of the population in arab gulf countries.they should consist of short , clear , and simple messages.they should promote a healthy lifestyle , especially the promotion of physical activity.they should be based on current scientific and health information . step 3 ( determine the food consumption patterns)a systematic literature review of the studies published in english between 1990 and 2010 was carried out , using pubmed and google scholar search to find out the food consumption patterns and nutrition situation in the arab gulf countries . the key words used were food consumption , nutrition , diet , anaemia , iodine deficiency disorders , cardiovascular disease , diabetes , vitamin d deficiency and undernutrition for each country . over 800 papers and reports were identified primarily . about 120 papers that possibly addressed food and nutrition in the arab gulf states were then identified to be included in the main review . however , for the purpose of this report , 40 papers were selected from these 120 papers to give general background on the food and nutrition status in these states.there has been a drastic change in food consumption patterns in the arab gulf countries . this change includes both quantitative and qualitative change in diet . the structure of diet has shifted towards a high - energy - density diet with more fat and added sugar in foods , more saturated fat ( mostly from animal origin ) and lower intake of complex carbohydrates , dietary fiber , fruit , and vegetables . for example , the total per capita energy intake exceeds 3000 kcal in all arab gulf countries , and the fat represents 2535% of total energy . however , animal fat represents 4052% of total per capita fat intake in the region .the intake of animal source foods is growing steady and it replaced many typical diets in the region . in general , poultry and eggs were more consumed compare to red meat , and fish less than that of poultry and eggs . data from fao food balance sheets indicated that the daily per capita availability of poultry meat during 20032005 ranged from 106163 grams in the arab gulf countries , compared to 273 grams in other countries in the middle east . there was a slight increase in fish intake during 19902005 ; however , the average daily per capita availability of fish was still low compared to red meat and poultry ( ranged from 24 to 52 grams ) . although the total production of milk and dairy products is increasing in the gulf region during the past decade , the per capita intake of these foods is still below the daily requirements . the daily per capita availability of milk and dairy products has decreased by 20% and 33% in kuwait and uae , respectively , and increased by 15% in saudi arabia , during 19902005 .the intake of fiber - rich foods is particularly important , as the relationship between dietary fiber and prevention of some chronic diseases is well established . since fiber is only available in foods of plant origin , the intake of fruit , vegetables , and complex carbohydrates is a good indicator for fiber intake . the consumption of fruit and vegetables by people in this region is below the recommended allowances . the who / emro reported that more than 85% of adults in the arab gulf countries consumed fruit and vegetables below 5 servings per day . additionally , the consumption of whole grain in this region is decreasing markedly , with more dependence on refined cereals . in saudi arabia , for example , it was found that the main contribution to fiber intake came from vegetable - based foods ( 31% ) , followed by cereal and their products ( 26% ) , and fruit and their products .food rich in salt is highly consumed in the region . data available from food composition of dishes commonly consumed in the arab gulf countries showed a high content of sodium in these dishes . the high use of table salt , spices , and pickles , in addition to the salinity of water are contributing factors for the high intake of sodium in these countries . among children and adolescents , the high consumption of fast foods and french fries is playing a great role in the increasing intake of sodium among these age groups .high intake of foods rich in added sugar , particularly among children and adolescents , has been reported by many studies in the region [ 1719 ] . potential health problems associated with overconsumption of food rich in free sugar such as overweight , dental caries , and potential enamel erosion are well documented . in saudi arabia , for example , carbonated beverages and canned fruit drinks represented 26% and 25% of daily fluid consumption by adolescents , respectively . step 4 ( determine the food and nutritional status)undernutrition , manifested by stunting , wasting , and underweight , is still a problem of concern among preschool children ( 15 years ) in the region . the prevalence of stunting in these children ranged from 18% to 22% whereas wasting ranged from 6% to 13% , and underweight ranged from 7% to 23% [ 8 , 22].despite the high per capita income in the arab gulf countries , anemia especially iron deficiency anemia , remains an important nutrition problem . according to who criteria for hemoglobin , the prevalence of anemia was 20%60% among preschool children , 1350% among school children and adolescents , and 23%54% in women of childbearing age . low intake of dietary iron as well as foods enhancing iron absorption is the most possible factor that contributes to iron deficiency anemia .vitamin a deficiency is less common compared to other micronutrient deficiencies . it was estimated that the prevalence of vitamin a deficiency among 072-month - old children ranged from 16% to 20% [ 22 , 25 ] . the prevalence of goiter in these countries ranged from 2% to 10% .although these countries have a sunny environment , vitamin d deficiency is one of the main public health problem . in qatar , for example , it was reported that 69% of children below 16 years had vitamin d deficiency . studies in saudi arabia revealed that 28% to 80% of adults had vitamin d deficiency . low sunlight exposure , low intake of dietary vitamin d and calcium , and short duration of breastfeeding during the first six months are the main risk factors for occurrence of this problem .the change in dietary habits , lifestyle , and life expectancy in the arab gulf countries has led to a remarkable change in disease trends . diet - related chronic diseases such as cardiovascular disease ( cvd ) , diabetes mellitus , hypertension , obesity , cancer , dental caries , and osteoporosis have become the main health problems . overweight and obesity have become epidemic among all age groups in the arab gulf countries , responsible for increased morbidity . it was estimated that 12%25% of school children aged 611 years were overweight and obese in this region . the proportion increased to 15%45% among adolescents ( 1118 years ) , and to 30% to 60% among adult men , and to 35% to 75% among adult women . inactivity , high consumption of high - energy - density foods , multipregnancy , and long duration of watching television or using the internet were reported as contributing factors for high prevalence of obesity [ 1 , 13 , 28].cardiovascular diseases ( cvd ) are the main cause of death in the region . health statistics revealed that 2830% of total deaths in the arab gulf countries were due to cvd . with changes in lifestyle , exposure to risk factors for cvd has become more prominent , such as diets high in saturated fat and low in dietary fiber , hypertension , diabetes , hyperlipidemia , smoking , and inactivity .the prevalence of diabetes among adults in the arab gulf countries is higher than that reported in western region . prediabetes and undiagnosed diabetes are particularly important since they can lead to chronic disease complication , if left untreated . in saudi arabia , for example , it was found that 28% of diabetes were unaware of their condition . age is one of the main factors associated with increasing prevalence of type 2 diabetes and impaired glucose tolerance among the gulf population . obesity , especially abdominal obesity , is the potent risk factors for the high proportion of type 2 diabetes . in general , diabetes is getting more attention by health authorities in the region , due to health and economic burden on government services .the high risk of diabetes and cvd associated with obesity in the arab gulf countries may lead to the metabolic syndrome [ 3436 ] . the prevalence of the metabolic syndrome ranged from 29.6% to 36.2% in men and 36% to 46% in women using international diabetes federation definition . overall , the prevalence of the metabolic syndrome in the arab gulf countries is 1015% higher than in most developed countries .data on the prevalence of diet - related cancer in the arab gulf are mostly hospital based . breast , bronchial , lung , stomach , colon , and liver cancers are the most common types of cancer which may be related to diet .osteoporosis is a growing public health problem in the arab gulf . studies in the region showed that a lower bone density was common , especially among women [ 3739 ] . the main risk factors are female sex , age , menopause , smoking and vitamin d and calcium deficiencies . the diseases may be toxic or infectious in nature and are caused by the ingestion of contaminated foods . these diseases can be grouped on the basis of causative agents : bacteria , fungi , viruses , parasites , and chemicals . the health consequence due to these diseases can be very severe and may lead to death , especially in individuals with low disease resistance , such as infants , young children , pregnant women , and the elderly . food - borne diseases in arab gulf countries are mainly caused by bacteria , specifically salmonellosis , hepatitis a , shigellosis , and poisoning with staphylococcus are the main constitutes of these diseases [ 41 , 42 ] . step 5 ( determine the lifestyle patterns that are associated with diet - related diseases)in addition to the changes in food consumption patterns , other changes in lifestyle , are also apparent and include smoking , a decrease in physical activity , an increase in alcohol consumption , and drug abuse . the increase in sedentary lifestyles is mainly due to the abundant use of cars , dependency on housemaids for home management , as well as due to spending a long time watching television and using the internet , particularly by children , teenagers , and young people [ 13 , 43 ] . the prevalence of adult physically activity for at least 150 min / week ( based on the international standard definition ) ranged from 30% to 42% for men and 26% to 29% for women . participation in physical activity in the gcc states was estimated to be considerably lower than those for many developed countries .an increase in smoking amongst both males and females was reported , with the prevalence of cigarette smoking ranging from 2050% among men , and 5% to 12% among women . the smoking of shisha ( water pipe ) has also increased sharply in the region and become more acceptable in the community . in addition , passive smoking complicated the problem , as many family members , especially women and children are regularly exposed to a smoking environment at home . it was estimated that 3040% of women in bahrain were exposed to a smoking environment in the home . step 6 ( formulating and testing the first draft of the fbdg)after collecting information on nutritional status and lifestyle , the first draft of the fbdg was formulated and reviewed by five nutrition experts in the region . the draft was also pretested on a small number of people in the arab gulf countries . several comments and suggestions were provided by the experts . the first draft of the arabic version was then modified and revised based on the comments received by both the experts and nutritionists . step 1 ( determine the purpose and goals for establishing fbdg)the arab gulf countries are facing great challenges to prevent and control several nutritional problems and diet - related chronic diseases . two types of nutritional and health problems occur : those associated with change in lifestyle , such as obesity , cardiovascular disease , diabetes , hypertension , cancer , dental caries , and osteoporosis and those associated with nutrient deficiencies , such as iron deficiency anemia , undernutrition among preschool children , and deficiencies of vitamin d and calcium . in addition , food - borne diseases are a problem of concern in these countries . therefore , the need for simple dietary guidelines to address the burden of these diseases is urgent , especially as some of these diseases contribute to more than 50% of total mortality in this region .there are no specific food - based dietary guidelines used by arab countries in general . nutritionists , dietitians and other health workers in the arabian gulf region have been relying on fbdg developed for other countries , such as usa , canada , and uk , to convey nutrition messages to the public.in oman , an attempt was made to establish guidelines to healthy eating . however , these guidelines lack some issues related to the prevention and control of diet - related chronic disease such as reduced intake of salt and added sugar in foods , weight maintenance , and avoiding alcoholic drinks and smoking . encouraging drinking of water and other liquids were given very little attention ; it was mentioned in conjunction with physical activity , without any further advices . in general , these guidelines were prepared for the people who are responsible to educate the public , but not directed to the public . in general our work is different than omani guidelines in two main issues : ( 1 ) the omani guidelines were prepared for the health workers and not for the public , and ( 2 ) the omani guidelines were focused in portion size of the food groups while the current guidelines focused on food - based advices.an attempt , therefore , has been made to develop fbdg specifically for arab gulf countries to provide simple and practical dietary guidelines to help prevent and control the main nutrition - related disorders and that take into account the socio - cultural , lifestyle , and dietary habits of the people in these countries . the arab gulf countries are facing great challenges to prevent and control several nutritional problems and diet - related chronic diseases . two types of nutritional and health problems occur : those associated with change in lifestyle , such as obesity , cardiovascular disease , diabetes , hypertension , cancer , dental caries , and osteoporosis and those associated with nutrient deficiencies , such as iron deficiency anemia , undernutrition among preschool children , and deficiencies of vitamin d and calcium . in addition , food - borne diseases are a problem of concern in these countries . therefore , the need for simple dietary guidelines to address the burden of these diseases is urgent , especially as some of these diseases contribute to more than 50% of total mortality in this region . there are no specific food - based dietary guidelines used by arab countries in general . nutritionists , dietitians and other health workers in the arabian gulf region have been relying on fbdg developed for other countries , such as usa , canada , and uk , to convey nutrition messages to the public . in oman however , these guidelines lack some issues related to the prevention and control of diet - related chronic disease such as reduced intake of salt and added sugar in foods , weight maintenance , and avoiding alcoholic drinks and smoking . encouraging drinking of water and other liquids were given very little attention ; it was mentioned in conjunction with physical activity , without any further advices . in general , these guidelines were prepared for the people who are responsible to educate the public , but not directed to the public . in general our work is different than omani guidelines in two main issues : ( 1 ) the omani guidelines were prepared for the health workers and not for the public , and ( 2 ) the omani guidelines were focused in portion size of the food groups while the current guidelines focused on food - based advices . an attempt , therefore , has been made to develop fbdg specifically for arab gulf countries to provide simple and practical dietary guidelines to help prevent and control the main nutrition - related disorders and that take into account the socio - cultural , lifestyle , and dietary habits of the people in these countries . step 2 ( characteristics of fbdg)to successfully promote healthy eating habits , a number of specific characteristics of fbdg were identified based on current knowledge and available information on food , nutrition , and health status in the countries of the region . these characteristics can be summarized as follows [ 2 , 6].the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries.they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods.they should consider the local dietary habits and food consumption patterns in the arab gulf countries.they should take into account food safety situation . they should consider the cultural and religious background of the population in arab gulf countries.they should consist of short , clear , and simple messages.they should promote a healthy lifestyle , especially the promotion of physical activity.they should be based on current scientific and health information . to successfully promote healthy eating habits , a number of specific characteristics of fbdg were identified based on current knowledge and available information on food , nutrition , and health status in the countries of the region . these characteristics can be summarized as follows [ 2 , 6].the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries.they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods.they should consider the local dietary habits and food consumption patterns in the arab gulf countries.they should take into account food safety situation . they should consider the cultural and religious background of the population in arab gulf countries.they should consist of short , clear , and simple messages.they should promote a healthy lifestyle , especially the promotion of physical activity.they should be based on current scientific and health information . the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries . they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods . they should consider the local dietary habits and food consumption patterns in the arab gulf countries . they should consider the cultural and religious background of the population in arab gulf countries . step 3 ( determine the food consumption patterns)a systematic literature review of the studies published in english between 1990 and 2010 was carried out , using pubmed and google scholar search to find out the food consumption patterns and nutrition situation in the arab gulf countries . the key words used were food consumption , nutrition , diet , anaemia , iodine deficiency disorders , cardiovascular disease , diabetes , vitamin d deficiency and undernutrition for each country . over 800 papers and reports about 120 papers that possibly addressed food and nutrition in the arab gulf states were then identified to be included in the main review . however , for the purpose of this report , 40 papers were selected from these 120 papers to give general background on the food and nutrition status in these states.there has been a drastic change in food consumption patterns in the arab gulf countries . the structure of diet has shifted towards a high - energy - density diet with more fat and added sugar in foods , more saturated fat ( mostly from animal origin ) and lower intake of complex carbohydrates , dietary fiber , fruit , and vegetables . for example , the total per capita energy intake exceeds 3000 kcal in all arab gulf countries , and the fat represents 2535% of total energy . however , animal fat represents 4052% of total per capita fat intake in the region .the intake of animal source foods is growing steady and it replaced many typical diets in the region . in general , poultry and eggs were more consumed compare to red meat , and fish less than that of poultry and eggs . data from fao food balance sheets indicated that the daily per capita availability of poultry meat during 20032005 ranged from 106163 grams in the arab gulf countries , compared to 273 grams in other countries in the middle east . there was a slight increase in fish intake during 19902005 ; however , the average daily per capita availability of fish was still low compared to red meat and poultry ( ranged from 24 to 52 grams ) . although the total production of milk and dairy products is increasing in the gulf region during the past decade , the per capita intake of these foods is still below the daily requirements . the daily per capita availability of milk and dairy products has decreased by 20% and 33% in kuwait and uae , respectively , and increased by 15% in saudi arabia , during 19902005 .the intake of fiber - rich foods is particularly important , as the relationship between dietary fiber and prevention of some chronic diseases is well established . since fiber is only available in foods of plant origin , the intake of fruit , vegetables , and complex carbohydrates is a good indicator for fiber intake . the consumption of fruit and vegetables by people in this region is below the recommended allowances . the who / emro reported that more than 85% of adults in the arab gulf countries consumed fruit and vegetables below 5 servings per day . additionally , the consumption of whole grain in this region is decreasing markedly , with more dependence on refined cereals . in saudi arabia , for example , it was found that the main contribution to fiber intake came from vegetable - based foods ( 31% ) , followed by cereal and their products ( 26% ) , and fruit and their products .food rich in salt is highly consumed in the region . data available from food composition of dishes commonly consumed in the arab gulf countries showed a high content of sodium in these dishes . the high use of table salt , spices , and pickles , in addition to the salinity of water are contributing factors for the high intake of sodium in these countries . among children and adolescents , the high consumption of fast foods and french fries is playing a great role in the increasing intake of sodium among these age groups .high intake of foods rich in added sugar , particularly among children and adolescents , has been reported by many studies in the region [ 1719 ] . potential health problems associated with overconsumption of food rich in free sugar such as overweight , dental caries , and potential enamel erosion are well documented . in saudi arabia , for example , carbonated beverages and canned fruit drinks represented 26% and 25% of daily fluid consumption by adolescents , respectively . a systematic literature review of the studies published in english between 1990 and 2010 was carried out , using pubmed and google scholar search to find out the food consumption patterns and nutrition situation in the arab gulf countries . the key words used were food consumption , nutrition , diet , anaemia , iodine deficiency disorders , cardiovascular disease , diabetes , vitamin d deficiency and undernutrition for each country . over 800 papers and reports were identified primarily . about 120 papers that possibly addressed food and nutrition in the arab gulf states were then identified to be included in the main review . however , for the purpose of this report , 40 papers were selected from these 120 papers to give general background on the food and nutrition status in these states . there has been a drastic change in food consumption patterns in the arab gulf countries . the structure of diet has shifted towards a high - energy - density diet with more fat and added sugar in foods , more saturated fat ( mostly from animal origin ) and lower intake of complex carbohydrates , dietary fiber , fruit , and vegetables . for example , the total per capita energy intake exceeds 3000 kcal in all arab gulf countries , and the fat represents 2535% of total energy . however , animal fat represents 4052% of total per capita fat intake in the region . the intake of animal source foods is growing steady and it replaced many typical diets in the region . in general , poultry and eggs were more consumed compare to red meat , and fish less than that of poultry and eggs . data from fao food balance sheets indicated that the daily per capita availability of poultry meat during 20032005 ranged from 106163 grams in the arab gulf countries , compared to 273 grams in other countries in the middle east . there was a slight increase in fish intake during 19902005 ; however , the average daily per capita availability of fish was still low compared to red meat and poultry ( ranged from 24 to 52 grams ) . although the total production of milk and dairy products is increasing in the gulf region during the past decade , the per capita intake of these foods is still below the daily requirements . the daily per capita availability of milk and dairy products has decreased by 20% and 33% in kuwait and uae , respectively , and increased by 15% in saudi arabia , during 19902005 . the intake of fiber - rich foods is particularly important , as the relationship between dietary fiber and prevention of some chronic diseases is well established . since fiber is only available in foods of plant origin , the intake of fruit , vegetables , and complex carbohydrates is a good indicator for fiber intake . the consumption of fruit and vegetables by people in this region is below the recommended allowances . the who / emro reported that more than 85% of adults in the arab gulf countries consumed fruit and vegetables below 5 servings per day . additionally , the consumption of whole grain in this region is decreasing markedly , with more dependence on refined cereals . in saudi arabia , for example , it was found that the main contribution to fiber intake came from vegetable - based foods ( 31% ) , followed by cereal and their products ( 26% ) , and fruit and their products . data available from food composition of dishes commonly consumed in the arab gulf countries showed a high content of sodium in these dishes . the high use of table salt , spices , and pickles , in addition to the salinity of water are contributing factors for the high intake of sodium in these countries . among children and adolescents , the high consumption of fast foods and french fries is playing a great role in the increasing intake of sodium among these age groups . high intake of foods rich in added sugar , particularly among children and adolescents , has been reported by many studies in the region [ 1719 ] . potential health problems associated with overconsumption of food rich in free sugar such as overweight , dental caries , and potential enamel erosion are well documented . in saudi arabia , for example , carbonated beverages and canned fruit drinks represented 26% and 25% of daily fluid consumption by adolescents , respectively . step 4 ( determine the food and nutritional status)undernutrition , manifested by stunting , wasting , and underweight , is still a problem of concern among preschool children ( 15 years ) in the region . the prevalence of stunting in these children ranged from 18% to 22% whereas wasting ranged from 6% to 13% , and underweight ranged from 7% to 23% [ 8 , 22].despite the high per capita income in the arab gulf countries , anemia especially iron deficiency anemia , remains an important nutrition problem . according to who criteria for hemoglobin , the prevalence of anemia was 20%60% among preschool children , 1350% among school children and adolescents , and 23%54% in women of childbearing age . low intake of dietary iron as well as foods enhancing iron absorption is the most possible factor that contributes to iron deficiency anemia .vitamin a deficiency is less common compared to other micronutrient deficiencies . it was estimated that the prevalence of vitamin a deficiency among 072-month - old children ranged from 16% to 20% [ 22 , 25 ] . the prevalence of goiter in these countries ranged from 2% to 10% .although these countries have a sunny environment , vitamin d deficiency is one of the main public health problem . in qatar , for example , it was reported that 69% of children below 16 years had vitamin d deficiency . studies in saudi arabia revealed that 28% to 80% of adults had vitamin d deficiency . low sunlight exposure , low intake of dietary vitamin d and calcium , and short duration of breastfeeding during the first six months are the main risk factors for occurrence of this problem .the change in dietary habits , lifestyle , and life expectancy in the arab gulf countries has led to a remarkable change in disease trends . diet - related chronic diseases such as cardiovascular disease ( cvd ) , diabetes mellitus , hypertension , obesity , cancer , dental caries , and osteoporosis have become the main health problems . overweight and obesity have become epidemic among all age groups in the arab gulf countries , responsible for increased morbidity . it was estimated that 12%25% of school children aged 611 years were overweight and obese in this region . the proportion increased to 15%45% among adolescents ( 1118 years ) , and to 30% to 60% among adult men , and to 35% to 75% among adult women . inactivity , high consumption of high - energy - density foods , multipregnancy , and long duration of watching television or using the internet were reported as contributing factors for high prevalence of obesity [ 1 , 13 , 28].cardiovascular diseases ( cvd ) are the main cause of death in the region . health statistics revealed that 2830% of total deaths in the arab gulf countries were due to cvd . with changes in lifestyle , exposure to risk factors for cvd has become more prominent , such as diets high in saturated fat and low in dietary fiber , hypertension , diabetes , hyperlipidemia , smoking , and inactivity .the prevalence of diabetes among adults in the arab gulf countries is higher than that reported in western region . prediabetes and undiagnosed diabetes are particularly important since they can lead to chronic disease complication , if left untreated . in saudi arabia , for example , it was found that 28% of diabetes were unaware of their condition . age is one of the main factors associated with increasing prevalence of type 2 diabetes and impaired glucose tolerance among the gulf population . obesity , especially abdominal obesity , is the potent risk factors for the high proportion of type 2 diabetes . in general , diabetes is getting more attention by health authorities in the region , due to health and economic burden on government services .the high risk of diabetes and cvd associated with obesity in the arab gulf countries may lead to the metabolic syndrome [ 3436 ] . the prevalence of the metabolic syndrome ranged from 29.6% to 36.2% in men and 36% to 46% in women using international diabetes federation definition . overall , the prevalence of the metabolic syndrome in the arab gulf countries is 1015% higher than in most developed countries .data on the prevalence of diet - related cancer in the arab gulf are mostly hospital based . breast , bronchial , lung , stomach , colon , and liver cancers are the most common types of cancer which may be related to diet .osteoporosis is a growing public health problem in the arab gulf . studies in the region showed that a lower bone density was common , especially among women [ 3739 ] . the main risk factors are female sex , age , menopause , smoking and vitamin d and calcium deficiencies . the diseases may be toxic or infectious in nature and are caused by the ingestion of contaminated foods . these diseases can be grouped on the basis of causative agents : bacteria , fungi , viruses , parasites , and chemicals . the health consequence due to these diseases can be very severe and may lead to death , especially in individuals with low disease resistance , such as infants , young children , pregnant women , and the elderly . food - borne diseases in arab gulf countries are mainly caused by bacteria , specifically salmonellosis , hepatitis a , shigellosis , and poisoning with staphylococcus are the main constitutes of these diseases [ 41 , 42 ] . undernutrition , manifested by stunting , wasting , and underweight , is still a problem of concern among preschool children ( 15 years ) in the region . the prevalence of stunting in these children ranged from 18% to 22% whereas wasting ranged from 6% to 13% , and underweight ranged from 7% to 23% [ 8 , 22 ] . despite the high per capita income in the arab gulf countries , anemia especially iron deficiency anemia , remains an important nutrition problem . according to who criteria for hemoglobin , the prevalence of anemia was 20%60% among preschool children , 1350% among school children and adolescents , and 23%54% in women of childbearing age . low intake of dietary iron as well as foods enhancing iron absorption is the most possible factor that contributes to iron deficiency anemia . it was estimated that the prevalence of vitamin a deficiency among 072-month - old children ranged from 16% to 20% [ 22 , 25 ] . they are mostly prevalent in the mountain area . the prevalence of goiter in these countries ranged from 2% to 10% . although these countries have a sunny environment , vitamin d deficiency is one of the main public health problem . in qatar , for example , it was reported that 69% of children below 16 years had vitamin d deficiency . studies in saudi arabia revealed that 28% to 80% of adults had vitamin d deficiency . low sunlight exposure , low intake of dietary vitamin d and calcium , and short duration of breastfeeding during the first six months are the main risk factors for occurrence of this problem . the change in dietary habits , lifestyle , and life expectancy in the arab gulf countries has led to a remarkable change in disease trends . diet - related chronic diseases such as cardiovascular disease ( cvd ) , diabetes mellitus , hypertension , obesity , cancer , dental caries , and osteoporosis have become the main health problems . overweight and obesity have become epidemic among all age groups in the arab gulf countries , responsible for increased morbidity . it was estimated that 12%25% of school children aged 611 years were overweight and obese in this region . the proportion increased to 15%45% among adolescents ( 1118 years ) , and to 30% to 60% among adult men , and to 35% to 75% among adult women . inactivity , high consumption of high - energy - density foods , multipregnancy , and long duration of watching television or using the internet were reported as contributing factors for high prevalence of obesity [ 1 , 13 , 28 ] . health statistics revealed that 2830% of total deaths in the arab gulf countries were due to cvd . with changes in lifestyle , exposure to risk factors for cvd has become more prominent , such as diets high in saturated fat and low in dietary fiber , hypertension , diabetes , hyperlipidemia , smoking , and inactivity . the prevalence of diabetes among adults in the arab gulf countries is higher than that reported in western region . prediabetes and undiagnosed diabetes are particularly important since they can lead to chronic disease complication , if left untreated . in saudi arabia , for example , it was found that 28% of diabetes were unaware of their condition . age is one of the main factors associated with increasing prevalence of type 2 diabetes and impaired glucose tolerance among the gulf population . obesity , especially abdominal obesity , is the potent risk factors for the high proportion of type 2 diabetes . in general , diabetes is getting more attention by health authorities in the region , due to health and economic burden on government services . the high risk of diabetes and cvd associated with obesity in the arab gulf countries may lead to the metabolic syndrome [ 3436 ] . the prevalence of the metabolic syndrome ranged from 29.6% to 36.2% in men and 36% to 46% in women using international diabetes federation definition . overall , the prevalence of the metabolic syndrome in the arab gulf countries is 1015% higher than in most developed countries . data on the prevalence of diet - related cancer in the arab gulf are mostly hospital based . it was estimated that about 1012% of total deaths were due to cancer . breast , bronchial , lung , stomach , colon , and liver cancers are the most common types of cancer which may be related to diet . studies in the region showed that a lower bone density was common , especially among women [ 3739 ] . the main risk factors are female sex , age , menopause , smoking and vitamin d and calcium deficiencies . the diseases may be toxic or infectious in nature and are caused by the ingestion of contaminated foods . these diseases can be grouped on the basis of causative agents : bacteria , fungi , viruses , parasites , and chemicals . the health consequence due to these diseases can be very severe and may lead to death , especially in individuals with low disease resistance , such as infants , young children , pregnant women , and the elderly . food - borne diseases in arab gulf countries are mainly caused by bacteria , specifically salmonellosis , hepatitis a , shigellosis , and poisoning with staphylococcus are the main constitutes of these diseases [ 41 , 42 ] . step 5 ( determine the lifestyle patterns that are associated with diet - related diseases)in addition to the changes in food consumption patterns , other changes in lifestyle , are also apparent and include smoking , a decrease in physical activity , an increase in alcohol consumption , and drug abuse . the increase in sedentary lifestyles is mainly due to the abundant use of cars , dependency on housemaids for home management , as well as due to spending a long time watching television and using the internet , particularly by children , teenagers , and young people [ 13 , 43 ] . the prevalence of adult physically activity for at least 150 min / week ( based on the international standard definition ) ranged from 30% to 42% for men and 26% to 29% for women . participation in physical activity in the gcc states was estimated to be considerably lower than those for many developed countries .an increase in smoking amongst both males and females was reported , with the prevalence of cigarette smoking ranging from 2050% among men , and 5% to 12% among women . the smoking of shisha ( water pipe ) has also increased sharply in the region and become more acceptable in the community . in addition , passive smoking complicated the problem , as many family members , especially women and children are regularly exposed to a smoking environment at home . it was estimated that 3040% of women in bahrain were exposed to a smoking environment in the home . in addition to the changes in food consumption patterns , other changes in lifestyle , are also apparent and include smoking , a decrease in physical activity , an increase in alcohol consumption , and drug abuse . the increase in sedentary lifestyles is mainly due to the abundant use of cars , dependency on housemaids for home management , as well as due to spending a long time watching television and using the internet , particularly by children , teenagers , and young people [ 13 , 43 ] . the prevalence of adult physically activity for at least 150 min / week ( based on the international standard definition ) ranged from 30% to 42% for men and 26% to 29% for women . participation in physical activity in the gcc states was estimated to be considerably lower than those for many developed countries . an increase in smoking amongst both males and females was reported , with the prevalence of cigarette smoking ranging from 2050% among men , and 5% to 12% among women . the smoking of shisha ( water pipe ) has also increased sharply in the region and become more acceptable in the community . in addition , passive smoking complicated the problem , as many family members , especially women and children are regularly exposed to a smoking environment at home . it was estimated that 3040% of women in bahrain were exposed to a smoking environment in the home . step 6 ( formulating and testing the first draft of the fbdg)after collecting information on nutritional status and lifestyle , the first draft of the fbdg was formulated and reviewed by five nutrition experts in the region . the draft was also pretested on a small number of people in the arab gulf countries . the first draft of the arabic version was then modified and revised based on the comments received by both the experts and nutritionists . after collecting information on nutritional status and lifestyle , the first draft of the fbdg was formulated and reviewed by five nutrition experts in the region . the draft was also pretested on a small number of people in the arab gulf countries . the first draft of the arabic version was then modified and revised based on the comments received by both the experts and nutritionists . the food - based dietary guidelines ( fbdg ) for arab gulf countries the human body needs more than 40 nutrients to maintain good health and prevent disease . however , there is no single food which contains all these nutrients in their required entirety and in the right quantity to maintain a healthy body . therefore , increasing the variety of foods consumed is important to ensure an adequacy to intake of these nutrients in our meals , breakfast , lunch , supper , and snacks . as each type of food can be rich in certain nutrients but poor in others , it is important to eat food from all five food groups , as listed in these guidelines , to ensure a varied diet ( table 1 ) . select one or more type of food and try to combine them in your favorite way in every meal . for example , for lunch , you could select rice from the cereal group to eat with fish ( meat group ) in addition to vegetables and salad ( vegetable group ) , along with a glass of fruit juice ( fruit group ) . fruit and vegetables must comprise a basic part of your daily diet to maintain a healthy body . their consumption can contribute to the prevention of several diet - related chronic diseases [ 4850 ] . studies indicate that the majority of the population of arab gulf countries does not consume sufficient quantities of fruit and vegetables , particularly children and teenagers , and this deprives them of essential nutrients . it is crucial to vary your daily intake of fruit and vegetables to obtain the maximum nutritional and health benefits of such foods . it is recommended to eat at least three servings of vegetables and two of fruit daily , as shown in ( table 1 ) . meat , fish , and poultry are the main source of dietary balanced protein and are rich in essential nutrients , especially iron , zinc , and other vitamins . communities in the arab gulf region frequently suffer from anemia , mainly iron deficiency anemia . take one to two servings of fish , skinless poultry , or lean meat daily ( as prescribed in the food groups ) . increased fish intake is especially important as scientific information showed that the consumption of fish or fish oil containing omega-3 polyunsaturated fatty acids reduces the risk of coronary heart disease . at the same time , red meat consumption should be restricted to not more than 0.5 kg per week , as high consumption of red meat may increase the risk of stomach and colon cancer . limit your intake of processed meat products that are rich in fat and salt such as sausages and mortadella , and eat low - fat processed meat . reduce the intake of liver , cerebrums , and kidneys due to their high cholesterol content . also , reduce the intake of salted fish and fish sauces such as mihiyawa ( mishawa ) and tareeh as they contain a great amount of salt . legumes are a rich source of protein , some vitamins , and minerals , and dietary fiber . legumes include , among others , beans , lentils , cowpeas , kidney beans , lupine , green peas , and soybeans . on the other hand , nuts such as walnuts , hazelnuts , almonds , and pine nuts , as well as seeds such as watermelon seeds , muskmelon , and sunflower are very rich sources of energy for their high fat content , which ranges between 30% to 60% . nuts and seeds are also important sources of certain vitamins , minerals , phytochemicals , and unsaturated fatty acids that are recommended for their protective role against heart disease . the intake of nuts and seeds is recommended , at least , once to twice a week , using the serving size prescribed in the food groups . reduce the intake of salty nuts and seeds for their high content of sodium and instead choose unsalted roasted nuts . nevertheless , it should be noted that overconsumption of nuts can contribute to an increase in calories . arab gulf communities rely on grain - based foods , mainly rice , as the main food in addition to wheat ( which is processed to produce bread , macaroni , pastries , cakes , and other products ) . it is important to consume adequate amounts of these foods particularly those made from whole grains unfortunately , many people do not consume an adequate amount of food that consists of complex carbohydrates such as whole - wheat products , although it is recommended that at least 55% of calories should come from carbohydrates . try to concentrate on foods based on whole grains especially bread , biscuits , and other bakery products . cereal - based foods such as cakes , biscuits , and pastries which can have high levels of added fats and sugars are not included in this recommendation and should be regarded as occasional treats only . active adolescents and youths need more carbohydrates to compensate for daily exerted energy than nonactive adolescents . foods from grains such as rice , oats and wheat , in addition to carbohydrates supply the body with some vitamins , minerals , and dietary fiber . additionally , these foods are low in fat except when fat is added during processing or cooking . whole - grains are different from peeled grains in that the latter lose some of their nutrients and fiber , a fact that reduces their nutritional value . for those who find the taste of whole grains unpleasant or where they are not available , this can be compensated for by eating other types of food like fruit and cooked or fresh vegetables to ensure an adequate intake of dietary fiber . it is recommended that brown or whole grain bread be an essential part of your daily intake , as it is available and affordable . also , at least half of your daily intake of grains must be whole grains as much as possible . this term refers to grains to which vitamins and minerals are added to flour to increase their nutritional value . hence eat grains fortified with iron , folic acid , calcium , and vitamin d whenever possible . there is considerable interest in the production of grains fortified with vitamins and minerals that compensate for any deficiency resulting from the elimination of these nutrients during processing . the most common nutrients used in fortification are folic acid , iron , and some of the b vitamins . the main fortified grain - based foods available in the region are cornflakes , bread , and some types of biscuit . read the label on the product to ensure that the foods have been fortified with these nutrients . milk and dairy products , which contain many essential nutrients , are important for the body and for its development . dairy products are the best source of calcium , which is vital to strengthen bones and for a healthy nervous system . in general , young female adolescents and elderly people are more susceptible to a deficiency in calcium and this is one of the leading causes of osteoporosis a disease that is currently widely prevalent among arab gulf communities [ 38 , 59 , 60 ] . the consumption of milk and dairy products is important for building a healthy body , particularly during childhood and adolescence . studies of dairy products , particularly yogurt and cheese along with milk , have revealed a positive link between the consumption of dairy products and bone density and the existence of high mineral content . dairy products should not be avoided on the grounds that they lead to becoming overweight . children between 2 to 8 years old should consume about 2 cups of low - fat milk or the equivalent each day while nine year olds and above are advised to consume 3 cups of low - fat milk or the equivalent each day . for those who are allergic to milk or who are intolerant to lactose , yogurt or cheese ( low fat ) are suitable substitute , for milk . many foods and traditional dishes consumed in the arab gulf region are known for their considerable high fat content ( 5%20% ) , and this does not mean to exclude these dishes , but to reduce the amount consumed . fat provides energy , essential fatty acids necessary for development and fat helps in the absorption of certain vitamins . dietary fats can be classified into two types , based on their relation to heart disease and their effect on raising cholesterol levels in the blood . animal fat , which may raise blood cholesterol , is mainly found in meat , chicken skin , cheese , whole - milk butter , and liver . however , remember that fats contain plenty of energy , so excessive intake of food rich in fat may play an important factor for prevalence of obesity in this region . try to get energy requirements from foods of plant origins such as grains , legumes , seeds , and nuts . if you eat foods which contain a high amount of animal fat at one meal , try to eat foods low in this type of fat in the second meal . also try to replace meat with fish whenever possible and affordable . sugar is considered a carbohydrate and a source of energy . in general , all carbohydrates ( except dietary fiber ) are converted to sugars . natural sugars are found in many foods such as milk , fruit , certain vegetables , bread , grains , and potatoes . frequent intake of foods rich in added sugar , especially sweets , chocolates , and sugary drinks ( such as soft drinks and canned juices ) can contribute to tooth decay , unless the teeth are cleaned immediately after eating such foods . evidence from many research studies suggests that the proportion of tooth decay increases with an increase in the intake of sugar per day [ 8 , 63 ] . in order to combat teeth decay , you must reduce the intake of food rich in sugar , regularly clean your teeth , and use toothpaste containing fluoride . overconsumption of food rich in sugar leads to an increase in energy intake , which contributes to an increase in your weight . it is found naturally in many foods and it is also added to many foods for flavoring and preservation . it has been found that arab gulf people consume more sodium than they need . many gulf traditional dishes , canned , and fast foods contain high amounts of salt . studies show that an overintake of sodium is associated with high blood pressure , strokes , and contributes to heart attacks , heart failure , and kidney failure [ 64 , 65 ] . it is recommended not to exceed 5 g sodium per day . to achieve low intake of salt , people should consume fresh foods , foods normally processed without salt , and add low salt or avoid addition of salt to food . in case of using salt , use iodized salt . among the substitutes for salt are acidic ingredients such as vinegar , lemon , line , spices , and herbs . it accounts for more than 60% of our bodies and plays important roles in digestion , absorption , and transportation of nutrients in the body , as well as for elimination of waste products and thermoregulation . the quantity of water consumed may differ from one person to another and there are no fixed recommendations in this regard . factors like body size , weather , physical activity , and individual differences have an effect on the body 's need for fluids . usually , thirst is the common indicator of this need , but this mechanism does not always work fully and in many times you may need the water before you get the sign of thirst . therefore , a general sound recommendation is drinking water and other fluids daily in appropriate amount , especially in the gulf region , which is known for hot weather and high humidity . however , intake of fluids containing added sugar should be avoided as much as possible . drinking water and other fluids is not the only way to get the required intake of fluids ; many of the food consumed contains a good amount of water . for instance , about 85% to 95% of most fruit and vegetable weight is water and meat contains 45% to 65% of water while cheese contains 2535% water . obesity has become an epidemic in the arab gulf countries during the last two decades . studies indicate that more than half of adults in these countries are overweight or obese . meanwhile , the rate of overweight and obesity has risen three - fold among children and adolescents during the same period . the more weight a person has the more the chances of developing hypertension , cardiovascular disease , hypercholesterolemia , and certain kinds of cancers , osteoarthritis and other respiratory problems . bmi is calculated as body weight in kilograms divided by height in meters , squared . people with bmi ranging between 18.5 and 24.9 are considered to be at healthy weight , a bmi between 25.0 and 29.9 to be overweight , and a bmi of 30.0 and over to be obese . the major health objective of adults therefore should be to maintain a weight that sustains a healthy life and prevent extra weight gain . the way to achieve a healthy body weight is to balance energy intake ( food and drinks ) with energy expenditure ( physical activity ) . excess body fat can be reduced by reducing calorie intake and increasing physical activity . as for children and adolescents , studies indicate that maintaining a healthy weight for children and adolescents helps to minimize the risk of obesity and its complications in adulthood [ 8 , 68 ] . studies in the arab gulf countries indicate a serious decline in physical activity especially among women and adults and that these communities tend to be sedentary . it was shown that inactivity is one of the main factors contributing to high prevalence of obesity in the region and that children and adolescents have become less active [ 13 , 43 ] . physical activity and maintenance of optimum weight are two important issues for good health and both can benefit your health in different ways . children , adolescents , adults , and elderly people can enjoy wellbeing and improve their health status if they start daily physical activity . light physical activity includes moving the body about while moderate - intense physical activity is any bodily movement that requires the exertion of much more energy like walking for at least 30 minutes . a target of 30 minutes for adults and 60 minutes for children of moderate physical activity most days of the week , preferably daily , is recommended . if you have been practicing physical activity for 30 minutes a day , you can further improve your health by increasing the time of practice . you can practice physical activity at any suitable time , no matter the type or speed of activity of your choice . it is possible to divide time into two periods of 15 minutes each , or to three periods of 10 minutes each . you might see people who prefer to engage in activity relevant to their daily routine , like mowing the lawn or using the stairs , while others prefer to engage in specific activity such as playing football , jogging , or walking . adults ( over 19 years ) who suffer from one of the following diseases or have special conditions related to these diseases are advised to seek a medical consultation to check their fitness before they engage in any vigorous activity : existence of chronic disease , for example , heart disease , hypertension , diabetes , osteoarthritis , and obesity . existence of risks leading to heart disease such as smoking , and a rise in blood cholesterol . indicators have revealed an increase in the proportion of smokers in the arab gulf countries particularly among adolescents . moreover , smoking shisha ( water pipe ) is widely practiced by a considerable proportion of the population at different ages and among different classes [ 45 , 74 ] . the arab gulf communities have begun to accept the smoking of shisha among women and adolescents believing wrongly that it is not as harmful as smoking cigarettes . it is well known that smoking is linked to many heart and respiratory system diseases , as well as to lung cancer , therefore not smoking is highly recommended . smoking shisha is actually worse than smoking cigarettes because the shisha 's tobacco contains greater quantities of nicotine and evidence shows that many of its toxicants are insoluble in the shisha 's bottled water . unfortunately , people who are exposed to a smoking environment are also exposed to the same health implications as the smokers themselves . studies have revealed that a passive smoker is not different from a smoker with regard to the risk of smoking - related diseases [ 7678 ] . valid advice is not only the cessation of smoking , but also to stay away from smoking environments where there are cigarettes and shisha or any other type of smoking . importation of alcoholic beverages the percentage of those who drink alcohol is on the rise , especially among teenagers and young people . in other countries , alcoholic beverages are forbidden , hence these countries witness less alcoholism , nevertheless , economic open - market policies and globalization have contributed to the rise in the number of alcohol drinkers in both environments . alcohol intake is associated with many diseases , mainly the risks of cirrhosis ( hepatitis ) , hypertension , and esophagus cancer and pancreas infection . in addition , alcoholism damages the heart and cerebrum , increases traffic accident rates , and negatively affects attentiveness at work [ 7982 ] . alcohol intake during pregnancy has an adverse impact on children 's development and behavior and can result in deformity and mental retardation . alcoholic beverages may contribute to family disagreements and problems such as divorce , violence , and children 's misbehavior . although some dietary institutions advice moderate intakes of alcohol and point out some of alcohol 's healthy pros , it is worth mentioning that the great majority of local gulf communities are muslim ( more than 98% ) and therefore drinking all types of alcohol is prohibited . recently , emerging food - borne illnesses have begun to constitute a serious concern for medical authorities in all the arab gulf countries . the symptoms of such illnesses range from mild to serious and include abdominal cramps , diarrhea , nausea , vomiting and dehydration , and other severe systems of dysfunction such as paralysis and meningitis . microbial food poisoning is one of the most common types of food - borne diseases that affect many people as a result of eating contaminated foods or foods containing poisons . symptoms include stomach ache , flatulence , and diarrhea which affect the health of susceptible groups , namely , children and elderly people . to avoid microbial illnesses , wash your hands before and after cooking or eating especially when dealing with raw meat . also , clean surfaces that are directly in contact with food , vegetables , and fruit and do not use these surfaces for chopping up , cutting , or slicing raw meat , chicken , and poultry . it is also important to separate fresh foods from cooked or ready - to - serve foods while shopping , preparing , or freezing . food should be cooked at a safe temperature , particularly fish , poultry , eggs , meat and its products . furthermore , it is highly recommended that perishable foods are instantly stored in the refrigerator or freezer and that people follow the proper method of thawing out frozen items before they are cooked . other important advice is to avoid nonpasteurized milk and raw eggs or dishes which contain these ingredients . the current fbdg are useful guides for the communities in the arab gulf countries to promote their healthy eating and lifestyle to reduce the incidence of nutrition - related diseases among them . however , such guidelines need to be revised periodically ( every 3 to 5 years ) with the change of scientific evidence and research regarding the risk factors for nutrition - related diseases . we hope that these guidelines will stimulate other investigators in other arab countries to establish their own ones .

the concept of food - based dietary guidelines ( fbdg ) has been promoted by several international organizations . however , there are no fbdg for the countries in the arab region . as the arab gulf countries share similar a socioeconomic and nutrition situation , an attempt was made to develop fbdg for these countries . this paper summarizes the steps taken to develope such guidelines by the arab center for nutrition . the fbdg were developed through 6 steps : ( 1 ) determination of the purpose and goals for establishing fbdg , ( 2 ) characteristics of fbdg , ( 3 ) determination of the food consumption patterns , ( 4 ) review the current nutrition situation , ( 5 ) determination of the lifestyle patterns that are associated with diet - related diseases and ( 6 ) formulating the fbdg . the fbdg consist of 14 simple and practical pieces of advice taking into consideration the sociocultural status and nutritional problems in the arab gulf countries . the fbdg can be a useful tool in educating the public in healthy eating and prevention of diet - related chronic diseases .

1. Introduction 2. Methods 3. Findings and Discussion 4. Conclusion

the arab gulf countries , namely , bahrain , kuwait , oman , qatar , saudi arabia , and united arab emirates have undergone a rapid change in their socio - economic situation , food consumption patterns , and lifestyle and health status during the past four decades . communicable diseases have almost diminished and diet - related chronic diseases have become the main health problems the concept of food - based dietary guidelines ( fbdg ) has been promoted by the food and agriculture organization ( fao ) and the world health organization ( who ) of the united nations since the 1992 international conference on nutrition in rome , italy . in 1997 , the first workshop on diet , nutrition and a healthy lifestyle in the arab gulf countries was held in manama , bahrain . two main recommendations were proposed : establishing food - based dietary guidelines ( fbdg ) for the arab gulf countries , and promoting a healthy lifestyle in these countries . a simplified arabic version of the fbdg for the arab gulf countries was published in a booklet to be used by the public . it was distributed to many health authorities in the region , and it is also available in the website of the arab center of nutrition for more dissemination . the main objectives of this paper , therefore , are to summarize the steps taken to develope the food - based dietary guidelines for the arab gulf countries , and to explore the scientific bases of these guidelines . developing food - based dietary guidelines ( fbdg ) for the arab gulf countries step 1 ( determine the purpose and goals for establishing fbdg)the arab gulf countries are facing great challenges to prevent and control several nutritional problems and diet - related chronic diseases . therefore , the need for simple dietary guidelines to address the burden of these diseases is urgent , especially as some of these diseases contribute to more than 50% of total mortality in this region .there are no specific food - based dietary guidelines used by arab countries in general . nutritionists , dietitians and other health workers in the arabian gulf region have been relying on fbdg developed for other countries , such as usa , canada , and uk , to convey nutrition messages to the public.in oman , an attempt was made to establish guidelines to healthy eating . however , these guidelines lack some issues related to the prevention and control of diet - related chronic disease such as reduced intake of salt and added sugar in foods , weight maintenance , and avoiding alcoholic drinks and smoking . in general our work is different than omani guidelines in two main issues : ( 1 ) the omani guidelines were prepared for the health workers and not for the public , and ( 2 ) the omani guidelines were focused in portion size of the food groups while the current guidelines focused on food - based advices.an attempt , therefore , has been made to develop fbdg specifically for arab gulf countries to provide simple and practical dietary guidelines to help prevent and control the main nutrition - related disorders and that take into account the socio - cultural , lifestyle , and dietary habits of the people in these countries . step 2 ( characteristics of fbdg)to successfully promote healthy eating habits , a number of specific characteristics of fbdg were identified based on current knowledge and available information on food , nutrition , and health status in the countries of the region . these characteristics can be summarized as follows [ 2 , 6].the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries.they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods.they should consider the local dietary habits and food consumption patterns in the arab gulf countries.they should take into account food safety situation . they should consider the cultural and religious background of the population in arab gulf countries.they should consist of short , clear , and simple messages.they should promote a healthy lifestyle , especially the promotion of physical activity.they should be based on current scientific and health information . step 3 ( determine the food consumption patterns)a systematic literature review of the studies published in english between 1990 and 2010 was carried out , using pubmed and google scholar search to find out the food consumption patterns and nutrition situation in the arab gulf countries . about 120 papers that possibly addressed food and nutrition in the arab gulf states were then identified to be included in the main review . however , for the purpose of this report , 40 papers were selected from these 120 papers to give general background on the food and nutrition status in these states.there has been a drastic change in food consumption patterns in the arab gulf countries . data from fao food balance sheets indicated that the daily per capita availability of poultry meat during 20032005 ranged from 106163 grams in the arab gulf countries , compared to 273 grams in other countries in the middle east . the daily per capita availability of milk and dairy products has decreased by 20% and 33% in kuwait and uae , respectively , and increased by 15% in saudi arabia , during 19902005 .the intake of fiber - rich foods is particularly important , as the relationship between dietary fiber and prevention of some chronic diseases is well established . the who / emro reported that more than 85% of adults in the arab gulf countries consumed fruit and vegetables below 5 servings per day . data available from food composition of dishes commonly consumed in the arab gulf countries showed a high content of sodium in these dishes . the prevalence of stunting in these children ranged from 18% to 22% whereas wasting ranged from 6% to 13% , and underweight ranged from 7% to 23% [ 8 , 22].despite the high per capita income in the arab gulf countries , anemia especially iron deficiency anemia , remains an important nutrition problem . low sunlight exposure , low intake of dietary vitamin d and calcium , and short duration of breastfeeding during the first six months are the main risk factors for occurrence of this problem .the change in dietary habits , lifestyle , and life expectancy in the arab gulf countries has led to a remarkable change in disease trends . diet - related chronic diseases such as cardiovascular disease ( cvd ) , diabetes mellitus , hypertension , obesity , cancer , dental caries , and osteoporosis have become the main health problems . overweight and obesity have become epidemic among all age groups in the arab gulf countries , responsible for increased morbidity . health statistics revealed that 2830% of total deaths in the arab gulf countries were due to cvd . with changes in lifestyle , exposure to risk factors for cvd has become more prominent , such as diets high in saturated fat and low in dietary fiber , hypertension , diabetes , hyperlipidemia , smoking , and inactivity .the prevalence of diabetes among adults in the arab gulf countries is higher than that reported in western region . in general , diabetes is getting more attention by health authorities in the region , due to health and economic burden on government services .the high risk of diabetes and cvd associated with obesity in the arab gulf countries may lead to the metabolic syndrome [ 3436 ] . overall , the prevalence of the metabolic syndrome in the arab gulf countries is 1015% higher than in most developed countries .data on the prevalence of diet - related cancer in the arab gulf are mostly hospital based . step 5 ( determine the lifestyle patterns that are associated with diet - related diseases)in addition to the changes in food consumption patterns , other changes in lifestyle , are also apparent and include smoking , a decrease in physical activity , an increase in alcohol consumption , and drug abuse . step 6 ( formulating and testing the first draft of the fbdg)after collecting information on nutritional status and lifestyle , the first draft of the fbdg was formulated and reviewed by five nutrition experts in the region . the draft was also pretested on a small number of people in the arab gulf countries . step 1 ( determine the purpose and goals for establishing fbdg)the arab gulf countries are facing great challenges to prevent and control several nutritional problems and diet - related chronic diseases . therefore , the need for simple dietary guidelines to address the burden of these diseases is urgent , especially as some of these diseases contribute to more than 50% of total mortality in this region .there are no specific food - based dietary guidelines used by arab countries in general . nutritionists , dietitians and other health workers in the arabian gulf region have been relying on fbdg developed for other countries , such as usa , canada , and uk , to convey nutrition messages to the public.in oman , an attempt was made to establish guidelines to healthy eating . however , these guidelines lack some issues related to the prevention and control of diet - related chronic disease such as reduced intake of salt and added sugar in foods , weight maintenance , and avoiding alcoholic drinks and smoking . in general our work is different than omani guidelines in two main issues : ( 1 ) the omani guidelines were prepared for the health workers and not for the public , and ( 2 ) the omani guidelines were focused in portion size of the food groups while the current guidelines focused on food - based advices.an attempt , therefore , has been made to develop fbdg specifically for arab gulf countries to provide simple and practical dietary guidelines to help prevent and control the main nutrition - related disorders and that take into account the socio - cultural , lifestyle , and dietary habits of the people in these countries . the arab gulf countries are facing great challenges to prevent and control several nutritional problems and diet - related chronic diseases . there are no specific food - based dietary guidelines used by arab countries in general . in oman however , these guidelines lack some issues related to the prevention and control of diet - related chronic disease such as reduced intake of salt and added sugar in foods , weight maintenance , and avoiding alcoholic drinks and smoking . in general our work is different than omani guidelines in two main issues : ( 1 ) the omani guidelines were prepared for the health workers and not for the public , and ( 2 ) the omani guidelines were focused in portion size of the food groups while the current guidelines focused on food - based advices . an attempt , therefore , has been made to develop fbdg specifically for arab gulf countries to provide simple and practical dietary guidelines to help prevent and control the main nutrition - related disorders and that take into account the socio - cultural , lifestyle , and dietary habits of the people in these countries . step 2 ( characteristics of fbdg)to successfully promote healthy eating habits , a number of specific characteristics of fbdg were identified based on current knowledge and available information on food , nutrition , and health status in the countries of the region . these characteristics can be summarized as follows [ 2 , 6].the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries.they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods.they should consider the local dietary habits and food consumption patterns in the arab gulf countries.they should take into account food safety situation . they should consider the cultural and religious background of the population in arab gulf countries.they should consist of short , clear , and simple messages.they should promote a healthy lifestyle , especially the promotion of physical activity.they should be based on current scientific and health information . to successfully promote healthy eating habits , a number of specific characteristics of fbdg were identified based on current knowledge and available information on food , nutrition , and health status in the countries of the region . these characteristics can be summarized as follows [ 2 , 6].the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries.they should be based on affordable and available foods , which are commonly consumed by the public , including traditional foods.they should consider the local dietary habits and food consumption patterns in the arab gulf countries.they should take into account food safety situation . they should consider the cultural and religious background of the population in arab gulf countries.they should consist of short , clear , and simple messages.they should promote a healthy lifestyle , especially the promotion of physical activity.they should be based on current scientific and health information . the guidelines should address the prevention and control of the main nutritional problems and diet - related diseases in the arab gulf countries . they should consider the local dietary habits and food consumption patterns in the arab gulf countries . step 3 ( determine the food consumption patterns)a systematic literature review of the studies published in english between 1990 and 2010 was carried out , using pubmed and google scholar search to find out the food consumption patterns and nutrition situation in the arab gulf countries . over 800 papers and reports about 120 papers that possibly addressed food and nutrition in the arab gulf states were then identified to be included in the main review . however , for the purpose of this report , 40 papers were selected from these 120 papers to give general background on the food and nutrition status in these states.there has been a drastic change in food consumption patterns in the arab gulf countries . data from fao food balance sheets indicated that the daily per capita availability of poultry meat during 20032005 ranged from 106163 grams in the arab gulf countries , compared to 273 grams in other countries in the middle east . the daily per capita availability of milk and dairy products has decreased by 20% and 33% in kuwait and uae , respectively , and increased by 15% in saudi arabia , during 19902005 .the intake of fiber - rich foods is particularly important , as the relationship between dietary fiber and prevention of some chronic diseases is well established . the who / emro reported that more than 85% of adults in the arab gulf countries consumed fruit and vegetables below 5 servings per day . data available from food composition of dishes commonly consumed in the arab gulf countries showed a high content of sodium in these dishes . a systematic literature review of the studies published in english between 1990 and 2010 was carried out , using pubmed and google scholar search to find out the food consumption patterns and nutrition situation in the arab gulf countries . about 120 papers that possibly addressed food and nutrition in the arab gulf states were then identified to be included in the main review . however , for the purpose of this report , 40 papers were selected from these 120 papers to give general background on the food and nutrition status in these states . there has been a drastic change in food consumption patterns in the arab gulf countries . data from fao food balance sheets indicated that the daily per capita availability of poultry meat during 20032005 ranged from 106163 grams in the arab gulf countries , compared to 273 grams in other countries in the middle east . the intake of fiber - rich foods is particularly important , as the relationship between dietary fiber and prevention of some chronic diseases is well established . the who / emro reported that more than 85% of adults in the arab gulf countries consumed fruit and vegetables below 5 servings per day . data available from food composition of dishes commonly consumed in the arab gulf countries showed a high content of sodium in these dishes . the prevalence of stunting in these children ranged from 18% to 22% whereas wasting ranged from 6% to 13% , and underweight ranged from 7% to 23% [ 8 , 22].despite the high per capita income in the arab gulf countries , anemia especially iron deficiency anemia , remains an important nutrition problem . low sunlight exposure , low intake of dietary vitamin d and calcium , and short duration of breastfeeding during the first six months are the main risk factors for occurrence of this problem .the change in dietary habits , lifestyle , and life expectancy in the arab gulf countries has led to a remarkable change in disease trends . diet - related chronic diseases such as cardiovascular disease ( cvd ) , diabetes mellitus , hypertension , obesity , cancer , dental caries , and osteoporosis have become the main health problems . health statistics revealed that 2830% of total deaths in the arab gulf countries were due to cvd . with changes in lifestyle , exposure to risk factors for cvd has become more prominent , such as diets high in saturated fat and low in dietary fiber , hypertension , diabetes , hyperlipidemia , smoking , and inactivity .the prevalence of diabetes among adults in the arab gulf countries is higher than that reported in western region . in general , diabetes is getting more attention by health authorities in the region , due to health and economic burden on government services .the high risk of diabetes and cvd associated with obesity in the arab gulf countries may lead to the metabolic syndrome [ 3436 ] . overall , the prevalence of the metabolic syndrome in the arab gulf countries is 1015% higher than in most developed countries .data on the prevalence of diet - related cancer in the arab gulf are mostly hospital based . despite the high per capita income in the arab gulf countries , anemia especially iron deficiency anemia , remains an important nutrition problem . the change in dietary habits , lifestyle , and life expectancy in the arab gulf countries has led to a remarkable change in disease trends . diet - related chronic diseases such as cardiovascular disease ( cvd ) , diabetes mellitus , hypertension , obesity , cancer , dental caries , and osteoporosis have become the main health problems . health statistics revealed that 2830% of total deaths in the arab gulf countries were due to cvd . the prevalence of diabetes among adults in the arab gulf countries is higher than that reported in western region . the high risk of diabetes and cvd associated with obesity in the arab gulf countries may lead to the metabolic syndrome [ 3436 ] . overall , the prevalence of the metabolic syndrome in the arab gulf countries is 1015% higher than in most developed countries . data on the prevalence of diet - related cancer in the arab gulf are mostly hospital based . step 5 ( determine the lifestyle patterns that are associated with diet - related diseases)in addition to the changes in food consumption patterns , other changes in lifestyle , are also apparent and include smoking , a decrease in physical activity , an increase in alcohol consumption , and drug abuse . in addition to the changes in food consumption patterns , other changes in lifestyle , are also apparent and include smoking , a decrease in physical activity , an increase in alcohol consumption , and drug abuse . step 6 ( formulating and testing the first draft of the fbdg)after collecting information on nutritional status and lifestyle , the first draft of the fbdg was formulated and reviewed by five nutrition experts in the region . the draft was also pretested on a small number of people in the arab gulf countries . after collecting information on nutritional status and lifestyle , the first draft of the fbdg was formulated and reviewed by five nutrition experts in the region . the draft was also pretested on a small number of people in the arab gulf countries . the food - based dietary guidelines ( fbdg ) for arab gulf countries the human body needs more than 40 nutrients to maintain good health and prevent disease . their consumption can contribute to the prevention of several diet - related chronic diseases [ 4850 ] . obesity has become an epidemic in the arab gulf countries during the last two decades . studies in the arab gulf countries indicate a serious decline in physical activity especially among women and adults and that these communities tend to be sedentary . indicators have revealed an increase in the proportion of smokers in the arab gulf countries particularly among adolescents . recently , emerging food - borne illnesses have begun to constitute a serious concern for medical authorities in all the arab gulf countries . the current fbdg are useful guides for the communities in the arab gulf countries to promote their healthy eating and lifestyle to reduce the incidence of nutrition - related diseases among them . however , such guidelines need to be revised periodically ( every 3 to 5 years ) with the change of scientific evidence and research regarding the risk factors for nutrition - related diseases .

cobalamin , c63h88o14n14pco ( figure 1 ) , participates in only two known mammalian enzymatic reactions . yet , these two cbl - dependent enzymes , cytosolic methionine synthase ( ms ) [ ec 2.1.1.13 ] , requiring methylcobalamin ( mecbl ) , and mitochondrial methylmalonyl - coa mutase ( mu ) [ ec 5.4.99.2 ] , requiring adenosylcobalamin ( adocbl ) [ 1 , 2 ] , are critically involved in key metabolic pathways essential for gene expression and regulation , via formation of s - adenosylmethionine ( sam ) and methylation , and in protein synthesis and catabolism , cellular respiration , and energy . activation of methionine synthase also ensures key antioxidant defense status , as it triggers concurrent activation of cystathionine -synthase ( cs ) , the pivotal enzyme at the homocysteine junction in the trans - sulfuration pathway to glutathione ( gsh ) . anaemia , and macrocytic or megaloblastic anaemia , as well as for subacute combined degeneration of the spinal cord . however , an increasing body of work suggests that cbl may also play a central role in the regulation of immunity and inflammation ( reviewed in ) . cbl confers significant protection in various animal models of shock , from anaphylaxis to trauma and sepsis [ 57 ] , and has remarkable organ / tissue protective effects when used clinically for the treatment of analogous inflammation in cn poisoning ( reviewed in ) . amongst cbl 's known immunological effects are an augmentation of the cd8+/cd4 + t - lymphocyte ratio and natural killer cell activity [ 9 , 10 ] , both significantly reduced in inflammatory pathology , with negative consequences in septic patients . interesting homeostatic links between cbl and pivotal cytokines are also emerging , indicative of complex but still incompletely defined regulatory circuits : mecbl lowers interleukin-6 ( il-6 ) expression in peripheral blood monocytes , whilst cbl deficiency raises circulating il-6 in humans and cbl physiological status regulates il-6 levels in rat cerebrospinal fluid . moreover , in both rodents and humans there appears to be an inverse relation between cbl physiological levels and tumour necrosis factor alpha ( tnf- ) serum levels . in vitro , a reasonable hypothesis is that such cbl / tnf-/il-6 regulation may be partly effected via cbl indirect regulation of the central immune regulatory transcription factor , nuclear factor kappa b ( nf-b ) . normal physiological levels of cbl in spinal fluid appear to correlate with nf-b quiescence , at least , in a non - inflammatory / non - immune challenge model . recently , a kinetic study reported that cob(ii)alamin reacts with superoxide at rates approaching superoxide dismutase . cncbl protects human aortic endothelial cells , and neuronal cells , in vitro , against superoxide induced injury [ 19 , 20 ] . given that oxidative stress is a major trigger of nf-b activation , this potential antioxidant effect of cbl could theoretically lead to nf-b inhibition . it may also be of critical local importance in vivo , as the phagocytic burst includes release of the cbl carrier , haptocorrin ( hc / tc 3 ) , in the immediate vicinity of nadph oxidase [ 8 , 21 ] one of the major biochemical sources of superoxide in immune challenge and inflammation . hc / tc3 , moreover , is upregulated by il-1 , itself expressed within fifteen minutes of inflammatory challenge . nevertheless , though antioxidant effects of cbls have been observed in vitro and may , indeed , be important in vivo , no systematic analysis of the in vivo mechanisms of cbl conferred protection against inflammation during acute immune challenge has hitherto been done . we wondered if a more comprehensive explanation for cbl effects on inflammation and immunity , and thence beneficial outcomes in sepsis and other forms of shock , may lie in a potential direct / indirect regulation by cbl of one or more of the several actions of nitric oxide ( no ) as a ubiquitous , cell - signal transduction molecule and second messenger for post - translational modification , whose targets include soluble guanylate cyclase .no is the product of three nitric oxide synthases ( nos ) : two constitutive , nnos ( neuronal nos ; nos i ) and enos ( endothelial nos ; nos iii ) , and one inducible , inos ( nos ii ) , at much higher levels of expression , with the potential to produce 1000-fold higher than normal amounts of no , during gestation , growth , and the immune response . cobalamins are known to have effects on no [ 2729 ] , but these have hitherto been thought to be a consequence of cbl / no scavenging effects [ 7 , 3035 ] demonstrable chemically and in vitro [ 36 , 37 ] , but biologically unproven in vivo and still controversial [ 3840 ] . nitrosylcobalamin has not been detected , to date , in vivo or in vitro , amongst naturally occurring intracellular cbls . nevertheless , if the hypothesis that cbl is involved in nos catalysis has any substance [ 42 , 43 ] , then it is conceivable that , in analogy to previously observed ferric - heme - no complex formation at the conclusion of nos catalysis , nocbl might be transiently formed , just prior to release of free no by the nos . such a theoretical transience and discrete localisation might account for the failure to detect nocbl in vivo to date . ubiquitous and continuous cbl scavenging of no , on the other hand , may pose biochemical hazards . for no has important antioxidant and cell - signalling actions which might be obstructed by hocbl 's previously proposed , indiscriminate no scavenging , or even just by a recently proposed , cbl structural - based , direct inhibition of the nos tout court and nothing else . there is some evidence that hocbl can discriminate between exogenous no donors and the natural endogenous donor , s - nitrosoglutathione , gsno , actually prolonging only gsno - induced , gastric fundus relaxations . moreover , there are also diverse indications that positive cbl status is allied to beneficial no activity : in diabetic rats , high cobalamin levels correlate with high nos protein levels , no activity , and increased erectile function ; cbl supplementation of vegetarians with low cbl status significantly increases enos no release in the brachial artery ; in the digestive tract of endotoxemic rats , the highest expression of inos is in the ileum , precisely where cbl is internalized , and both cbl and no are known to mediate cell protective effects via erk1/2 and akt [ 5054 ] . these protective effects of no and cbl include induction and regulation of heme oxygenase-1 ( ho-1 ) [ 52 , 5558 ] , which converts biliverdin to the powerful antioxidant bilirubin , and carbon monoxide . ( for a more comprehensive list of coincidences of cbl's / no 's positive actions , see table 1 and its related discussion in ) . thus , in these studies we explored an alternative hypothesis to that of cbl as just an no , or , indeed , superoxide , mop . we posited that the principal mechanism behind cbl 's beneficial , pleiotropic effects in inflammation may involve a biphasic regulation of nos expression and protein translation and the ensuing no synthesis , during the two distinct pro- and anti - inflammatory phases of the immune response . male c57bl/6 mice , weighing 20 to 25 g , were purchased from harlan , uk , and maintained on a standard chow pellet diet , containing standard amounts of cbl ( 50 g / kg vitamin b12/cncbl ) , with tap water supplied ad libitum . animals were kept in a 12:00 h light / dark cycle , and all were housed for 7 days prior to experimentation . all experiments were performed in accordance with uk home office regulations ( guidance on the operation of animals : scientific procedures act , 1986 ) . the coenzymes , 5-deoxyadenosylcobalamin , and methylcobalamin ; vitamin b12a , cyanocobalamin , and hydroxocobalamin ( cas 78091 - 12 - 0 ) were purchased from sigma - aldrich ( uk ) . glutathionylcobalamin and n - acetyl - cysteinyl - cobalamin were synthesized and supplied by professor nicola brasch ( kent state university , ohio , usa ) . all cbls ( and cbl - treated animal samples ) were protected from light during storage and handling , and were 98% to 99.5% pure . 5-deoxyadenosylcobalamin ( adocbl ) , methylcobalamin ( mecbl ) , hydroxocobalamin ( hocbl ) , glutathionylcobalamin ( gscbl ) , and n - acetyl - cysteinyl - cobalamin ( nac - cbl ) were all stored at 20c , and fresh solutions of them were made using sterile , pyrogen - free , phosphate - buffered saline ( pbs ; gibco ) , prior to the experiments . for the in vivo experiments , cobalamins were diluted at 10 ml / kg prior to treatments ( with pbs used as vehicle ) . raw 246.7 macrophage cells , stably transfected with nf-b luciferase reporter construct ( stratagene ) , were maintained in dulbecco 's modified eagle 's medium , supplemented with 10% ( v / v ) fetal bovine serum , 2 mm l - glutamine , 1 g / ml geneticin , and 50 g / ml g418 . cells ( 2 10 cells ) were seeded in 96-well plates and then preincubated for 1 h with increasing concentrations ( 110100 m ) of the five principally occurring , intracellular cbls . thereafter , at time 0 h , cells were stimulated with e. coli lps ( 0111 : b4 ; 1 g ) for 4 h and then processed for measurement of luciferase activity in a luminometer ( luminometer td-20/20 ; turner designs instruments ) . endotoxaemia was induced by the intraperitoneal injection of lps ( 0.1 mg / kg ) , alone ( non - lethal ) or , in the lethal endotoxaemia protocol , in combination with 1 g / kg d - galactosamine ( table 1 ) . sample collection in non - lethal endotoxaemia was carried out at both 4 and 24 h after lps challenge . animal survival , in all lethal endotoxaemia experiments , was monitored for a total of 5 days , and all data were analysed using chi - squared or kaplan - meier tests . times shown are in relation to time 0 h , when either lps alone or lps+d - gal was administered by intraperitoneal injection . individual cobalamins were injected into the peritoneum at the doses and times reported in table 1 . blood ( 500 l ) was centrifuged ( for 5 min , at 2500 rpm ) , and the plasma then collected for elisa analysis . 500 l of trizol reagent ( invitrogen ) was added to the remaining fraction . 20 l ) was treated with 2 u ( 1 l ) of turbo dnase 1 ( ambion , austin , tx ) , as described by the manufacturer , to remove any contaminating genomic dna . an aliquot of the dna - free rna ( 7.6 l ) was then transferred to a new rnase - free tube and reverse - transcribed into complementary dna ( cdna ) , using superscript iii reverse transcriptase ( invitrogen ) , as described by manufacturer . the following reagents were used : oligo dt primers ( invitrogen ) ; 1 l , 10 mm dntp ( bioline ) ; 4 l of 5x first - strand buffer ; 1 l , 0.1 m dtt ; 1 l ( 40 u ) rnaseout ; and 1 l ( 200 u ) of superscript iii reverse transcriptase ( invitrogen ) . after synthesis , cdna was quantified using a nanodrop nd-1000 and diluted ( 80 ng/l ) in molecular biology grade water and then loaded into 384-well plates for real - time pcr . real - time pcr assays were performed on the various samples in order to evaluate the expression of the following genes : gapdh , rpl32 , il-1 , cox-2 , inos , enos , tnf- , and hmgb1 ( table 2 ) . for each gene analyzed , reactions were performed using 1 l of the qiagen quantitect primer assay , added to 5 l power sybr green pcr master mix ( applied biosystems , warrington , uk ) and then diluted with 2 l molecular grade water . a final volume of 8 l was dispensed into each well and 2 l of diluted cdna ( 160 ng / reaction ) was added . each sample was tested in triplicate for each gene , and pcr reactions were performed using abi prism 7900 real - time pcr equipment . the thermal profile consisted of 95c for 15 min , then 40 cycles of 94c for 15 s , 55c for 30 s , and 72c for 30 s. this rest mcs software was utilized for the calculation of the relative difference between the test groups . liver tissues were harvested from ( n = 5 ) animals , after lps endotoxaemia , with or without hocbl treatment and then homogenized in lysis buffer , which contained a cocktail of protease inhibitors . protein concentrations prior to loading were determined using the bradford assay ( sigma ) : samples were mixed with 6x laemmli sample buffer , and equal protein amounts ( 100 g ) then underwent electrophoresis on a 10% polyacrylamide gel in running buffer ( 0.3% tris base , 1.44% glycine , and 0.1% sds in distilled water ) . this was followed by transfer of the proteins onto pvdf membranes in transfer buffer ( using 0.3% tris base , 1.44% glycine , and 20% methanol , in distilled water ) . membranes were blocked for 1 h with 5% nonfat milk solution in tbs containing 0.1% tween 20 . inos expression was assessed using a specific monoclonal antibody ( 1 : 1000 ; santa cruz , usa ) . the signal was amplified with hrp - linked anti - mouse secondary antibody ( 1 : 2000 ) and visualized by ecl ( western blotting detection reagent ; amersham biosciences , usa ) . densitometric analysis was performed using nih imagej software and normalised to tubulin loading controls in the same sample . animals ( n = 5 ) were challenged with lps and treated with cbls as described above . at 4 h and 24 h after lps challenge , lung and liver tissue samples were harvested , homogenized , and processed for determination of nos activity , as measured by nitrate / nitrite end - products of no . the ultrasensitive , nos assay used ( oxford biomedical research , oxford , mi , usa : ultrasensitive colorimetric nos assay : cat no . nb78 ) employs an nadph recycling system nadp , glucose-6-phosphate , glucose-6-phosphate dehydrogenase and the substrate , l - arginine , but not the cofactor , bh4,to ensure that nos operate linearly for up to 6 hours , as no - derived nitrate and nitrite accumulate . the assay kit can accurately measure as little as 1 pmol / millil ( ~1 millim ) no produced in aqueous solution . in these studies , the enzyme nitrate reductase was used to convert all nitrate to nitrite , then griess reagent employed to quantify nitrite levels , with the generation of a nitrite standard , as recommended by the supplier . after collection , blood was centrifuged and the plasma separated , under low lighting conditions , then stored at 80c until performance of the analyses . for determination of circulating tnf- and il-6 levels , using elisa assays , samples were diluted 1 : 10 in the assay diluent , as specified by the manufacturer ( r&d , uk ) . absorbance was plotted in a standard curve , and data expressed as the content of tnf- ( ng ) or il-6 ( pg ) per ml of plasma . unless otherwise stated , all reagents were purchased from sigma - aldrich , poole , uk . of 5 animals per group for the analyses in non - lethal endotoxaemia and , initially , 79 per group , then 12 per group , for lethal endotoxaemia survival , series i and ii , respectively . chi - square and kaplan - meier tests were used for the lethality studies . in all cases , a p < 0.05 was taken as significant . since cbl has been shown to prevent nf-b activation in a non - immune challenge model , and activation of nf-b leads to inos induction , we first looked at the effects of the five principally occurring , intracellular cbls , ( cncbl , hocbl , gscbl , and the two mammalian enzyme cofactors for mu and ms , respectively , adocbl and mecbl ) , on lps - induced nf-b activation in vitro , using a canonical reporter assay . although the various incoming forms of cbl are all reduced or dealkylated soon after cell entry , prior to ms / mu cofactor formation , the different incoming forms affect both the rate and ratio of formation of the two known active cofactors , adocbl / mecbl [ 6062 ] . theoretically , this variability in cbl cofactor formation may impact on the effects of cbls in immune challenge with respect to nf-b activation . raw 264.7 macrophage cells , stably transfected with nf-b luciferase reporter construct , were preincubated for 1 h with increasing concentrations ( 110100 m ) of cbls . upon lps stimulation , none of the five cbls significantly affected or inhibited lps - driven nf-b activation at 1 h , with no significant inhibitory effect on nf-b at a later time point ( 24 h ) . cncbl alone slightly stimulated nf-b activity , but only at the 1 h time point and when tested at the concentration of 1 m ( table 3 ) . in two separate experiments , each performed in triplicate , raw246.7 cells , stably transfected with nf-b luciferase reporter construct , were seeded in 96-well plates and preincubated for 1 h with increasing concentrations ( 110100 m ) of individual cbls , followed by stimulation with e. coli lps ( 1 g ) . at 1 h , and 24 h , following lps in the respective experiments , cells were processed for measurement of luciferase activity . basal values of fluorescence were 2.60 0.38 and 3.77 0.31 for 1 h and 24 h incubation , respectively . p < 0.05 versus lps alone . as there was no observable difference between the effects of alkyl and non - alkyl cbls on nf-b in vitro , we chose to focus these first investigations in vivo principally on hocbl , as a clinically licensed cbl form , known to be partially converted on cell entry to the two cbl cofactors , mecbl and adocbl , for ms and mcm , respectively . furthermore , at supraphysiological doses of 5 g i.v . , hocbl , as a clinical cyanide antidote , has shown remarkable protection against corollary inflammation ( analogous to the inflammation seen in sirs , sepsis , and septic shock ) , that goes beyond merely acting as a magnet for cn . we therefore next decided to see if the lethality survival protection also conferred by hocbl in a sepsis / endotoxaemia mouse model was reproducible in a different strain and in a more acute endotoxaemia model . some groups of animals were alternatively treated with the relatively novel , intracellular cbl , glutathionylcobalamin ( gscbl ) [ 61 , 64 ] whose clinical effects are untested in sepsis , or with n - acetyl - cysteinyl - cobalamin ( nac - cbl ) , a synthetic cobalamin , used as a non - endogenously occurring , thiol cbl comparison . to gain some information on potential clinical dosage , all cbls were tested in two distinct , high dosing regimes , with or without prophylactic pretreatment . in a severe sepsis protocol ( lps+d - gal ) , using c57bl/6 mice , we administered a relatively low dose of cbls ( 0.2 mg / kg i.p . ) , equivalent to a maximal concentration of approximately 1 m ( considering a total blood volume of 2.5 ml in the mouse , this concentration being well within the range tested in vitro ) . h prior to lps+d - gal and then given in repeated doses at + 1 , + 2 , + 6 , and + 22 h after lps+d - gal . alternatively , a high dose cbl protocol ( 40 mg / kg i.p . ) was administered only twice , at + 2 and + 22 h after lps+d - gal , to assess its potential as a rescue regimen . the urine of all cbl - treated animals was red , within 1 h of administration , an indicator of rapid , high , systemic cbl saturation ( data not shown ) . lps+d - gal mice rapidly reached 88.9% mortality by 8 h. this did not change further up to 24 h. animals treated with the relatively low - dose regimen of gscbl or nac - cbl , were protected in the early , 48 h time frame ( figure 2(a ) ) . during this period all cbl - treated animals also exhibited less huddling and pilo - erection ( data not shown ) . at 8 h after lps+d - gal , all relatively low - dose cbl treatments afforded 25% survival , p < 0.05 versus lps+d - gal alone ( figure 2(a ) ) . however , only low - dose hocbl treatment maintained this level of protection up to 24 h ( figure 2(a ) ) . ( indeed , as regards the long - term outcomes , 8 h seemed to be a watershed time point at which the outcome was determined for all groups . ) paradoxically , in view of its early protective effects at the lower dose , the high - dose gscbl regimen was less protective within the first 8 h. high - dose nac - cbl , which again provided some degree of protection in the first 6 h , was not significantly different from controls at 24 h. in contrast , high - dose gscbl and hocbl , despite the lesser protection of the former in the first hours , offered a consistent 28.60% survival up to 24 h , p < 0.01 versus lps+d - gal alone ( figure 2(b ) ) . later , at 72 h following lps+d - gal , in the gscbl high - dose group , mortality was equal to that observed in the nac - cbl high dose group , 85.72% , close to that of lps+d - gal control animals , though this increase in mortality was a late event : with 28.60% survival to 54 h in this group , perhaps indicative of the general cbl protective trend . nonetheless , the 25% and 28.60% , respectively , of mice that were alive at 24 h , in each of the two distinct , low- or high - dose , hocbl - treated groups , exhibited continued survival up to 72 h , /p < 0.05 versus lps+d - gal alone / p < 0.01 versus lps+d - gal alone ( figures 2(a ) and 2(b ) ) and beyond ( data not shown ) . ii . since these initial endotoxaemia studies might be considered underpowered , we repeated the lethality survival experiments using larger groups of mice ( n = 12 ) and focussing on hocbl alone , as having previously shown the most consistent protective effects . this time , given the trend towards improved survival seen at the higher hocbl dose , two distinct ultra - high doses of hocbl ( 40 mg / kg and 80 mg / kg ) were tested , with a more concise dose / time frame , + 2 h and + 4 h only for the 40 mg / kg , and , in the case of the 80 mg / kg dose , a single bolus administration at + 2 h. the significant survival advantage of hocbl treatment results demonstrated over 5 days , in figures 3(a ) and 3(b ) , not only shows that our hocbl data is consistently reproducible , but also that increasing the dose of hocbl significantly increases survival , from 25% up to 33.333% : p < 0.01 for hocbl ( 80 mg / kg ) by using the kaplan - meyer test . by comparison , at 24 h in the lps - only group , there was 90% mortality . to gain information about the mechanisms behind the consistent protection afforded by hocbl , and to observe any potential impact on the nos , then the expression of inflammatory mediator genes in liver and lung was analysed , in both the pro- and anti - inflammatory phases of the immune response , at the 4 h and 24 h time points . the early effects of hocbl treatment on enos mrna appeared organ dependent , with significant promotion of enos mrna in the lung and attenuation in the liver ( figures 4(a ) and 4(b ) ) . for enos , in lps - only animals we observed a decrease in the lung of 2.9 0.1 , whereas there was an increase of 2.1 0.1-fold change in lps+hocbl - treated animals ( figure 4(a ) ) . paradoxically , in the liver of lps - only treated animals , there was an increase of enos expression of up to ~15-fold compared to up to ~4-fold change only in lps+hocbl treated animals ( figure 4(b ) ) . liver and lung inos and cox-2 gene expression levels were increased in lps - only treated animals when compared to that of pbs - only injected mice , whose value was set as 1 . however , as for enos , the effects of hocbl on inos expression were once more organ selective , failing to inhibit the rise in inos mrna in the lung , but attenuating it in the liver ( figures 4(c ) and 4(d ) ) . strikingly , in spite of hocbl 's failure to completely inhibit inos expression , hocbl was a consistent inhibitor of cox-2 mrna in both liver and lung , bringing its degree of expression back to and below that of pbs - injected mice ( figures 4(e ) and 4(f ) ) . hocbl treatment also had a consistent regulatory effect on il-1 expression , which was moderately and significantly decreased in lung and completely inhibited in liver ( figures 4(g ) and 4(h ) ) . to determine efficiency of translation of the post - lps increased nos mrna , we assessed enos and inos protein expression by western blot in 4 h liver samples . as predicted by our hypothesis that sepsis may involve a failure in translation of the nos , this revealed that whilst in the lps - only challenged group there was a significant depression of enos protein translation , that was at odds with its high mrna expression , hocbl significantly promoted enos protein translation , above the levels of both pbs control and lps - only treatment groups ( figure 5(a ) ) . a similar paradoxical pattern was observed in hepatic inos protein translation , with significant depression of inos protein translation in the lps - only challenged group , and promotion of inos protein in the hocbl+lps treated group ( figure 5(b ) ) . we confirmed that these effects of hocbl on nos protein promotion were not random or artifactual , but were specific to cbl , by repeating the lps non - lethal endotoxaemia experiment using either gscbl or nac - cbl treatment and performing western blots for enos / inos protein . once again , we observed a significant early promotion of enos / inos protein by these other cbls , when compared to lps only ( figures 5(a ) and 5(b ) ) . however , when nos activity was measured ( using a nitrite production assay ) both in the early post - lps challenge , pro - inflammatory phase and in the late anti - inflammatory , resolution phase , a further paradoxical result emerged , suggesting that hocbl may exert some post - translational modification of nos activity . levels of nitrite at 4 h showed an inverse relation to levels of nos protein , with significantly higher levels of nitrite in the lps - only enos / inos - depressed group and significantly lower levels of nitrite being generated in the hocbl / enos / inos - promoted group ( figure 6(a ) ) . ( that this was a general , reproducible cbl effect was confirmed , as stated previously , by also doing the western blot with 4 h gscbl / nac - cbl - treated liver samples and also running the nos activity assay with both thiol - cbl treated samples . ) here we again observed a correlation between gscbl / nac - cbl promoted high nos protein in the western blots and decreased nitrite in the nos activity assay ( figure 7 ) . at 24 h following lps , levels of nos - derived nitrite , as measured in tissue samples , were even higher than at 4 h in both the lps - only and hocbl - treated groups . nevertheless , hocbl consistently showed relatively less nitrite production than lps only , in both lung and liver tissue samples ( figures 6(b ) and 6(c ) ) . since high inos expression / protein and no activity in the sepsis literature are associated with high tnf- , il-6 , and ensuing toxicity , we evaluated how hocbl might impact upon systemic levels of tnf- and il-6 triggered by lps . levels of il-6 protein were not significantly lower in the hocbl - treated group ( figure 8(a ) ) however , hocbl treatment significantly ( p < 0.05 ) attenuated the post - lps - induced increase in circulating plasma tnf- , as measured at the 4 h time point , ( ~50% reduction : figure 8(b ) ) . consistent with its effects on plasma tnf- , hocbl also showed some protection from the inhibitory effects of lps on tnf- mrna in the lung and significant attenuation of tnf- mrna in liver ( figures 8(c ) and 8(d ) ) . in the resolution phase of the immune response to lps , the effects of hocbl treatment on nos expression displayed a degree of organ selectivity , though most notable with respect to inos expression . whilst hocbl did not change lps - induced enos mrna inhibition in the lung , it significantly attenuated its inhibition in the liver , from 75- to 58 fold - change , respectively , for lps only and lps+hocbl ( figures 9(a ) and 9(b ) ) . hocbl effects on inos mrna in the lung were more distinctive , with ~80% inhibition compared to lps - only ( vehicle group ) . in the liver , hocbl treatment attenuated the lps - induced inhibition of inos mrna by ~40% ( figures 9(c ) and 9(d ) ) . the consistent hocbl tissue inhibition of cox-2 mrna , seen at the early pro - inflammatory phase time point of 4 h , persisted at 24 h , showing a significantly greater degree of inhibition than lps - only : 7- versus 2.5-fold for lps - only in the lung ; 115- versus 50-fold for lps - only in liver ( figures 9(e ) and 9(f ) ) . given the early regulatory impact of hocbl on nos / no activity , and cox-2 , il-1 , and tnf- , we expected to see related downstream beneficial effects on expression of the late ( 18 h ) effector of endotoxaemia , high mobility group box 1 ( hmgb1 ) . this prediction was confirmed . where lps - only presented an inconsistent picture , hocbl treatment consistently inhibited hmgb1 mrna : in the lung , from an increase of 2.5 in lps - only to a near threefold decrease ( setting levels of expression even lower than those observed in the control group , taken as a value of 1 ) ; and in the liver , to a more significant degree , even beyond the remarkable inhibitory effect of lps - only ( figures 9(g ) and 9(h ) ) . to conclude the 24 h gene expression analyses , tissue levels of il-1 and tnf- mrna were also quantified using rt - pcr . in resolution , hocbl treatment significantly increased inhibition of hepatic il-1 ( in line with its inhibitory effect at 4 h ) and inhibition of tnf- in the lung , whilst also , paradoxically , decreasing lps inhibition of tnf- in the liver ( table 4 ) . of note was the fact that the late effects of hocbl on tnf- mirrored the degree of late inos expression , in both lung and liver , as , indeed , did lps - only ( figures 9(c ) and 9(d ) and table 4 ) . our studies present a picture of complex and far - reaching homeostatic regulation of the activation , expression , and translation of nos , no synthesis , and inflammatory mediators by hocbl during the immune response . we propose that this regulation accounts for the noted survivals in rodent endotoxaemia , both in our more acute , septic shock models i and ii ( a modest but significant 25%/28.60% , and 25%/33.333% , survival ) and in a previous sub - acute , sepsis model ( performed with cncbl / hocbl the regulation we show here may also explain the observed organ / tissue - protective effects of hocbl in the clinical treatment of cn poisoning and the ensuing shock , which appear to go beyond what may be expected from the cbl binding of cn alone . furthermore , given the supraphysiological , saturating doses of cbl used in our studies , if cbl had been acting just as an no scavenger , or even as a nos inhibitor , it seems unlikely that it would have permitted the increasing rise in nos activity ( as indexed by nitrite ) , both early and late , or that its effects would be so subtle , complicated , and ultimately beneficial . the thiol cbls , on the other hand , present some mysteries , about which one can only give speculative answers at this point . the survival advantage exhibited in the early hours by comparatively low dose gscbl / nac - cbl treated animals , but not with hocbl treatment , may be due to the known higher reduction potential / lability of thiol cbls , resulting in more rapidly available intracellular cbl . cbl is likely to be , at least in part , functionally deficient in sepsis , as indexed by the down - regulation of the cbl receptors , megalin and cubilin , in the kidneys of endotoxaemic mice , the kidney being known , as a cbl homeostatic regulator , to reduce its cbl uptake in states of cbl deficiency . pertinently , megalin is down - regulated via the lps - induced erk1/2 signaling pathway , through which , as noted earlier , both cbl and no achieve their coincidentally regulatory , beneficial effects . although the beneficial supply of some extra gsh , as a consequence of thiol cbl lability , might be proposed as an alternative explanation for early survival protection , it is noteworthy that , at the higher dose , gscbl showed no early hours protection . moreover , in a new series of endotoxaemia lethality , in vivo survival studies that compared protective effects of all endogenous cbls against those of gscbl and nac - cbl , we observed that though gscbl / nac - cbl still consistently conferred the same early / pre-8-hour protection compared to the 4 other cbls , paradoxically , both thiol cbls repeatedly produced poor long - term survival outcomes , equivalent to or worse than lps - only , whereas hocbl / cncbl / mecbl / adocbl all consistently showed significant protection , with cncbl / mecbl in particular , producing even better survival results than the current hocbl - centred study ( brancaleone , dalli et al . gscbl is certainly known to produce a more rapid early increase in ms activity ( together with fourfold greater formation of adocbl ) , when compared to hocbl . such an increase in ms activity , normally rapidly deactivated by oxidative stress [ 6870 ] , would increase synthesis of the methyl donor , s - adenosylmethionine ( sam ) , which inhibits lps - induced gene expression by modulating histone methylation . whilst this may have initial short - term benefits , as observed , there are also negative long - term consequences from excessive inhibition of the necessary , pro - inflammatory gene expression at this stage . this may be why ms expression and activity are transiently decreased by 25% and 30% early on in the normal pro - inflammatory phase of the immune response to lps , as well as to allow for gsh synthesis modulation . the consequent , equally paradoxical failure of the other thiol cbl , nac - cbl , to significantly increase survival beyond 8 h , at both high- and low - dose protocols , may also be attributable to the fact that nac is particularly unstable as a cbl ligand and may therefore have acted independently of cbl as nac alone . further , though nac can ( 1 ) act as an antioxidant by increasing gsh levels ( 2 ) it can equally act as a pro - oxidant , increasing disulfides , gssg , and ( 3 ) is counter - indicated in sepsis , since , whilst nac enhances phagocytosis , it also suppresses the bactericidal respiratory burst in icu patients , with potential negative outcomes . indeed , consistent with this independent , paradoxical observation , additional data from our in vivo endotoxaemia model shows that in the early 4 h phase of the immune response nac - cbl , in contrast to hocbl / gscbl , significantly increases circulating pmn , specifically granulocytes , yet decreases the intensity of cd11b expression ( see supplementary data available online at http://dx.doi.org/10.1155/2013/741804 ( and researchgate ) ) . the adhesion molecule / complement receptor , cd11b , is a marker of neutrophil activation , and its high expression normally correlates with a strong respiratory burst . it is conceivable then that such nac - promoted suppressive effects ultimately outweighed the very early benefits in survival protection conferred by nac - cbl . the initial in vitro observation that all major endogenous cbls do not inhibit nf-b activation , even at 24 h , may appear surprising , particularly in view of the cbl beneficial outcomes in vivo . however , it has previously been shown in vivo that early inhibition of nf-b in immune challenge increases and prolongs inflammation , and that persisting late nf-b activation ( 24/48 h ) permits its resolution . moreover , this failure to inhibit nf-b by cbl during inflammation , with positive outcomes , has now also been observed by a group who were aware of our findings and , independently , in a cbl cancer model by marguerite et al . further , since activation of nf-b is linked to induction of inos , and since inadequately low levels of no and , indeed , inos gene knockout in mice and nos inhibitors in clinical trials [ 79 , 80 ] have been implicated in sepsis morbidity and mortality , we adopted the hypothesis that nos translation may , in contradiction to the common view , actually be depressed in sepsis and nos catalytic activity uncoupled or malfunctioning . previously observed high no in sepsis is believed to comprise a greater ratio of the more toxic no species , such as peroxynitrite , onoo- , which inhibits enos , as opposed to more antioxidant / cytotoxic no forms , such as s - nitrosothiols / gsno [ 84 , 85 ] or no itself ; although overhigh levels of gsno also have negative effects in sepsis - like inflammation . since cbl is known to promote gsh [ 3 , 8688 ] , whose synthesis is induced simultaneously with that of inos , it should theoretically alter the ratio of gsno / no to onoo- and related species , so that the more positive actions of no predominate ( figure 10 ) . therefore , we predicted that cbl would not inhibit inos expression and translation early on . this proved correct , with significant hocbl ( and gscbl / nac - cbl ) early promotion of both inos and enos proteins and significantly lower lps - only inos / enos protein . since enos is known to be depressed in sepsis , with adverse cardiovascular consequences , this early effect of cbl may have positive clinical implications . there is an apparent contradiction in the data showing relatively low inos / enos mrna leading to strikingly high protein translation in the lps+hocbl treated animals , in direct contrast to the lps - only group , where strikingly high inos / enos mrna yielded much lower levels of inos / enos protein . that this is not an artefact , but a specific cbl effect , also observed by others , is seen by the comparable inverse results achieved with the two , thiol cbls . ( these collective cbl results were also mirrored by decreasing nitrite / nos activity , in inverse proportion to the ascending levels of hocbl / gscbl / nac - cbl inos protein . ) we propose that this paradox may actually be an index of cbl / nos regulation in endotoxaemia and that the high mrna levels in lps - only animals may be due to the observed phenomenon of relaxed control of rna synthesis when sam / methyl groups are deficient , as in folate or cbl deficiency , thus consequential on the functional cbl deficiency of endotoxaemia / sepsis , and more permanent ms inactivation by lps , discussed earlier . furthermore , it is theoretically possible that abnormal cell function in sepsis may result in much of the mrna produced by the lps - only group being masked and unavailable for efficient translation . it is possible also that the translated protein may be unstable and degrade at a faster rate . this is certainly known to be the case with enos mrna in hypoxia and in the presence of high tnf- , both characteristic of sepsis . in contrast , given cbl 's impact on the two coenzymes , ms and mu , upon cbl treatment , the septic cell should afford a degree of metabolic normality and thus economic efficiency in transcription / translation . a remarkable study , over half a century ago , demonstrated that cbl is capable of reactivating a diversity of key enzymes after acute oxidant stress , most of them also negatively affected in sepsis , including glucose-6-phosphate - dehydrogenase , lactate dehydrogenase , lysine , ornithine , and glutamic decarboxylase . this last is critical for the supply of alpha - ketoglutarate in the krebs cycle , which is depressed in sepsis , with consequent lower atp production that is associated clinically with increased mortality . in turn , supply of alpha - ketoglutarate determines the availability of l - arginine , glutamate , and glutamine , all of which also decrease in sepsis , with adverse consequences , in the case of l - arginine especially for the nos . observed low levels of l - arginine in sepsis are associated with increased reactive nitrogen species , including high onoo- , and reactive oxygen species production during nos catalysis , interfering with the normal , more beneficial no cell signalling , necessary for the efficient resolution of the pro - inflammatory phase of the immune response . inos - derived no is known to have a direct regulatory correlation to levels of tnf- [ 100 , 101 ] . this inos / no / tnf- regulation seems operative here with hocbl treatment , not lps - only . since hocbl permitted a moderate rise in no ( at least , as measured by nos nitrite end - products ) , in tandem with moderate levels of tnf- mrna and protein , and since cbl status has an inverse relation to tnf- levels , it seems reasonable to conclude that such cbl / tnf- regulation occurs downstream of hocbl / nos / no regulation . additional evidence for this in our studies may be seen even in the resolution phase of the immune response , where hocbl - related levels of inos expression showed a direct correlation to those of tnf- , even to the degree of inhibition , in both lung and liver tissues ( figures 9(c ) and 9(d)/table 3 ) . importantly , however , in the early pro - inflammatory phase hocbl inos / no regulation does not completely inhibit tnf- : 50% reduction being observed in our experiments . this is a critical point as anti - tnf- mab treatment increases sepsis mortality in the clinic , since some degree of tnf- production and consequent early pro - inflammatory signalling is essential for an effective immune response . also noteworthy , and crucially consistent with such hocbl / inos / tnf- regulation , il-6 is essential for induction of acute phase proteins , whilst simultaneously also decreasing pro - inflammatory cytokines and increasing anti - inflammatory factors . il-6 regulation of pro - inflammatory factors includes regulation of tnf- and il-1 , expression of the latter also determining that of cox-2 , involved in arachidonic acid - derived prostaglandin and leukotriene synthesis . il-1 is rapidly expressed ( ~15 min post lps ) , whereas inos , which may be induced by il-1 , is not fully expressed until 6 h after lps . we have observed that treatment with high doses of endogenous cbls ( hocbl / gscbl ) promotes inos mrna expression as early as 2 h following lps ( unpublished data ) , possibly fast forwarding the immune response . this , together with the hocbl - promoted high nos protein , controlled rise in no synthesis , and thence a moderate production of tnf-/il-6 may form a feedback loop accounting for the tight hocbl regulation of il-1 , and consequently also cox-2 , as seen at 4 h after lps ( figure 10 scheme ) . cobalamin - promoted nos / no early regulation of tnf-/il-6/il-1/cox-2 seems also to be consistent with , and accounts for , the later inhibition of hmgb1 mrna . if expressed at > 18 h and then released extracellularly , hmgb1 can trigger further late release of tnf- , il-1 , and inflammatory products from cox-2 , inos , and excessive ros and rni species , leading to pathology . it is known that the nervous system can modulate circulating tnf- levels via release of acetylcholine by the vagus nerve . but our studies show that cbl essential for acetylcholine synthesis is the first known endogenous inhibitor of late hmgb1 mrna expression . both nicotine and ethyl - pyruvate have been used to block extracellular release of hmgb1 but , in addition to the fact that hocbl appears to impact on hmgb1 much further upstream , at least in tissues , neither drug is endowed with the safety profile of cbl and , more pertinently , neither is known to exert such a central , endogenous regulation of the immune response . but , on the evidence of the general anti - inflammatory regulation observed in our studies , we predict that extracellular release of hmgb1 , from macrophages and pmn , should be negligible with hocbl treatment . the theory that cbl may impact on the nos indirectly , through the contribution of its two known mammalian coenzymatic functions to nos substrate and cofactor assembly and , indeed , to assembly of the nos protein itself , may further explain our findings , including the cbl - promoted high nos protein ( figure 10 scheme ) . furthermore , a deficiency of any of the nos substrates and cofactors ( the likely result of cbl functional deficiency in endotoxaemia ) is known to result in less tightly coupled nos activity and increased free radical generation [ 112 , 113 ] , with a corollary increase in inflammatory mediators and prolonged period of nos activity , indexed by our observed higher lps - only nos nitrite levels . ( in a forthcoming study we will also analyse more exactly how cbl may shift the ratio of no / gsno / onoo and related species ) . it may also be that cbl , as adocbl and its radical , takes a direct , active part in nos catalysis , as a third mammalian cbl cofactor . from this perspective , the high nos protein seen with high cbl administration may be a classical instance of the cofactor promoting coenzyme assembly . such a central , direct cbl / nos , catalytic interaction would further reduce excess production of toxic forms of no , as well as superoxide and other related ros and rni species . the consequent , more precise , pro- and anti - inflammatory signalling should again result in a shorter , more effective period of nos activity , thus lower detectable nitrite levels ( as seen ) with the beneficial signalling and antioxidant effects of no predominant . however , direct cbl scavenger interactions , in discrete intracellular compartments , with primarily toxic rnis , such as onoo-/onooh / no2 , with which cbl interacts ex vivo , , may also play a part and can not be ruled out as contributing to a more complex picture behind our observed results ( figure 10 ) . these novel observations on the mechanism behind cobalamin protection in endotoxaemia suggest that we may be looking at the ideal natural , selective and collective regulator of the nos , and thence of cytokines and other pivotal factors , in immune challenge and sepsis . in fact , it is now accepted that anti - inflammatory therapies ( based on blocking a specific mediator ) fail toutcourt in sepsis and that a more modulatory approach , which regulates the homeostatic inflammatory response , ( in itself beneficial ) , could be successful . thus , our findings may have significant clinical implications , not only for the treatment of sepsis , but also for other analogous inflammation - driven conditions , such as cancer and malaria , where nos / no deregulation , and consequent loss of control over key inflammatory mediators , are equally pathogenic [ 115 , 116 ] .

background . nos/no inhibitors are potential therapeutics for sepsis , yet they increase clinical mortality . however , there has been no in vivo investigation of the ( in vitro ) no scavenger , cobalamin 's ( cbl ) endogenous effects on nos/no / inflammatory mediators during the immune response to sepsis . methods . we used quantitative polymerase chain reaction ( qpcr ) , elisa , western blot , and nos griess assays , in a c57bl/6 mouse , acute endotoxaemia model . results . during the immune response , pro - inflammatory phase , parenteral hydroxocobalamin ( hocbl ) treatment partially inhibits hepatic , but not lung , inos mrna and promotes lung enos mrna , but attenuates the lps hepatic rise in enos mrna , whilst paradoxically promoting high inos / enos protein translation , but relatively moderate no production . hocbl / nos/no regulation is reciprocally associated with lower 4 h expression of tnf- , il-1 , cox-2 , and lower circulating tnf- , but not il-6 . in resolution , 24 h after lps , hocbl completely abrogates a major late mediator of sepsis mortality , high mobility group box 1 ( hmgb1 ) mrna , inhibits inos mrna , and attenuates lps - induced hepatic inhibition of enos mrna , whilst showing increased , but still moderate , nos activity , relative to lps only . experiments ( lps+d - galactosamine ) hocbl afforded significant , dose - dependent protection in mice conclusions . hocbl produces a complex , time- and organ - dependent , selective regulation of nos/no during endotoxaemia , corollary regulation of downstream inflammatory mediators , and increased survival . this merits clinical evaluation .

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

we wondered if a more comprehensive explanation for cbl effects on inflammation and immunity , and thence beneficial outcomes in sepsis and other forms of shock , may lie in a potential direct / indirect regulation by cbl of one or more of the several actions of nitric oxide ( no ) as a ubiquitous , cell - signal transduction molecule and second messenger for post - translational modification , whose targets include soluble guanylate cyclase .no is the product of three nitric oxide synthases ( nos ) : two constitutive , nnos ( neuronal nos ; nos i ) and enos ( endothelial nos ; nos iii ) , and one inducible , inos ( nos ii ) , at much higher levels of expression , with the potential to produce 1000-fold higher than normal amounts of no , during gestation , growth , and the immune response . moreover , there are also diverse indications that positive cbl status is allied to beneficial no activity : in diabetic rats , high cobalamin levels correlate with high nos protein levels , no activity , and increased erectile function ; cbl supplementation of vegetarians with low cbl status significantly increases enos no release in the brachial artery ; in the digestive tract of endotoxemic rats , the highest expression of inos is in the ileum , precisely where cbl is internalized , and both cbl and no are known to mediate cell protective effects via erk1/2 and akt [ 5054 ] . we posited that the principal mechanism behind cbl 's beneficial , pleiotropic effects in inflammation may involve a biphasic regulation of nos expression and protein translation and the ensuing no synthesis , during the two distinct pro- and anti - inflammatory phases of the immune response . 5-deoxyadenosylcobalamin ( adocbl ) , methylcobalamin ( mecbl ) , hydroxocobalamin ( hocbl ) , glutathionylcobalamin ( gscbl ) , and n - acetyl - cysteinyl - cobalamin ( nac - cbl ) were all stored at 20c , and fresh solutions of them were made using sterile , pyrogen - free , phosphate - buffered saline ( pbs ; gibco ) , prior to the experiments . real - time pcr assays were performed on the various samples in order to evaluate the expression of the following genes : gapdh , rpl32 , il-1 , cox-2 , inos , enos , tnf- , and hmgb1 ( table 2 ) . at 4 h and 24 h after lps challenge , lung and liver tissue samples were harvested , homogenized , and processed for determination of nos activity , as measured by nitrate / nitrite end - products of no . since cbl has been shown to prevent nf-b activation in a non - immune challenge model , and activation of nf-b leads to inos induction , we first looked at the effects of the five principally occurring , intracellular cbls , ( cncbl , hocbl , gscbl , and the two mammalian enzyme cofactors for mu and ms , respectively , adocbl and mecbl ) , on lps - induced nf-b activation in vitro , using a canonical reporter assay . , hocbl , as a clinical cyanide antidote , has shown remarkable protection against corollary inflammation ( analogous to the inflammation seen in sirs , sepsis , and septic shock ) , that goes beyond merely acting as a magnet for cn . in a severe sepsis protocol ( lps+d - gal ) , using c57bl/6 mice , we administered a relatively low dose of cbls ( 0.2 mg / kg i.p . ) paradoxically , in view of its early protective effects at the lower dose , the high - dose gscbl regimen was less protective within the first 8 h. high - dose nac - cbl , which again provided some degree of protection in the first 6 h , was not significantly different from controls at 24 h. in contrast , high - dose gscbl and hocbl , despite the lesser protection of the former in the first hours , offered a consistent 28.60% survival up to 24 h , p < 0.01 versus lps+d - gal alone ( figure 2(b ) ) . nonetheless , the 25% and 28.60% , respectively , of mice that were alive at 24 h , in each of the two distinct , low- or high - dose , hocbl - treated groups , exhibited continued survival up to 72 h , /p < 0.05 versus lps+d - gal alone / p < 0.01 versus lps+d - gal alone ( figures 2(a ) and 2(b ) ) and beyond ( data not shown ) . this time , given the trend towards improved survival seen at the higher hocbl dose , two distinct ultra - high doses of hocbl ( 40 mg / kg and 80 mg / kg ) were tested , with a more concise dose / time frame , + 2 h and + 4 h only for the 40 mg / kg , and , in the case of the 80 mg / kg dose , a single bolus administration at + 2 h. the significant survival advantage of hocbl treatment results demonstrated over 5 days , in figures 3(a ) and 3(b ) , not only shows that our hocbl data is consistently reproducible , but also that increasing the dose of hocbl significantly increases survival , from 25% up to 33.333% : p < 0.01 for hocbl ( 80 mg / kg ) by using the kaplan - meyer test . to gain information about the mechanisms behind the consistent protection afforded by hocbl , and to observe any potential impact on the nos , then the expression of inflammatory mediator genes in liver and lung was analysed , in both the pro- and anti - inflammatory phases of the immune response , at the 4 h and 24 h time points . paradoxically , in the liver of lps - only treated animals , there was an increase of enos expression of up to ~15-fold compared to up to ~4-fold change only in lps+hocbl treated animals ( figure 4(b ) ) . however , as for enos , the effects of hocbl on inos expression were once more organ selective , failing to inhibit the rise in inos mrna in the lung , but attenuating it in the liver ( figures 4(c ) and 4(d ) ) . as predicted by our hypothesis that sepsis may involve a failure in translation of the nos , this revealed that whilst in the lps - only challenged group there was a significant depression of enos protein translation , that was at odds with its high mrna expression , hocbl significantly promoted enos protein translation , above the levels of both pbs control and lps - only treatment groups ( figure 5(a ) ) . a similar paradoxical pattern was observed in hepatic inos protein translation , with significant depression of inos protein translation in the lps - only challenged group , and promotion of inos protein in the hocbl+lps treated group ( figure 5(b ) ) . however , when nos activity was measured ( using a nitrite production assay ) both in the early post - lps challenge , pro - inflammatory phase and in the late anti - inflammatory , resolution phase , a further paradoxical result emerged , suggesting that hocbl may exert some post - translational modification of nos activity . levels of nitrite at 4 h showed an inverse relation to levels of nos protein , with significantly higher levels of nitrite in the lps - only enos / inos - depressed group and significantly lower levels of nitrite being generated in the hocbl / enos / inos - promoted group ( figure 6(a ) ) . at 24 h following lps , levels of nos - derived nitrite , as measured in tissue samples , were even higher than at 4 h in both the lps - only and hocbl - treated groups . since high inos expression / protein and no activity in the sepsis literature are associated with high tnf- , il-6 , and ensuing toxicity , we evaluated how hocbl might impact upon systemic levels of tnf- and il-6 triggered by lps . levels of il-6 protein were not significantly lower in the hocbl - treated group ( figure 8(a ) ) however , hocbl treatment significantly ( p < 0.05 ) attenuated the post - lps - induced increase in circulating plasma tnf- , as measured at the 4 h time point , ( ~50% reduction : figure 8(b ) ) . in the resolution phase of the immune response to lps , the effects of hocbl treatment on nos expression displayed a degree of organ selectivity , though most notable with respect to inos expression . whilst hocbl did not change lps - induced enos mrna inhibition in the lung , it significantly attenuated its inhibition in the liver , from 75- to 58 fold - change , respectively , for lps only and lps+hocbl ( figures 9(a ) and 9(b ) ) . in the liver , hocbl treatment attenuated the lps - induced inhibition of inos mrna by ~40% ( figures 9(c ) and 9(d ) ) . the consistent hocbl tissue inhibition of cox-2 mrna , seen at the early pro - inflammatory phase time point of 4 h , persisted at 24 h , showing a significantly greater degree of inhibition than lps - only : 7- versus 2.5-fold for lps - only in the lung ; 115- versus 50-fold for lps - only in liver ( figures 9(e ) and 9(f ) ) . given the early regulatory impact of hocbl on nos / no activity , and cox-2 , il-1 , and tnf- , we expected to see related downstream beneficial effects on expression of the late ( 18 h ) effector of endotoxaemia , high mobility group box 1 ( hmgb1 ) . in resolution , hocbl treatment significantly increased inhibition of hepatic il-1 ( in line with its inhibitory effect at 4 h ) and inhibition of tnf- in the lung , whilst also , paradoxically , decreasing lps inhibition of tnf- in the liver ( table 4 ) . our studies present a picture of complex and far - reaching homeostatic regulation of the activation , expression , and translation of nos , no synthesis , and inflammatory mediators by hocbl during the immune response . furthermore , given the supraphysiological , saturating doses of cbl used in our studies , if cbl had been acting just as an no scavenger , or even as a nos inhibitor , it seems unlikely that it would have permitted the increasing rise in nos activity ( as indexed by nitrite ) , both early and late , or that its effects would be so subtle , complicated , and ultimately beneficial . cbl is likely to be , at least in part , functionally deficient in sepsis , as indexed by the down - regulation of the cbl receptors , megalin and cubilin , in the kidneys of endotoxaemic mice , the kidney being known , as a cbl homeostatic regulator , to reduce its cbl uptake in states of cbl deficiency . moreover , in a new series of endotoxaemia lethality , in vivo survival studies that compared protective effects of all endogenous cbls against those of gscbl and nac - cbl , we observed that though gscbl / nac - cbl still consistently conferred the same early / pre-8-hour protection compared to the 4 other cbls , paradoxically , both thiol cbls repeatedly produced poor long - term survival outcomes , equivalent to or worse than lps - only , whereas hocbl / cncbl / mecbl / adocbl all consistently showed significant protection , with cncbl / mecbl in particular , producing even better survival results than the current hocbl - centred study ( brancaleone , dalli et al . such an increase in ms activity , normally rapidly deactivated by oxidative stress [ 6870 ] , would increase synthesis of the methyl donor , s - adenosylmethionine ( sam ) , which inhibits lps - induced gene expression by modulating histone methylation . whilst this may have initial short - term benefits , as observed , there are also negative long - term consequences from excessive inhibition of the necessary , pro - inflammatory gene expression at this stage . this may be why ms expression and activity are transiently decreased by 25% and 30% early on in the normal pro - inflammatory phase of the immune response to lps , as well as to allow for gsh synthesis modulation . indeed , consistent with this independent , paradoxical observation , additional data from our in vivo endotoxaemia model shows that in the early 4 h phase of the immune response nac - cbl , in contrast to hocbl / gscbl , significantly increases circulating pmn , specifically granulocytes , yet decreases the intensity of cd11b expression ( see supplementary data available online at http://dx.doi.org/10.1155/2013/741804 ( and researchgate ) ) . however , it has previously been shown in vivo that early inhibition of nf-b in immune challenge increases and prolongs inflammation , and that persisting late nf-b activation ( 24/48 h ) permits its resolution . further , since activation of nf-b is linked to induction of inos , and since inadequately low levels of no and , indeed , inos gene knockout in mice and nos inhibitors in clinical trials [ 79 , 80 ] have been implicated in sepsis morbidity and mortality , we adopted the hypothesis that nos translation may , in contradiction to the common view , actually be depressed in sepsis and nos catalytic activity uncoupled or malfunctioning . this proved correct , with significant hocbl ( and gscbl / nac - cbl ) early promotion of both inos and enos proteins and significantly lower lps - only inos / enos protein . there is an apparent contradiction in the data showing relatively low inos / enos mrna leading to strikingly high protein translation in the lps+hocbl treated animals , in direct contrast to the lps - only group , where strikingly high inos / enos mrna yielded much lower levels of inos / enos protein . ( these collective cbl results were also mirrored by decreasing nitrite / nos activity , in inverse proportion to the ascending levels of hocbl / gscbl / nac - cbl inos protein . ) observed low levels of l - arginine in sepsis are associated with increased reactive nitrogen species , including high onoo- , and reactive oxygen species production during nos catalysis , interfering with the normal , more beneficial no cell signalling , necessary for the efficient resolution of the pro - inflammatory phase of the immune response . since hocbl permitted a moderate rise in no ( at least , as measured by nos nitrite end - products ) , in tandem with moderate levels of tnf- mrna and protein , and since cbl status has an inverse relation to tnf- levels , it seems reasonable to conclude that such cbl / tnf- regulation occurs downstream of hocbl / nos / no regulation . additional evidence for this in our studies may be seen even in the resolution phase of the immune response , where hocbl - related levels of inos expression showed a direct correlation to those of tnf- , even to the degree of inhibition , in both lung and liver tissues ( figures 9(c ) and 9(d)/table 3 ) . importantly , however , in the early pro - inflammatory phase hocbl inos / no regulation does not completely inhibit tnf- : 50% reduction being observed in our experiments . this is a critical point as anti - tnf- mab treatment increases sepsis mortality in the clinic , since some degree of tnf- production and consequent early pro - inflammatory signalling is essential for an effective immune response . also noteworthy , and crucially consistent with such hocbl / inos / tnf- regulation , il-6 is essential for induction of acute phase proteins , whilst simultaneously also decreasing pro - inflammatory cytokines and increasing anti - inflammatory factors . il-6 regulation of pro - inflammatory factors includes regulation of tnf- and il-1 , expression of the latter also determining that of cox-2 , involved in arachidonic acid - derived prostaglandin and leukotriene synthesis . we have observed that treatment with high doses of endogenous cbls ( hocbl / gscbl ) promotes inos mrna expression as early as 2 h following lps ( unpublished data ) , possibly fast forwarding the immune response . this , together with the hocbl - promoted high nos protein , controlled rise in no synthesis , and thence a moderate production of tnf-/il-6 may form a feedback loop accounting for the tight hocbl regulation of il-1 , and consequently also cox-2 , as seen at 4 h after lps ( figure 10 scheme ) . if expressed at > 18 h and then released extracellularly , hmgb1 can trigger further late release of tnf- , il-1 , and inflammatory products from cox-2 , inos , and excessive ros and rni species , leading to pathology . both nicotine and ethyl - pyruvate have been used to block extracellular release of hmgb1 but , in addition to the fact that hocbl appears to impact on hmgb1 much further upstream , at least in tissues , neither drug is endowed with the safety profile of cbl and , more pertinently , neither is known to exert such a central , endogenous regulation of the immune response . furthermore , a deficiency of any of the nos substrates and cofactors ( the likely result of cbl functional deficiency in endotoxaemia ) is known to result in less tightly coupled nos activity and increased free radical generation [ 112 , 113 ] , with a corollary increase in inflammatory mediators and prolonged period of nos activity , indexed by our observed higher lps - only nos nitrite levels . the consequent , more precise , pro- and anti - inflammatory signalling should again result in a shorter , more effective period of nos activity , thus lower detectable nitrite levels ( as seen ) with the beneficial signalling and antioxidant effects of no predominant . these novel observations on the mechanism behind cobalamin protection in endotoxaemia suggest that we may be looking at the ideal natural , selective and collective regulator of the nos , and thence of cytokines and other pivotal factors , in immune challenge and sepsis . in fact , it is now accepted that anti - inflammatory therapies ( based on blocking a specific mediator ) fail toutcourt in sepsis and that a more modulatory approach , which regulates the homeostatic inflammatory response , ( in itself beneficial ) , could be successful . thus , our findings may have significant clinical implications , not only for the treatment of sepsis , but also for other analogous inflammation - driven conditions , such as cancer and malaria , where nos / no deregulation , and consequent loss of control over key inflammatory mediators , are equally pathogenic [ 115 , 116 ] .

during the last five decades , nonclassical thermoelasticity theories involving hyperbolic type heat transport equations admitting finite speed of thermal signals have been formulated . according to these theories , heat propagation is to be viewed as a wave phenomenon rather than a diffusion phenomenon . in order to overcome the paradox of an infinite speed of thermal wave inherent in cte and ccte ( classical coupled theory of thermoelasticity ) , efforts were made to modify coupled thermoelasticity , on different grounds , to obtain a wave - type heat conduction equation by different researchers . lord and shulman formulated the generalized thermoelasticity theory introducing one relaxation time in fourier 's law of heat conduction equation and thus transforming the heat conduction equation into a hyperbolic type . green and lindsay used the theory of two different relaxation times in the constitutive relations for the stress tensor and the entropy equation . later green and naghdi [ 35 ] have proposed three models , labeled as types i , ii , and iii . when they are linearized , type i is the same as the classical heat equation ( based on fourier 's law ) whereas the linearized versions of type - ii and type - iii theories permit propagation of thermal waves at finite speed . the entropy flux vector in type - ii ( i.e. , thermoelasticity without energy dissipation ) and type - iii ( i.e. , thermoelasticity with energy dissipation ) models is determined in terms of the potential that also determines stresses . when fourier conductivity is dominant , then the temperature equation reduces to classical fourier 's law of heat conduction and when the effect of conductivity is negligible , then the equation has undamped thermal wave solutions without energy dissipation . tzou introduced two - phase - lag models to both the heat flux vector and the temperature gradient . according to this model , classical fourier 's law q=-k has been replaced by q(p , t+q)=-k(p , t+t ) , where the temperature gradient at a point p of the material at time t + t corresponds to the heat flux vector q at the same point at time t + q . here the delay time t is interpreted as that caused by the microstructural interactions and is called the phase - lag of the temperature gradient . the other delay time q is interpreted as the relaxation time due to the fast transient effects of thermal inertia and is called the phase - lag of the heat flux . if q = and t = 0 , tzou refers to the model as single - phase - lag model . recently several researchers have attempted to solve their problems on the basis of the theory of dual - phase - lag model . roychoudhuri has studied one - dimensional thermoelastic wave propagation in an elastic half - space in the context of dual - phase - lag model . the exponential stability and condition of the delay parameters in the dual - phase - lag theory under this model have been studied by quintanilla . [ 10 , 11 ] have studied the well - posedness and solution structure of the dual - phase - lag heat conduction equation . wang and mingtian have studied the thermal oscillation and resonance in dual - phase - lag heat conduction equation . ailawalia and budhiraja have studied a problem of dual - phase - lag model with internal heat source . gurtin and williams [ 14 , 15 ] have suggested that there is no a priori ground for assuming that the second law of thermodynamics for continuous bodies involves only a single temperature ; that is , it is more logical to assume a second law in which the entropy contribution due to heat conduction is governed by one temperature , that of the heat supply by another . chen and gurtin and chen et al . [ 17 , 18 ] have formulated a theory of heat conduction in deformable bodies , which depends on two distinct temperatures : the conductive temperature and the thermodynamic temperature . for time - independent situations , the differences between these two temperatures are proportional to the heat supply , and in the absence of any heat supply , the two temperatures are identical . for time - dependent problems , however , and for wave propagation problems in particular , the two temperatures are , in general , different , independent of the presence of a heat supply . the key element that sets the two - temperature thermoelasticity ( 2tt ) apart from the classical theory of thermoelasticity ( cte ) is the material parameter a()0 , called the temperature discrepancy . specifically , if a = 0 , then = and the field equations of the 2tt reduce to those of cte . the linearized version of the two - temperature theory ( 2tt ) has been studied by many authors . warren and chen have investigated the wave propagation in the two - temperature theory of thermoelasticity . the existence , structural stability , and spatial behavior of the solution in 2tt have been discussed by quintanilla . it should be pointed out that both cte and 2tt suffer from the so - called paradox of heat conduction , that is , the prediction that a thermal disturbance at some point in a body is felt instantly , but unequally , throughout the body . [ 23 , 24 ] have studied problems on two - temperature green naghdi iii and dual - phase - lag model with variable thermal conductivity . , das and kanoria , and banik and kanoria have studied on two - temperature generalized thermoelasticity . [ 29 , 30 ] have established variational and reciprocal principles and some theorems in two - temperature generalized thermoelasticity . have solved a dynamic problem on green - naghdi ( type iii ) half - space with two - temperature theory . in this work , we have studied the thermoelastic stress , strain , displacement , conductive temperature , and the thermodynamic temperature in an infinite , isotropic , homogeneous elastic half - space under thermal shock using two - temperature dual - phase - lag generalized thermoelasticity in the context of fractional heat conduction equation . the governing equations of two - temperature generalized thermoelasticity theory are formed in the laplace transform domain which are then solved by state space approach . the inversion of the transformed solutions is carried out numerically , applying a method based on a fourier series expansion technique . a complete and comprehensive analysis of the results is presented and the effects of fractional order parameter , two - type temperature , and variable thermal conductivity are discussed . differential equations of fractional order have been the focus of many studies due to their frequent appearance in various applications in fluid mechanics , viscoelasticity , biology , physics , and engineering . the most important advantage of using fractional differential equations in these and other applications is their nonlocal property . it is well - known that the integer order differential operator is a local operator but the fractional order differential operator is nonlocal . this means that the next state of a system depends not only upon its current state , but also upon all of its historical states . this is more realistic , and this is one reason why fractional calculus has become more and more popular [ 3335 ] . one can state that the whole theory of fractional derivatives and integrals was established in the second half of the nineteenth century . the first application of fractional derivatives was given by abel who applied fractional calculus in the solution of an integral equation that arises in the formulation of the tautochrone problem . the generalization of the concept of derivative and integral to a noninteger order has been subjected to several approaches , and some various alternative definitions of fractional derivatives have appeared [ 3639 ] . in the last few years , fractional calculus has been applied successfully in various areas to modify many existing models of physical processes , for example , chemistry , biology , modeling and identification , electronics , wave propagation , and viscoelasticity . youssef introduced the formula of heat conduction given by ( 1)qi+0qit=ki1,i , 0<2 , where the notation i is the riemann - liouville fractional integral , introduced as a natural generalization of the well - known n - fold repeated integral if(t ) written in a convolution - type form as in which is written as follows : ( 2)inf(t)=1(n)0t(t)n1f()d , 0<n2,=f(t ) , n=0 , where (n ) is the gamma function . sur and kanoria have employed the theory to study problems on functionally graded and viscoelastic material . ezzat and el - karamany [ 44 , 45 ] established a new model of fractional heat conduction equation by using the new taylor series expansion of time - fractional order , developed by jumarie as ( 3)qi+0!qit=k,i , 0<1 . el - karamany and ezzat introduced two general models of fractional heat conduction law for a nonhomogeneous anisotropic elastic solid . uniqueness and reciprocal theorems are proved , and the convolutional variational principle is established and used to prove a uniqueness theorem with no restriction on the elasticity or thermal conductivity tensors except symmetry conditions . for fractional thermoelasticity not involving two - temperature , el - karamany and ezzat established the uniqueness , reciprocal theorems and convolution variational principle . for two - temperature theory the formula of heat conduction has been replaced by ( 4)qi+0qit=ki1,i , 0<2 , where is the conductive temperature . two - temperature fractional order thermoelasticity problem of ls and green naghdi models ( types ii and iii ) have been solved by sur and kanoria . several researchers have solved different problems [ 5054 ] using fractional order generalized thermoelasticity theory . more detailed discussion on the subject is available in the books of hetnarski and eslami , eslami et al . , and ignaczak and ostoja - starzewski . the constitutive equations are ( 5)ij=2eij+(e)ij , i , j=1,2,3 , where eij = ( 1/2)(ui , j + uj , i ) and e = ekk . in the context of two - temperature dual - phase - lag ( 2tdpl ) generalized thermoelasticity theory , the equation of motion in the absence of body forces and the heat conduction equation in absence of heat sources for a linearly isotropic generalized thermoelastic solid based on the theory of fractional integral are , respectively , given by ( 6)ui=(+)uj , ji+ui , jj,i , i , j=1,2,3,(7)[ki1(1+tt+t222t2),i],i = ( 1+qt+q222t2)(k+t0e ) , where is the density , and are lam 's constants , k is thermal conductivity , = ( 3 + 2)t , t being the coefficient of linear thermal expansion , t0 is the reference temperature , and = k/ce , ce being the specific heat at constant strain . the relation between conductive temperature ( ) and thermodynamic temperature ( ) is given by ( 8)=a,ii i=1,2,3 , where a(0 ) is the two - temperature parameter , called temperature discrepancy . we will consider the thermal conductivity as a linear function of thermodynamical temperature as follows : ( 9)k()=k0[1+k1 ] , where k0 is a constant which is equal to the thermal conductivity of the material when it does not depend on thermodynamical temperature ( ) and k1 is a nonpositive small parameter . substituting from ( 8) into ( 9 ) , we get ( 10)k()=k0[1+k1ak1,ii]or k()=k0[1+k1]ak0k1,iior k()=k()ak0k1,ii . we now use the following mapping : ( 11)~=1k00k()d,(12)~=1k00k()d. differentiating ( 11 ) with respect to xi , we get ( 13)k0~,i = k(),i . differentiating again the above equation , we obtain ( 14)k0~,ii=[k(),i],i . differentiating ( 12 ) with respect to xi , we get ( 15)k0~,i = k(),i . differentiating ( 12 ) with respect to t , we get ( 16)k0~=k(). substituting from ( 14 ) and ( 16 ) into ( 7 ) , we obtain ( 17)i1(1+tt+t222t2)~,ii = ( 1+qt+q222t2)(~+t0k0e ) + ak0k12[i1(1+tt+t222t2)(,i)2 ] . neglecting the last term on right hand side of the above equation due to nonlinearity , we get ( 18)i1(1+tt+t222t2)~,ii = ( 1+qt+q222t2)(~+t0k0e ) . using ( 15 ) in ( 6 ) , we get ( 19)ui=(+)uj , ji+ui , jjk0k()~,i . for linearity we can approximate last equation to the following form : ( 20)ui=(+)uj , ji+ui , jj~,i . now to transform ( 8) by using ( 11 ) and ( 12 ) , we first replace the dummy variable i with k and then differentiating with respect to xi and finally multiplying by k( ) we get ( 21)k(),ik(),i = ak(),kki , i , k=1,2,3 . now substituting from ( 10 ) into ( 21 ) we have ( 22)k(),ik(),i = ak(),kki+ak0k12[{(,i)2},ia{(,kk)2},i ] . for linearity we can approximate the last equation as ( 23)k(),ik(),i = ak(),kki . retaining only the linear terms , ( 14 ) can be written as ( 24)k0~,kki = k(),kki . now substituting from ( 13 ) , ( 15 ) , and ( 24 ) into ( 23 ) we have ( 25)~,i~,i = a~,kki , i , k=1,2,3 . now integrating with respect to xi we get ( 26)~~=a~,ii , i=1,2,3 . we consider a half space ( 0 x < ) with x - axis pointing to the medium . this half - space is subjected to thermal and mechanical loads on the bounding plane ( x = 0 ) that depends on the time t and is linearly quiescent . we will consider one - dimensional thermoelastic deformation of the body so that the displacement components can be taken in the following form : ( 27)(ux , uy , uz)=(u(x , t),0,0 ) . the strain displacement relation is ( 28)exx=ux and the constitutive relation ( 5 ) takes the form ( 29)xx=(+2)ux. the equation of motion , heat transport equation , and relation between conductive temperature and thermodynamic temperature can be written as ( 30)u=(+2)2ux2~x , i1(1+tt+t222t2)2~x2 = ( 1+qt+q222t2)(~+t0k0e),~~=a2~x2 . we now use the following nondimensional variables , to make the above equations nondimensional : ( 31)x=c0x , u=c0u , t=c02t,t=c02t , q=c02q , xx=xx+2,=t0 , ~=~t0 , =t0,~=~t0 , c02=+2. then the corresponding nondimensional equations , after omitting the primes , are ( 32)xx=ux2,u=2ux22~x , i1(1+tt+t222t2)2~x2 = ( 1+qt+q222t2)(~+1e),~~=2~x2 , where ( 33)1=k0 , 2=t0(+2 ) , =ac022 . initial and regularity conditions for the problem are given by ( 34)u===0 at t=0 for x0,ut=t=t=0 at t=0 for x0,u===0 as x. applying the laplace transform defined by ( 35)f(s)=0estf(t)dt , re(s)>0 to both the sides of ( 32 ) , we obtain ( 36)xx = e2,(37)d2edx2=s2e+2d2~dx2,(38)(1+ts+12t2s2)d2~dx2 = s(1+qs+12q2s2)(~+1e),(39)~~=d2~dx2 . eliminating ~- from ( 37)(39 ) we get ( 40)d2~dx2=1~+11e,d2edx2=2~+3e , where ( 41)a=(1+qs+(1/2)q2s21+ts+(1/2)t2s2),1=as1+as , 2=12(11)[1+112],3=s2+112(11)[1+112 ] . the equations ( 40 ) can be written in the form of a vector matrix differential equations as follows : ( 42)d2v(x , s)dx2=a(s)v(x , s ) , where ( 43)v(x , s)=[~(x , s)e(x , s ) ] , a(s)=[11123 ] . the formal solution of system ( 42 ) bounded at infinity can be written as ( 44)v(x , s)=exp[a(s)x]v(0,s ) , where ( 45)v(0,s)=[~(0,s)e(0,s)]=[~0e0],0=(0,t ) , e0=e(0,t ) . we will use the well - known cayley - hamilton theorem to find the form of the matrix ( 46)exp[a(s)x ] . the characteristic equation for the matrix a(s ) can be written as ( 47)k2k(1+3)+(13112)=0 . the roots of this equation , namely , k1 and k2 , satisfy the following relations : ( 48)k1+k2=1+3,k1k2=13112 . the taylor series expansion for the matrix exponential in ( 44 ) is given by ( 49)exp[a(s)x]=n=0[a(s)x]nn!. using cayley - hamilton theorem , we can express a and higher powers of the matrix a in terms of i and a , where i is the unit matrix of the second order . thus the infinite series in ( 49 ) can be reduced to the form ( 50)exp[a(s)x]=a0(x , s)i+a1(x , s)a(s ) , where a0 and a1 are coefficients depending on s and x. by cayley - hamilton theorem , the characteristic roots k1 and k2 of the matrix a must satisfy ( 50 ) ; thus we have ( 51)exp(k1x)=a0+a1k1,exp(k2x)=a0+a1k2 . by solving the above linear system of equations , hence from ( 50 ) we get ( 53)exp[a(s)x]=lij(x , s ) , i , j=1,2 , where ( 54)l11=ek2x(k11)ek1x(k21)k1k2,l12=11(ek1xek2x)k1k2,l21=2(ek1xek2x)k1k2,l22=ek1x(3k2)ek2x(3k1)k1k2 . using ( 53 ) we can write the solution in ( 44 ) in the following form : ( 55)v(x , s)=lij(x , s)v(0,s ) . hence the solution for ~-(x , s ) and e-(x , s ) can be obtained from ( 55 ) as follows : ( 56)~(x , s)=1k1k2[ek1x{11e0(k21)~0}mmmmmm ek2x{11e0(k11)~0}],(57)e(x , s)=1k1k2[ek1x{2~0(k23)e0}mmmmmm ek2x{2~0(k13)e0 } ] . using ( 56 ) , the solution for ~- can be obtained from ( 39 ) as follows : ( 58)~=1k1k2[ek1x{11e0(k21)~0}(1k1)mmmmmmek2x{11e0(k11)~0}(1k2 ) ] . we will consider the bounding plane of the medium at x = 0 subjected to thermal shock in the following nondimensional form : ( 59)(0,t)=1h(t ) , where 1 is constant . now applying the laplace transform to ( 59 ) we get ( 60)(0,s)=1s . using ( 9 ) , ( 11 ) , and ( 60 ) we get ( 61)~(0,s)=ls , where l = 1 + ( k1/2s)1 . the mechanical boundary condition is taken in the form ( 62)e(0,t)=0 . this implies ( 63)e(0,s)=e0=0 . applying the boundary conditions ( 61 ) and ( 63 ) to ( 56)(58 ) displacement component u- can be obtained from ( 28 ) using ( 65 ) in the following form : ( 67)u=l2s(k1k2)[ek1xk1ek2xk2 ] . using ( 9 ) , ( 11 ) , and ( 64 ) the solution for - can be obtained as follows : ( 68)=1k1[(1k1)ek2x]2k1ls(k1k2))1/2(1 + 2k1ls(k1k2)mmmnmm[(1k2)ek1xmmmmmmm(1k1)ek2x]2k1ls(k1k2))1/21 ] , k1<0=ls(k1k2)[(1k2)ek1xmmmmmmmm(1k1)ek2x ] , k1=0 . again using ( 9 ) , ( 12 ) , and ( 66 ) the solution for - can be obtained in the following form : ( 69)=ls(k1k2)[(1k2)(1k1)ek1xmmmmmmmm(1k1)(1k2)ek2x]=~0(say ) , k1=0=1k1[1 + 2k1~01 ] , k1<0 . the solution for -xx can be obtained from ( 36 ) using ( 65 ) and ( 69 ) in the following form : ( 70)xx = l2s(k1k2)[ek1xek2x]xx 2k1[1 + 2k1~01 ] , k1<0;xx = l2s(k1k2)[ek1xek2x]2~0,mmmmmmmmmmmmmmnmmmk1=0 . it is difficult to find the analytical inverse of laplace transform of the complicated solutions for the displacement , thermodynamic temperature , conductive temperature , stress , and strain in laplace transform domain . let f-(x , s ) be the laplace transform of a function f(x , t ) . then the inversion formula for laplace transform can be written as ( 71)f(x , t)=12idid+iestf(x , s)ds , where d is an arbitrary real number greater than real parts of all the singularities of f-(x , s ) . taking s = d + iw , the preceding integral takes the form ( 72)f(x , t)=edt2eitwf(x , d+iw)dw . expanding the function h(x , t ) = ef(x , t ) in a fourier series in the interval [ 0,2 t ] we obtain the approximate formula ( 73)f(x , t)=f(x , t)+ed , where ( 74)f(x , t)=12c0+k=1ck for 0t2t , ck = edtt[eikt / t f(x , d+iktt ) ] . the discretization error ed can be made arbitrary small by choosing d large enough . since the infinite series in ( 74 ) can be summed up to a finite number n of terms , the approximate value of f(x , t ) becomes ( 75)fn(x , t)=12c0+k=1nck for 0t2 t . using the preceding formula to evaluate f(x , t ) we introduce a truncation error et that must be added to the discretization error to produce total approximation error . the korrektur method uses the following formula to evaluate the function f(x , t ) : ( 76)f(x , t)=f(x , t)e2dtf(x,2t+t)+ed , where the discretization error |ed | |ed| . thus , the approximate value of f(x , t ) becomes ( 77)fnk(x , t)=fn(x , t)e2dtfn(x,2t+t ) , where n is an integer such that n < n. we will now describe the -algorithm that is used to accelerate the convergence of the series in ( 75 ) . let n = 2q + 1 , where q is a natural number , and let sm = k=1ck be the sequence of partial sum of series in ( 75 ) . we define the -sequence by ( 78)0,m=0 , 1,m = sm,p+1,m=p1,m+1 + 1p , m+1p , m , p=1,2,3, . it can be shown that the sequence ( 79)1,1,3,1,5,1, ,n,1 converges to f(x , t ) + ed ( c0/2 ) faster than the sequence of partial sums sm , m = 1,2 , 3, . the actual procedure used to invert to get the solution for strain ( e ) , thermal displacement component ( u ) , conductive temperature ( ) , thermodynamic temperature ( ) , and thermal stress ( xx ) in the space time domain we have applied laplace inversion formula to ( 65 ) , ( 67 ) , ( 68 ) , ( 69 ) , and ( 70 ) , respectively , which have been done numerically using a method based on fourier series expansion technique . the numerical code has been prepared using fortran 77 programming language . for computational purpose copper material has been taken into consideration . the values of the material constants are taken as follows : ( 80)=7.761010 nm2,=3.861010 nm2,=8954 kg m3,k0=386 wm1 k1,ce=383.1 jkg1 k1 , t0=293 k,t=1.78105 k1 , 1=1.618,2=0.01041 , =0.1,q=0.02 , t=0.015 . figures 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , and 15 are drawn to represent the variation of said thermophysical quantities versus the space variable x for different k1 ( = 0,2,4 ) , ( = 0.5,1.0,1.6 ) , and ( = 0.0,0.1 ) . here = 0.5 , 1.0 , and 1.6 corresponds to week conductivity , normal conductivity , and super conductivity , respectively ; = 0.0 and 0.1 corresponds to one - temperature and two - temperature theory , respectively . figures 15 show the effect of k1 on the said five thermophysical quantities for two - temperature theory ( = 0.1 ) and fractional order parameter = 0.5 . from these figures it is clear that magnitude of all the quantities , that is , thermodynamic temperature , conductive temperature , displacement u , stress component , and strain component e , is greater for smaller magnitude of k1 . figures 610 show the effect of for two - temperature ( = 0.1 ) theory and k1 = 2 on those five quantities . figures 69 show that the magnitude of thermodynamic temperature , conductive temperature , displacement component u , and stress component has greater value for smaller magnitude of . but from figure 10 it is observed that in the region 0.0 x < 0.3 ( approximate ) strain component has larger value for = 1.0 than for = 0.5 , which is again larger than for = 1.6 . figures 1115 are drawn to compare between the results of one - temperature ( = 0.0 ) theory and two - temperature ( = 0.1 ) theory for = 0.5 and k1 = 2 for five different thermophysical quantities . figures 11 , 13 , 14 , and 15 show that the magnitude of , u , , e is greater for one - temperature ( = 0.0 ) case than two - temperature ( = 0.1 ) case . it is observed that at the boundary plane x = 0 , = 1 ( figures 2 , 7 , and 12 ) , and e = 0 ( figures 5 , 10 , and 15 ) , which satisfies our theoretical boundary condition . it ensures the correctness of the numerical code used . in figures 610 the results for figures 16 and 17 represent the variation of the thermodynamic temperature and conductive temperature against time t for different value of k1 , namely , k1 = 2 , 5 , 8 , when = 0.1 , = 0.5 , and x = 0.2 . it is observed from the figures that the magnitudes of and are oscillatory in nature and the magnitude of peek of oscillation decreases with time . state space approach has been applied to solve a generalized thermoelastic problem of an isotropic , half - space with variable thermal conductivity . the boundary ( x = 0 ) of the half - space is subjected to thermal and mechanical loads . the problem has been studied using the two - temperataure dual - phase - lag model of generalized thermoelasticity in consideration of fractional order heat conduction equation.(1)the phenomenon of finite speeds of propagation is observed in all depicted figures . this is expected since the thermal wave travels with finite speed.(2)the effects of the fractional parameter on all the studied fields are very significant.(3)the value of k1 has an essential role in changing the value of the distributions.(4)significant differences in the physical quantities are observed between the one - temperature theory and the two - temperature theory . the two - temperature theory is more realistic than the one - temperature theory in the case of generalized thermoelasticity.(5)initially ( at t = 0 ) the conductive temperature has the value of 1 and strain has the value of 0 . the value of k1 has an essential role in changing the value of the distributions . significant differences in the physical quantities are observed between the one - temperature theory and the two - temperature theory . the two - temperature theory is more realistic than the one - temperature theory in the case of generalized thermoelasticity . initially ( at t = 0 ) the conductive temperature has the value of 1 and strain has the value of 0 .

a new theory of two - temperature generalized thermoelasticity is constructed in the context of a new consideration of dual - phase - lag heat conduction with fractional orders . the theory is then adopted to study thermoelastic interaction in an isotropic homogenous semi - infinite generalized thermoelastic solids with variable thermal conductivity whose boundary is subjected to thermal and mechanical loading . the basic equations of the problem have been written in the form of a vector - matrix differential equation in the laplace transform domain , which is then solved by using a state space approach . the inversion of laplace transforms is computed numerically using the method of fourier series expansion technique . the numerical estimates of the quantities of physical interest are obtained and depicted graphically . some comparisons of the thermophysical quantities are shown in figures to study the effects of the variable thermal conductivity , temperature discrepancy , and the fractional order parameter .

1. Introduction 2. Development of Fractional Order Theory 3. Basic Formulation 4. Formulation of the Problem 5. Method of Solution 6. Numerical Inversion of Laplace Transform 7. Numerical Results and Discussion 8. Conclusion

in order to overcome the paradox of an infinite speed of thermal wave inherent in cte and ccte ( classical coupled theory of thermoelasticity ) , efforts were made to modify coupled thermoelasticity , on different grounds , to obtain a wave - type heat conduction equation by different researchers . lord and shulman formulated the generalized thermoelasticity theory introducing one relaxation time in fourier 's law of heat conduction equation and thus transforming the heat conduction equation into a hyperbolic type . green and lindsay used the theory of two different relaxation times in the constitutive relations for the stress tensor and the entropy equation . , thermoelasticity with energy dissipation ) models is determined in terms of the potential that also determines stresses . tzou introduced two - phase - lag models to both the heat flux vector and the temperature gradient . according to this model , classical fourier 's law q=-k has been replaced by q(p , t+q)=-k(p , t+t ) , where the temperature gradient at a point p of the material at time t + t corresponds to the heat flux vector q at the same point at time t + q . here the delay time t is interpreted as that caused by the microstructural interactions and is called the phase - lag of the temperature gradient . the other delay time q is interpreted as the relaxation time due to the fast transient effects of thermal inertia and is called the phase - lag of the heat flux . if q = and t = 0 , tzou refers to the model as single - phase - lag model . recently several researchers have attempted to solve their problems on the basis of the theory of dual - phase - lag model . roychoudhuri has studied one - dimensional thermoelastic wave propagation in an elastic half - space in the context of dual - phase - lag model . the exponential stability and condition of the delay parameters in the dual - phase - lag theory under this model have been studied by quintanilla . [ 10 , 11 ] have studied the well - posedness and solution structure of the dual - phase - lag heat conduction equation . wang and mingtian have studied the thermal oscillation and resonance in dual - phase - lag heat conduction equation . ailawalia and budhiraja have studied a problem of dual - phase - lag model with internal heat source . gurtin and williams [ 14 , 15 ] have suggested that there is no a priori ground for assuming that the second law of thermodynamics for continuous bodies involves only a single temperature ; that is , it is more logical to assume a second law in which the entropy contribution due to heat conduction is governed by one temperature , that of the heat supply by another . [ 17 , 18 ] have formulated a theory of heat conduction in deformable bodies , which depends on two distinct temperatures : the conductive temperature and the thermodynamic temperature . for time - independent situations , the differences between these two temperatures are proportional to the heat supply , and in the absence of any heat supply , the two temperatures are identical . for time - dependent problems , however , and for wave propagation problems in particular , the two temperatures are , in general , different , independent of the presence of a heat supply . the key element that sets the two - temperature thermoelasticity ( 2tt ) apart from the classical theory of thermoelasticity ( cte ) is the material parameter a()0 , called the temperature discrepancy . specifically , if a = 0 , then = and the field equations of the 2tt reduce to those of cte . the linearized version of the two - temperature theory ( 2tt ) has been studied by many authors . warren and chen have investigated the wave propagation in the two - temperature theory of thermoelasticity . the existence , structural stability , and spatial behavior of the solution in 2tt have been discussed by quintanilla . [ 23 , 24 ] have studied problems on two - temperature green naghdi iii and dual - phase - lag model with variable thermal conductivity . , das and kanoria , and banik and kanoria have studied on two - temperature generalized thermoelasticity . [ 29 , 30 ] have established variational and reciprocal principles and some theorems in two - temperature generalized thermoelasticity . have solved a dynamic problem on green - naghdi ( type iii ) half - space with two - temperature theory . in this work , we have studied the thermoelastic stress , strain , displacement , conductive temperature , and the thermodynamic temperature in an infinite , isotropic , homogeneous elastic half - space under thermal shock using two - temperature dual - phase - lag generalized thermoelasticity in the context of fractional heat conduction equation . the governing equations of two - temperature generalized thermoelasticity theory are formed in the laplace transform domain which are then solved by state space approach . the inversion of the transformed solutions is carried out numerically , applying a method based on a fourier series expansion technique . a complete and comprehensive analysis of the results is presented and the effects of fractional order parameter , two - type temperature , and variable thermal conductivity are discussed . differential equations of fractional order have been the focus of many studies due to their frequent appearance in various applications in fluid mechanics , viscoelasticity , biology , physics , and engineering . it is well - known that the integer order differential operator is a local operator but the fractional order differential operator is nonlocal . this is more realistic , and this is one reason why fractional calculus has become more and more popular [ 3335 ] . one can state that the whole theory of fractional derivatives and integrals was established in the second half of the nineteenth century . the first application of fractional derivatives was given by abel who applied fractional calculus in the solution of an integral equation that arises in the formulation of the tautochrone problem . the generalization of the concept of derivative and integral to a noninteger order has been subjected to several approaches , and some various alternative definitions of fractional derivatives have appeared [ 3639 ] . in the last few years , fractional calculus has been applied successfully in various areas to modify many existing models of physical processes , for example , chemistry , biology , modeling and identification , electronics , wave propagation , and viscoelasticity . youssef introduced the formula of heat conduction given by ( 1)qi+0qit=ki1,i , 0<2 , where the notation i is the riemann - liouville fractional integral , introduced as a natural generalization of the well - known n - fold repeated integral if(t ) written in a convolution - type form as in which is written as follows : ( 2)inf(t)=1(n)0t(t)n1f()d , 0<n2,=f(t ) , n=0 , where (n ) is the gamma function . sur and kanoria have employed the theory to study problems on functionally graded and viscoelastic material . ezzat and el - karamany [ 44 , 45 ] established a new model of fractional heat conduction equation by using the new taylor series expansion of time - fractional order , developed by jumarie as ( 3)qi+0!qit=k,i , 0<1 . el - karamany and ezzat introduced two general models of fractional heat conduction law for a nonhomogeneous anisotropic elastic solid . uniqueness and reciprocal theorems are proved , and the convolutional variational principle is established and used to prove a uniqueness theorem with no restriction on the elasticity or thermal conductivity tensors except symmetry conditions . for fractional thermoelasticity not involving two - temperature , el - karamany and ezzat established the uniqueness , reciprocal theorems and convolution variational principle . for two - temperature theory the formula of heat conduction has been replaced by ( 4)qi+0qit=ki1,i , 0<2 , where is the conductive temperature . two - temperature fractional order thermoelasticity problem of ls and green naghdi models ( types ii and iii ) have been solved by sur and kanoria . several researchers have solved different problems [ 5054 ] using fractional order generalized thermoelasticity theory . , and ignaczak and ostoja - starzewski . in the context of two - temperature dual - phase - lag ( 2tdpl ) generalized thermoelasticity theory , the equation of motion in the absence of body forces and the heat conduction equation in absence of heat sources for a linearly isotropic generalized thermoelastic solid based on the theory of fractional integral are , respectively , given by ( 6)ui=(+)uj , ji+ui , jj,i , i , j=1,2,3,(7)[ki1(1+tt+t222t2),i],i = ( 1+qt+q222t2)(k+t0e ) , where is the density , and are lam 's constants , k is thermal conductivity , = ( 3 + 2)t , t being the coefficient of linear thermal expansion , t0 is the reference temperature , and = k/ce , ce being the specific heat at constant strain . the relation between conductive temperature ( ) and thermodynamic temperature ( ) is given by ( 8)=a,ii i=1,2,3 , where a(0 ) is the two - temperature parameter , called temperature discrepancy . we will consider the thermal conductivity as a linear function of thermodynamical temperature as follows : ( 9)k()=k0[1+k1 ] , where k0 is a constant which is equal to the thermal conductivity of the material when it does not depend on thermodynamical temperature ( ) and k1 is a nonpositive small parameter . this half - space is subjected to thermal and mechanical loads on the bounding plane ( x = 0 ) that depends on the time t and is linearly quiescent . we will consider one - dimensional thermoelastic deformation of the body so that the displacement components can be taken in the following form : ( 27)(ux , uy , uz)=(u(x , t),0,0 ) . the strain displacement relation is ( 28)exx=ux and the constitutive relation ( 5 ) takes the form ( 29)xx=(+2)ux. initial and regularity conditions for the problem are given by ( 34)u===0 at t=0 for x0,ut=t=t=0 at t=0 for x0,u===0 as x. applying the laplace transform defined by ( 35)f(s)=0estf(t)dt , re(s)>0 to both the sides of ( 32 ) , we obtain ( 36)xx = e2,(37)d2edx2=s2e+2d2~dx2,(38)(1+ts+12t2s2)d2~dx2 = s(1+qs+12q2s2)(~+1e),(39)~~=d2~dx2 . the equations ( 40 ) can be written in the form of a vector matrix differential equations as follows : ( 42)d2v(x , s)dx2=a(s)v(x , s ) , where ( 43)v(x , s)=[~(x , s)e(x , s ) ] , a(s)=[11123 ] . we will use the well - known cayley - hamilton theorem to find the form of the matrix ( 46)exp[a(s)x ] . using cayley - hamilton theorem , we can express a and higher powers of the matrix a in terms of i and a , where i is the unit matrix of the second order . thus the infinite series in ( 49 ) can be reduced to the form ( 50)exp[a(s)x]=a0(x , s)i+a1(x , s)a(s ) , where a0 and a1 are coefficients depending on s and x. by cayley - hamilton theorem , the characteristic roots k1 and k2 of the matrix a must satisfy ( 50 ) ; thus we have ( 51)exp(k1x)=a0+a1k1,exp(k2x)=a0+a1k2 . we will consider the bounding plane of the medium at x = 0 subjected to thermal shock in the following nondimensional form : ( 59)(0,t)=1h(t ) , where 1 is constant . now applying the laplace transform to ( 59 ) we get ( 60)(0,s)=1s . the mechanical boundary condition is taken in the form ( 62)e(0,t)=0 . again using ( 9 ) , ( 12 ) , and ( 66 ) the solution for - can be obtained in the following form : ( 69)=ls(k1k2)[(1k2)(1k1)ek1xmmmmmmmm(1k1)(1k2)ek2x]=~0(say ) , k1=0=1k1[1 + 2k1~01 ] , k1<0 . it is difficult to find the analytical inverse of laplace transform of the complicated solutions for the displacement , thermodynamic temperature , conductive temperature , stress , and strain in laplace transform domain . let f-(x , s ) be the laplace transform of a function f(x , t ) . then the inversion formula for laplace transform can be written as ( 71)f(x , t)=12idid+iestf(x , s)ds , where d is an arbitrary real number greater than real parts of all the singularities of f-(x , s ) . expanding the function h(x , t ) = ef(x , t ) in a fourier series in the interval [ 0,2 t ] we obtain the approximate formula ( 73)f(x , t)=f(x , t)+ed , where ( 74)f(x , t)=12c0+k=1ck for 0t2t , ck = edtt[eikt / t f(x , d+iktt ) ] . thus , the approximate value of f(x , t ) becomes ( 77)fnk(x , t)=fn(x , t)e2dtfn(x,2t+t ) , where n is an integer such that n < n. we will now describe the -algorithm that is used to accelerate the convergence of the series in ( 75 ) . the actual procedure used to invert to get the solution for strain ( e ) , thermal displacement component ( u ) , conductive temperature ( ) , thermodynamic temperature ( ) , and thermal stress ( xx ) in the space time domain we have applied laplace inversion formula to ( 65 ) , ( 67 ) , ( 68 ) , ( 69 ) , and ( 70 ) , respectively , which have been done numerically using a method based on fourier series expansion technique . figures 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , and 15 are drawn to represent the variation of said thermophysical quantities versus the space variable x for different k1 ( = 0,2,4 ) , ( = 0.5,1.0,1.6 ) , and ( = 0.0,0.1 ) . here = 0.5 , 1.0 , and 1.6 corresponds to week conductivity , normal conductivity , and super conductivity , respectively ; = 0.0 and 0.1 corresponds to one - temperature and two - temperature theory , respectively . figures 15 show the effect of k1 on the said five thermophysical quantities for two - temperature theory ( = 0.1 ) and fractional order parameter = 0.5 . from these figures it is clear that magnitude of all the quantities , that is , thermodynamic temperature , conductive temperature , displacement u , stress component , and strain component e , is greater for smaller magnitude of k1 . figures 610 show the effect of for two - temperature ( = 0.1 ) theory and k1 = 2 on those five quantities . but from figure 10 it is observed that in the region 0.0 x < 0.3 ( approximate ) strain component has larger value for = 1.0 than for = 0.5 , which is again larger than for = 1.6 . figures 1115 are drawn to compare between the results of one - temperature ( = 0.0 ) theory and two - temperature ( = 0.1 ) theory for = 0.5 and k1 = 2 for five different thermophysical quantities . figures 11 , 13 , 14 , and 15 show that the magnitude of , u , , e is greater for one - temperature ( = 0.0 ) case than two - temperature ( = 0.1 ) case . it is observed that at the boundary plane x = 0 , = 1 ( figures 2 , 7 , and 12 ) , and e = 0 ( figures 5 , 10 , and 15 ) , which satisfies our theoretical boundary condition . it ensures the correctness of the numerical code used . in figures 610 the results for figures 16 and 17 represent the variation of the thermodynamic temperature and conductive temperature against time t for different value of k1 , namely , k1 = 2 , 5 , 8 , when = 0.1 , = 0.5 , and x = 0.2 . state space approach has been applied to solve a generalized thermoelastic problem of an isotropic , half - space with variable thermal conductivity . the boundary ( x = 0 ) of the half - space is subjected to thermal and mechanical loads . the problem has been studied using the two - temperataure dual - phase - lag model of generalized thermoelasticity in consideration of fractional order heat conduction equation. (2)the effects of the fractional parameter on all the studied fields are very significant. (4)significant differences in the physical quantities are observed between the one - temperature theory and the two - temperature theory . the two - temperature theory is more realistic than the one - temperature theory in the case of generalized thermoelasticity. significant differences in the physical quantities are observed between the one - temperature theory and the two - temperature theory . the two - temperature theory is more realistic than the one - temperature theory in the case of generalized thermoelasticity .

the enzyme phosphodiesterase 5 ( pde5 ) hydrolyzes cyclic guanosine monophosphate ( cgmp ) to guanosine monophosphate ( gmp ) , which functions in smooth muscle relaxation . pde5 regulates cgmp levels , and inhibition of this regulator causes an increased level of cgmp , resulting in smooth muscle relaxation . cgmp is expressed in skeletal muscle , visceral smooth muscle , and vascular smooth muscle , including coronary vessels , as well as in the corpus cavernosum of the penis . sildenafil , a selective inhibitor of pde5 , was developed as a potential alternative agent to oral nitrates for the treatment of stable angina pectoris , but it has a nitrate - like hemodynamic effect . during clinical trials , penile erection was reported as a side effect , which led to the use of sildenafil as an agent for treating erectile dysfunction . in addition to its vasodilator effect on erectile dysfunction , sildenafil has several potential effects on various systems such as the urological , cardiovascular , and pulmonary systems and the central nervous system ( cns ) . sildenafil also improves cardiac output and exercise performance in athletes at high altitude , but not at sea level . nitric oxide ( no ) is a mediator in the central nervous system and endothelium and is synthesized from l - arginine by the enzyme no synthase . the no - cgmp pathway might be implicated in the regulation of certain cns functions , including locomotor activity and anxiety . numerous experimental studies and clinical observations indicate that sildenafil has some effects on the cns , but its mechanism of action is not precisely known . it has been reported that sildenafil has an anxiogenic effect in rats resulting from the promotion of the no - cgmp pathway following inhibition of the hydrolysis of cgmp to gmp . there are no studies to date showing increased athletic performance following acute sildenafil administration , although sildenafil might play a role in increasing oxygen exchange in pulmonary capillaries . since the enhancing or diminishing effects of sildenafil on locomotor activity and/or anxiety will alter athletic performance , it is important to investigate the effects of sildenafil on locomotor activity and anxiety . locomotor activity and the anxiety level of athletes may have an impact on sports performance . the purpose of the present study was to evaluate the effects of sildenafil ( 3 and 10 mg / kg ) on locomotor activity and anxiety in the open field and elevated plus maze ( epm ) tests in young mice and to compare the effects between young and aged mice . according to our literature search , this is the first study comparing the effects of sildenafil on locomotor activity and anxiety between young and aged mice . male , inbred balb / c byj mice ( uludag university , bursa , turkey ) , 6 weeks old upon arrival to the laboratory , were used in this study . the animals were kept in the laboratory for 2 weeks before the experiments were initiated . eight - week - old animals were used as young mice , and 9-month - old animals were used as aged animals . animals were maintained under standard laboratory conditions ( 12-h light : 12-h dark cycle , lights on at 07 : 00 h , t=211c ) . all procedures described in this paper were conducted in accordance with the european community council directive for the ethical treatment of animals ( 86/609/eec ) , and approval from the university medical school animal research ethics committee was obtained ( number 314/2012 ) . the animals were treated with sildenafil ( 3 or 10 mg / kg , dissolved in saline added with 1% dmso ) obtained from sigma ( st . sildenafil was administered acutely and intraperitoneally ( i.p . ) , 30 min . before the open field and epm tests , in a volume of 0.1 ml/10 g body weight . locomotor activity was measured using an open field test in a square arena ( 404040 cm box ) . the animal was placed in the center of the apparatus , and its behaviors were recorded for 5 min using the etovision - xt video tracking system . the locomotor activity was evaluated by measuring the total distance traveled in the apparatus and the speed of the animals . anxiety - related behavior was measured by the elevated plus - maze ( epm ) test . experiments were conducted in a dimly lit , semi - soundproof room that was illuminated with a table lamp ( 80 lux ) . the maze was made of wood and consisted of 2 open arms ( 29 cm long 5 cm wide ) and 2 closed arms ( 29 cm 5 cm with 15 cm high walls ) forming a square cross , with a 5-cm square centerpiece . to avoid falls , the open arms and central platform were painted white and the closed arms were painted black . each mouse was placed at the center of the maze , facing one of the open arms , and was allowed to explore the maze . during a 5-min test period , the number of entries into either the open or the closed arms of the maze ( defined as the entry of all 4 limbs into the arms ) and the time spent on the open arms were recorded . the open - arm activity was evaluated as follows : 1 ) the time spent in the open arms relative to the total time spent in the plus maze ( 300 s ) , expressed as a percentage and 2 ) the number of entries into the open arms relative to the total number of entries into both the open and closed arms , expressed as a percentage . if the values for both measured parameters changed in the same direction compared to the control values ( i.e. , if both the time spent in open arms and the number of open arm entries were increased or if both were decreased ) and the change in 1 of the parameters was statistically significant , then an effect on anxiety was considered to have occurred . in the current study , the time spent in the open arms and the number of open arm entries always seemed to change in the same direction . the results of the total distance moved and the speed of the animals in the open field test , the percent of time spent in the open arms and the percent and total number of entries to the open arms in the epm test , the total number of arms entries , and the total time spent in open arms were all compared using a one - way anova , followed by dunnett s post hoc test when significant differences were detected . male , inbred balb / c byj mice ( uludag university , bursa , turkey ) , 6 weeks old upon arrival to the laboratory , were used in this study . the animals were kept in the laboratory for 2 weeks before the experiments were initiated . eight - week - old animals were used as young mice , and 9-month - old animals were used as aged animals . animals were maintained under standard laboratory conditions ( 12-h light : 12-h dark cycle , lights on at 07 : 00 h , t=211c ) . all procedures described in this paper were conducted in accordance with the european community council directive for the ethical treatment of animals ( 86/609/eec ) , and approval from the university medical school animal research ethics committee was obtained ( number 314/2012 ) . the animals were treated with sildenafil ( 3 or 10 mg / kg , dissolved in saline added with 1% dmso ) obtained from sigma ( st . sildenafil was administered acutely and intraperitoneally ( i.p . ) , 30 min . before the open field and epm tests , in a volume of 0.1 ml/10 g body weight . locomotor activity was measured using an open field test in a square arena ( 404040 cm box ) . the animal was placed in the center of the apparatus , and its behaviors were recorded for 5 min using the etovision - xt video tracking system . the locomotor activity was evaluated by measuring the total distance traveled in the apparatus and the speed of the animals . anxiety - related behavior was measured by the elevated plus - maze ( epm ) test . experiments were conducted in a dimly lit , semi - soundproof room that was illuminated with a table lamp ( 80 lux ) . the maze was made of wood and consisted of 2 open arms ( 29 cm long 5 cm wide ) and 2 closed arms ( 29 cm 5 cm with 15 cm high walls ) forming a square cross , with a 5-cm square centerpiece . to avoid falls , the open arms and central platform were painted white and the closed arms were painted black . each mouse was placed at the center of the maze , facing one of the open arms , and was allowed to explore the maze . during a 5-min test period , the number of entries into either the open or the closed arms of the maze ( defined as the entry of all 4 limbs into the arms ) and the time spent on the open arms were recorded . the open - arm activity was evaluated as follows : 1 ) the time spent in the open arms relative to the total time spent in the plus maze ( 300 s ) , expressed as a percentage and 2 ) the number of entries into the open arms relative to the total number of entries into both the open and closed arms , expressed as a percentage . any animal that fell off the maze was excluded from the experiment . if the values for both measured parameters changed in the same direction compared to the control values ( i.e. , if both the time spent in open arms and the number of open arm entries were increased or if both were decreased ) and the change in 1 of the parameters was statistically significant , then an effect on anxiety was considered to have occurred . in the current study , the time spent in the open arms and the number of open arm entries always seemed to change in the same direction . the results of the total distance moved and the speed of the animals in the open field test , the percent of time spent in the open arms and the percent and total number of entries to the open arms in the epm test , the total number of arms entries , and the total time spent in open arms were all compared using a one - way anova , followed by dunnett s post hoc test when significant differences were detected . the data are expressed as mean values sem . a significant difference between the groups was observed when the effect of sildenafil on the percent of time spent in the open arms was evaluated in the epm test [ f(2,44)=14.61 , p<0.0001 ; figure 1a ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; figure 1a ) . additionally , a significant difference between the groups was observed when the effect of sildenafil on the percent and total number of entries to the open arms was evaluated in the epm test [ f(2.44)=7.45 , p=0.0017 ; figure 1b ] . at the higher dose ( 10 mg / kg ) , sildenafil significantly decreased the percent and total number of entries to the open arms compared to the control group ( p<0.01 ) , while only a partial and non - significant ( p>0.05 ) effect was observed for the lower dose ( 3 mg / kg ) of sildenafil ( figure 1b ) . a significant difference between the groups was observed when the effect of sildenafil on the total number of arm entries was evaluated in the epm test [ f(2.44)=7.68 , p=0.0014 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total number of arm entries compared to the control group ( p<0.05 , p<0.01 ; respectively ; table 1 ) . a significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) was evaluated in the epm test [ f(2,44)=14.12 , p<0.0001 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the time spent in open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; table 1 ) . there was no significant difference between the groups when the effects of sildenafil on the percent of time spent in the open arms was evaluated in aged animals in the epm test [ f(3,43)=2.02 , p=0.12 ; figure 2a ] . sildenafil ( 3 and 10 mg / kg ) partially decreased the percent of time spent in the open arms in aged animals , but the effect was not significant ( p>0.05 ; figure 2a ) . there was a significant difference between the groups when the effects of sildenafil on the percent and total number of entries to the open arms was evaluated in aged mice in the epm test [ f(3,43)=12.57 , p<0.0001 ; figure 2b ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent and total number of entries to the open arms in aged animals ( p<0.01 ; p<0.001 ; respectively ) , while there was no significant difference between young control and aged animals ( p>0.05 ; figure 2b ) . a significant difference between the groups was observed ( table 2 ) when the total number of arm entries in aged animals was evaluated in the epm test [ f(3,43)=17.39 , p<0.0001 ; table 2 ] . there was a significant difference between young control and aged animals ( p<0.001 ; table 2 ) . no significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) in aged animals was evaluated in the epm test [ f(3,43)=2.08 , p=0.11 ; table 2 ] . a significant difference between the groups was observed in the evaluation of the effects of sildenafil on the total distance moved in the open field test [ f(2,29)=20.74 , p<0.0001 ; figure 3a ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total distance moved compared to the control group ( p<0.001 ; figure 3a ) . there was also a significant difference between the groups when the effect of sildenafil on the speed of the animals was evaluated in the open field test [ f(2,29)=13.68 , p<0.0001 ; figure 3b ] ; sildenafil ( 3 and 10 mg / kg ) significantly increased the speed of the animals in the open field test ( p<0.001 ; figure 3b ) . there was a significant difference between the groups when the effect of sildenafil on the total distance moved was evaluated in aged animals in the open field test [ f(3,46)=5.12 , p=0.004 ; figure 4a ] . the total distance moved significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4a ) , and sildenafil ( 3 and 10 mg / kg ) did not increase the total distance moved in aged animals in the open field test ( p>0.05 ; figure 4a ) . a significant difference between the groups was also observed when the effect of sildenafil on the speed of the aged animals was evaluated in the open field test [ f(3,46)=5.19 , p=0.0038 ; figure 4b ] . the speed of the animals significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4b ) , and sildenafil ( 3 and 10 mg / kg ) did not change the speed of the aged animals in the open field test ( p>0.05 ; figure 4b ) . a significant difference between the groups was observed when the effect of sildenafil on the percent of time spent in the open arms was evaluated in the epm test [ f(2,44)=14.61 , p<0.0001 ; figure 1a ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; figure 1a ) . additionally , a significant difference between the groups was observed when the effect of sildenafil on the percent and total number of entries to the open arms was evaluated in the epm test [ f(2.44)=7.45 , p=0.0017 ; figure 1b ] . at the higher dose ( 10 mg / kg ) , sildenafil significantly decreased the percent and total number of entries to the open arms compared to the control group ( p<0.01 ) , while only a partial and non - significant ( p>0.05 ) effect was observed for the lower dose ( 3 mg / kg ) of sildenafil ( figure 1b ) . a significant difference between the groups was observed when the effect of sildenafil on the total number of arm entries was evaluated in the epm test [ f(2.44)=7.68 , p=0.0014 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total number of arm entries compared to the control group ( p<0.05 , p<0.01 ; respectively ; table 1 ) . a significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) was evaluated in the epm test [ f(2,44)=14.12 , p<0.0001 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the time spent in open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; table 1 ) . there was no significant difference between the groups when the effects of sildenafil on the percent of time spent in the open arms was evaluated in aged animals in the epm test [ f(3,43)=2.02 , p=0.12 ; figure 2a ] . sildenafil ( 3 and 10 mg / kg ) partially decreased the percent of time spent in the open arms in aged animals , but the effect was not significant ( p>0.05 ; figure 2a ) . there was a significant difference between the groups when the effects of sildenafil on the percent and total number of entries to the open arms was evaluated in aged mice in the epm test [ f(3,43)=12.57 , p<0.0001 ; figure 2b ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent and total number of entries to the open arms in aged animals ( p<0.01 ; p<0.001 ; respectively ) , while there was no significant difference between young control and aged animals ( p>0.05 ; figure 2b ) . a significant difference between the groups was observed ( table 2 ) when the total number of arm entries in aged animals was evaluated in the epm test [ f(3,43)=17.39 , p<0.0001 ; table 2 ] . there was a significant difference between young control and aged animals ( p<0.001 ; table 2 ) . no significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) in aged animals was evaluated in the epm test [ f(3,43)=2.08 , p=0.11 ; table 2 ] . a significant difference between the groups was observed in the evaluation of the effects of sildenafil on the total distance moved in the open field test [ f(2,29)=20.74 , p<0.0001 ; figure 3a ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total distance moved compared to the control group ( p<0.001 ; figure 3a ) . there was also a significant difference between the groups when the effect of sildenafil on the speed of the animals was evaluated in the open field test [ f(2,29)=13.68 , p<0.0001 ; figure 3b ] ; sildenafil ( 3 and 10 mg / kg ) significantly increased the speed of the animals in the open field test ( p<0.001 ; figure 3b ) . there was a significant difference between the groups when the effect of sildenafil on the total distance moved was evaluated in aged animals in the open field test [ f(3,46)=5.12 , p=0.004 ; figure 4a ] . the total distance moved significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4a ) , and sildenafil ( 3 and 10 mg / kg ) did not increase the total distance moved in aged animals in the open field test ( p>0.05 ; figure 4a ) . a significant difference between the groups was also observed when the effect of sildenafil on the speed of the aged animals was evaluated in the open field test [ f(3,46)=5.19 , p=0.0038 ; figure 4b ] . the speed of the animals significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4b ) , and sildenafil ( 3 and 10 mg / kg ) did not change the speed of the aged animals in the open field test ( p>0.05 ; figure 4b ) . this study revealed that sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms in young animals , but only the 10 mg / kg dose significantly decreased the percent and total number of entries to the open arms in young animals in the epm test . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent and total number of entries to the open arms in aged animals but had no effect on the percent of time spent in the open arms in aged animals in the epm test . the total distance moved and the speed of the animals significantly decreased in aged animals compared to the young control group . sildenafil significantly increased the total distance moved and the speed of the animals in the locomotor activity test in young animals , and it had no significant effect in aged mice . pde5 inhibitors , including sildenafil , influence the cardiovascular , pulmonary , and central nervous systems . these effects could affect athletic performance by improving cardiac output , pulmonary capillary oxygen exchange , and the excitation of some central nervous system pathways . sildenafil improves cardiac output and exercise performance in athletes at high altitude , but not at sea level . sildenafil has been examined as a potential doping agent , although no report has shown a positive effect on athletic performance in a normal sports environment . we attempted to evaluate the effects of sildenafil , administered at 3 and 10 mg / kg , on locomotor activity and anxiety in young and aged mice . kurt et al . reported decreased locomotor activity in the plus - maze test in mice administered a 3 mg / kg dose of sildenafil in mice aged 2 months . reported unchanged locomotor activity in mice administered 5 and 10 mg / kg sildenafil , but decreased activity in mice administered 20 mg / kg sildenafil or a combination of 20 mg / kg sildenafil and 30 mg / kg magnesium , in the forced swim test in mice aged 2 months . reported that sildenafil did not modify the behavior of animals in the open field test of locomotor activity in mice aged 34 months . the results of our locomotor activity test in young mice show that the administration of sildenafil ( 3 and 10 mg / kg ) increased the total distance moved and the speed of the animals , but it had no significant effect on locomotion in aged mice . the results of epm test are also correlated with the results of the locomotor activity test because sildenafil ( 3 and 10 mg / kg ) significantly increased total number of arms entries in young animals bu it had no effect on total number of arms entries in aged animals . the differences between our results and those of other studies could have been influenced by the study design and/or the age of the mice . milman et al . reported an association between sildenafil and various cns adverse effects , including aggressive behavior . there are conflicting reports regarding whether activating the no - cgmp pathway leads to increased or reduced anxiety - like behavior . the majority of studies suggest that inhibition of the no - cgmp pathway is anxiolytic and that its activation is anxiogenic , although other studies have published contradictory results . the effects of sildenafil on anxiety - related behavior and other behaviors such as aggressive behavior were recently assessed in a paper by dadomo et al . . in addition , a study reported that the same no - cgmp signaling modulators can have dual effects . the administration of sildenafil at low doses has been reported to produce both increases in and no effects on anxiety - like behaviors , using the elevated plus - maze test in mice . similarly , studies investigating the effects of no levels on anxiety - related responses have also reported contradictory results ; both increases and decreases in anxiety have been linked to the inhibition of no by nitric oxide synthase ( nos ) inhibitors . sildenafil and other pde-5 inhibitors have been shown to increase cgmp in the hippocampus in vitro . both animal and human studies have suggested that sildenafil may enhance the ability to focus attention and improve memory retention . administration of the no precursor l - arginine increases no synthesis in the brain , whereas the combined administration of l - arginine and sildenafil ( which inhibits cgmp degradation in the brain ) results in prominent anxiogenic - like effects . some other studies reported that acute administration of sildenafil has anxiogenic effects when administered 30 min before the behavioral tests , which could be attributed to activation of the no - cgmp pathway . similarly , the present study showed anxious behavior in both young and aged mice following the administration of 3 or 10 mg / kg sildenafil . in contrast , shahidi et al . reported that sildenafil - treated mice did not show anxiety - like effects in the elevated plus - maze test . the researchers performed the test 15 min after the administration of sildenafil at doses of 1 , 2 , and 10 mg / kg . in the present study liebenberg et al . reported the presence of significant concentrations of sildenafil in the prefrontal cortex and hippocampus following chronic administration , and the psychotropic actions of sildenafil can be ascribed to a central mode of action . the authors reported that chronic administration of pde5 inhibitors yields significant anxiolytic - like effects , highlighting the important role of pde5 and cgmp signaling in anxiety . however , it remains to be determined whether the anxiolytic effects of sildenafil observed following chronic administration are related to enhanced cgmp levels or to the indirect effects of sildenafil on no . similarly , prut and belzung indicated that chronic treatment with a selective inhibitor of pde-5 modifies mouse behavior in a manner consistent with that of anxiolytic agents . chronic administration of sildenafil might have a different effect on locomotor activity , behavior , and physical performance compared with acute administration ; thus , further investigation is needed . based on our results , we conclude that the acute administration of sildenafil increased locomotor activity in young mice but not in aged mice . although there is no reported evidence of enhanced athletic performance as a result of sildenafil use , the effects of sildenafil on the central nervous system shown by experimental studies such as the present one suggest a potential stimulatory effect of sildenafil on the performance of young athletes .

backgroundsildenafil is a selective pde5 inhibitor that increases cgmp levels in the target tissues and is an effective treatment agent for erectile dysfunction . the nitric oxide - cgmp pathway might be implicated in regulation of certain cns functions , including locomotor activity and anxiety.material/methodsthe aim of the current study was to investigate effects of sildenafil ( 3 and 10 mg / kg ) on anxiety and locomotor activity in open field and elevated plus maze ( epm ) tests in young and aged mice.resultssildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms compared to the control group in young animals in the epm test , but only the 10 mg / kg dose significantly decreased the percentage of total number of entries to the open arms in young animals . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percentage of total number of entries to the open arms in aged animals in the epm test , but it significantly increased total distance moved and speed of the animals in the locomotor activity test in young animals . the total distance moved and the speed of the animals significantly decreased in aged animals compared to the young control group , although sildenafil ( 3 and 10 mg / kg ) did not alter these parameters in aged mice.conclusionsour results show that sildenafil had anxiogenic effects in young as well as aged mice , but it enhanced locomotor activity only in the young mice in the epm test . thus , sildenafil seems to exert different effects on anxiety and locomotion in young and aged animals .

Background Material and Methods Animals Drugs and treatments Locomotor activity test Elevated plus maze test (EPM test) Statistics Results Effects of sildenafil treatment on anxiety in young animals in the EPM test Effects of sildenafil treatment on anxiety in aged animals in the EPM test Effects of sildenafil treatment on locomotion in young animals in the open field test Effects of sildenafil treatment on locomotion in aged animals in the open field test Discussion Conclusions

cgmp is expressed in skeletal muscle , visceral smooth muscle , and vascular smooth muscle , including coronary vessels , as well as in the corpus cavernosum of the penis . in addition to its vasodilator effect on erectile dysfunction , sildenafil has several potential effects on various systems such as the urological , cardiovascular , and pulmonary systems and the central nervous system ( cns ) . the no - cgmp pathway might be implicated in the regulation of certain cns functions , including locomotor activity and anxiety . since the enhancing or diminishing effects of sildenafil on locomotor activity and/or anxiety will alter athletic performance , it is important to investigate the effects of sildenafil on locomotor activity and anxiety . the purpose of the present study was to evaluate the effects of sildenafil ( 3 and 10 mg / kg ) on locomotor activity and anxiety in the open field and elevated plus maze ( epm ) tests in young mice and to compare the effects between young and aged mice . according to our literature search , this is the first study comparing the effects of sildenafil on locomotor activity and anxiety between young and aged mice . eight - week - old animals were used as young mice , and 9-month - old animals were used as aged animals . the animals were treated with sildenafil ( 3 or 10 mg / kg , dissolved in saline added with 1% dmso ) obtained from sigma ( st . the locomotor activity was evaluated by measuring the total distance traveled in the apparatus and the speed of the animals . during a 5-min test period , the number of entries into either the open or the closed arms of the maze ( defined as the entry of all 4 limbs into the arms ) and the time spent on the open arms were recorded . the open - arm activity was evaluated as follows : 1 ) the time spent in the open arms relative to the total time spent in the plus maze ( 300 s ) , expressed as a percentage and 2 ) the number of entries into the open arms relative to the total number of entries into both the open and closed arms , expressed as a percentage . , if both the time spent in open arms and the number of open arm entries were increased or if both were decreased ) and the change in 1 of the parameters was statistically significant , then an effect on anxiety was considered to have occurred . in the current study , the time spent in the open arms and the number of open arm entries always seemed to change in the same direction . the results of the total distance moved and the speed of the animals in the open field test , the percent of time spent in the open arms and the percent and total number of entries to the open arms in the epm test , the total number of arms entries , and the total time spent in open arms were all compared using a one - way anova , followed by dunnett s post hoc test when significant differences were detected . eight - week - old animals were used as young mice , and 9-month - old animals were used as aged animals . the animals were treated with sildenafil ( 3 or 10 mg / kg , dissolved in saline added with 1% dmso ) obtained from sigma ( st . the locomotor activity was evaluated by measuring the total distance traveled in the apparatus and the speed of the animals . during a 5-min test period , the number of entries into either the open or the closed arms of the maze ( defined as the entry of all 4 limbs into the arms ) and the time spent on the open arms were recorded . the open - arm activity was evaluated as follows : 1 ) the time spent in the open arms relative to the total time spent in the plus maze ( 300 s ) , expressed as a percentage and 2 ) the number of entries into the open arms relative to the total number of entries into both the open and closed arms , expressed as a percentage . if the values for both measured parameters changed in the same direction compared to the control values ( i.e. , if both the time spent in open arms and the number of open arm entries were increased or if both were decreased ) and the change in 1 of the parameters was statistically significant , then an effect on anxiety was considered to have occurred . in the current study , the time spent in the open arms and the number of open arm entries always seemed to change in the same direction . the results of the total distance moved and the speed of the animals in the open field test , the percent of time spent in the open arms and the percent and total number of entries to the open arms in the epm test , the total number of arms entries , and the total time spent in open arms were all compared using a one - way anova , followed by dunnett s post hoc test when significant differences were detected . a significant difference between the groups was observed when the effect of sildenafil on the percent of time spent in the open arms was evaluated in the epm test [ f(2,44)=14.61 , p<0.0001 ; figure 1a ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; figure 1a ) . additionally , a significant difference between the groups was observed when the effect of sildenafil on the percent and total number of entries to the open arms was evaluated in the epm test [ f(2.44)=7.45 , p=0.0017 ; figure 1b ] . at the higher dose ( 10 mg / kg ) , sildenafil significantly decreased the percent and total number of entries to the open arms compared to the control group ( p<0.01 ) , while only a partial and non - significant ( p>0.05 ) effect was observed for the lower dose ( 3 mg / kg ) of sildenafil ( figure 1b ) . a significant difference between the groups was observed when the effect of sildenafil on the total number of arm entries was evaluated in the epm test [ f(2.44)=7.68 , p=0.0014 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total number of arm entries compared to the control group ( p<0.05 , p<0.01 ; respectively ; table 1 ) . a significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) was evaluated in the epm test [ f(2,44)=14.12 , p<0.0001 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the time spent in open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; table 1 ) . there was no significant difference between the groups when the effects of sildenafil on the percent of time spent in the open arms was evaluated in aged animals in the epm test [ f(3,43)=2.02 , p=0.12 ; figure 2a ] . sildenafil ( 3 and 10 mg / kg ) partially decreased the percent of time spent in the open arms in aged animals , but the effect was not significant ( p>0.05 ; figure 2a ) . there was a significant difference between the groups when the effects of sildenafil on the percent and total number of entries to the open arms was evaluated in aged mice in the epm test [ f(3,43)=12.57 , p<0.0001 ; figure 2b ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent and total number of entries to the open arms in aged animals ( p<0.01 ; p<0.001 ; respectively ) , while there was no significant difference between young control and aged animals ( p>0.05 ; figure 2b ) . a significant difference between the groups was observed ( table 2 ) when the total number of arm entries in aged animals was evaluated in the epm test [ f(3,43)=17.39 , p<0.0001 ; table 2 ] . no significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) in aged animals was evaluated in the epm test [ f(3,43)=2.08 , p=0.11 ; table 2 ] . a significant difference between the groups was observed in the evaluation of the effects of sildenafil on the total distance moved in the open field test [ f(2,29)=20.74 , p<0.0001 ; figure 3a ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total distance moved compared to the control group ( p<0.001 ; figure 3a ) . there was also a significant difference between the groups when the effect of sildenafil on the speed of the animals was evaluated in the open field test [ f(2,29)=13.68 , p<0.0001 ; figure 3b ] ; sildenafil ( 3 and 10 mg / kg ) significantly increased the speed of the animals in the open field test ( p<0.001 ; figure 3b ) . there was a significant difference between the groups when the effect of sildenafil on the total distance moved was evaluated in aged animals in the open field test [ f(3,46)=5.12 , p=0.004 ; figure 4a ] . the total distance moved significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4a ) , and sildenafil ( 3 and 10 mg / kg ) did not increase the total distance moved in aged animals in the open field test ( p>0.05 ; figure 4a ) . a significant difference between the groups was also observed when the effect of sildenafil on the speed of the aged animals was evaluated in the open field test [ f(3,46)=5.19 , p=0.0038 ; figure 4b ] . the speed of the animals significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4b ) , and sildenafil ( 3 and 10 mg / kg ) did not change the speed of the aged animals in the open field test ( p>0.05 ; figure 4b ) . a significant difference between the groups was observed when the effect of sildenafil on the percent of time spent in the open arms was evaluated in the epm test [ f(2,44)=14.61 , p<0.0001 ; figure 1a ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; figure 1a ) . additionally , a significant difference between the groups was observed when the effect of sildenafil on the percent and total number of entries to the open arms was evaluated in the epm test [ f(2.44)=7.45 , p=0.0017 ; figure 1b ] . at the higher dose ( 10 mg / kg ) , sildenafil significantly decreased the percent and total number of entries to the open arms compared to the control group ( p<0.01 ) , while only a partial and non - significant ( p>0.05 ) effect was observed for the lower dose ( 3 mg / kg ) of sildenafil ( figure 1b ) . a significant difference between the groups was observed when the effect of sildenafil on the total number of arm entries was evaluated in the epm test [ f(2.44)=7.68 , p=0.0014 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total number of arm entries compared to the control group ( p<0.05 , p<0.01 ; respectively ; table 1 ) . a significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) was evaluated in the epm test [ f(2,44)=14.12 , p<0.0001 ; table 1 ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the time spent in open arms compared to the control group ( p<0.05 , p<0.001 ; respectively ; table 1 ) . there was no significant difference between the groups when the effects of sildenafil on the percent of time spent in the open arms was evaluated in aged animals in the epm test [ f(3,43)=2.02 , p=0.12 ; figure 2a ] . sildenafil ( 3 and 10 mg / kg ) partially decreased the percent of time spent in the open arms in aged animals , but the effect was not significant ( p>0.05 ; figure 2a ) . there was a significant difference between the groups when the effects of sildenafil on the percent and total number of entries to the open arms was evaluated in aged mice in the epm test [ f(3,43)=12.57 , p<0.0001 ; figure 2b ] . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent and total number of entries to the open arms in aged animals ( p<0.01 ; p<0.001 ; respectively ) , while there was no significant difference between young control and aged animals ( p>0.05 ; figure 2b ) . a significant difference between the groups was observed ( table 2 ) when the total number of arm entries in aged animals was evaluated in the epm test [ f(3,43)=17.39 , p<0.0001 ; table 2 ] . no significant difference between the groups was observed when the effect of sildenafil on time spent in open arms ( s ) in aged animals was evaluated in the epm test [ f(3,43)=2.08 , p=0.11 ; table 2 ] . a significant difference between the groups was observed in the evaluation of the effects of sildenafil on the total distance moved in the open field test [ f(2,29)=20.74 , p<0.0001 ; figure 3a ] . sildenafil ( 3 and 10 mg / kg ) significantly increased the total distance moved compared to the control group ( p<0.001 ; figure 3a ) . there was also a significant difference between the groups when the effect of sildenafil on the speed of the animals was evaluated in the open field test [ f(2,29)=13.68 , p<0.0001 ; figure 3b ] ; sildenafil ( 3 and 10 mg / kg ) significantly increased the speed of the animals in the open field test ( p<0.001 ; figure 3b ) . there was a significant difference between the groups when the effect of sildenafil on the total distance moved was evaluated in aged animals in the open field test [ f(3,46)=5.12 , p=0.004 ; figure 4a ] . the total distance moved significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4a ) , and sildenafil ( 3 and 10 mg / kg ) did not increase the total distance moved in aged animals in the open field test ( p>0.05 ; figure 4a ) . a significant difference between the groups was also observed when the effect of sildenafil on the speed of the aged animals was evaluated in the open field test [ f(3,46)=5.19 , p=0.0038 ; figure 4b ] . the speed of the animals significantly decreased in aged animals compared to the young control group ( p<0.01 ; figure 4b ) , and sildenafil ( 3 and 10 mg / kg ) did not change the speed of the aged animals in the open field test ( p>0.05 ; figure 4b ) . this study revealed that sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent of time spent in the open arms in young animals , but only the 10 mg / kg dose significantly decreased the percent and total number of entries to the open arms in young animals in the epm test . sildenafil ( 3 and 10 mg / kg ) significantly decreased the percent and total number of entries to the open arms in aged animals but had no effect on the percent of time spent in the open arms in aged animals in the epm test . the total distance moved and the speed of the animals significantly decreased in aged animals compared to the young control group . sildenafil significantly increased the total distance moved and the speed of the animals in the locomotor activity test in young animals , and it had no significant effect in aged mice . we attempted to evaluate the effects of sildenafil , administered at 3 and 10 mg / kg , on locomotor activity and anxiety in young and aged mice . reported decreased locomotor activity in the plus - maze test in mice administered a 3 mg / kg dose of sildenafil in mice aged 2 months . reported unchanged locomotor activity in mice administered 5 and 10 mg / kg sildenafil , but decreased activity in mice administered 20 mg / kg sildenafil or a combination of 20 mg / kg sildenafil and 30 mg / kg magnesium , in the forced swim test in mice aged 2 months . reported that sildenafil did not modify the behavior of animals in the open field test of locomotor activity in mice aged 34 months . the results of our locomotor activity test in young mice show that the administration of sildenafil ( 3 and 10 mg / kg ) increased the total distance moved and the speed of the animals , but it had no significant effect on locomotion in aged mice . the results of epm test are also correlated with the results of the locomotor activity test because sildenafil ( 3 and 10 mg / kg ) significantly increased total number of arms entries in young animals bu it had no effect on total number of arms entries in aged animals . the administration of sildenafil at low doses has been reported to produce both increases in and no effects on anxiety - like behaviors , using the elevated plus - maze test in mice . some other studies reported that acute administration of sildenafil has anxiogenic effects when administered 30 min before the behavioral tests , which could be attributed to activation of the no - cgmp pathway . similarly , the present study showed anxious behavior in both young and aged mice following the administration of 3 or 10 mg / kg sildenafil . reported that sildenafil - treated mice did not show anxiety - like effects in the elevated plus - maze test . the researchers performed the test 15 min after the administration of sildenafil at doses of 1 , 2 , and 10 mg / kg . however , it remains to be determined whether the anxiolytic effects of sildenafil observed following chronic administration are related to enhanced cgmp levels or to the indirect effects of sildenafil on no . based on our results , we conclude that the acute administration of sildenafil increased locomotor activity in young mice but not in aged mice .

all type 1 diabetic case subjects were recruited for the juvenile diabetes research foundation / wellcome trust diabetes and inflammation laboratory s genetic resource investigating diabetes british , type 1 diabetes collection ( www.childhood-diabetes.org.uk/grid.shtml ) . case subjects were diagnosed with type 1 diabetes under age 17 years ( mean age - at - diagnosis = 7.6 years ) and on average had diabetes for 6.8 years ( table 1 ) . the appropriate ethics committees approved the collection of all dna samples , and written consent was obtained from all individuals or parents of individuals who were too young to consent . distribution of phenotypes in type 1 diabetic case subjects overall and for each autoantibody subgroup the hla - drb1 , hla - dqb1 , hla - a , hla - b , and hla - c genes were typed at four - digit resolution using dynal reli sso assays ( invitrogen , paisley , u.k . ) . a subset of 2,960 case subjects was genotyped at hla - drb1 and hla - dqb1 using roche molecular systems sso reverse dot blot technology . as described previously , the mica - str was genotyped using pcr followed by capillary electrophoresis of the amplified product ( 18 ) ; the 2,420 snps from the type 1 diabetes genetics consortium ( t1dgc ) genome - wide association ( gwa ) study were genotyped using the illumina 550 k infinium platform ( 19 ) , and 1,687 snps from the wellcome trust case - control consortium gwa study were genotyped using the affymetrix genechip human mapping 500 k array set ( 20 ) . three hundred and twenty - eight snps that passed quality control were common to both platforms . follow - up and tag snps were genotyped blind to case - control status using the taqman 5 nuclease assay ( applied biosystems , warrington , u.k . ) according to manufacturer s protocols . presence of each of the autoantibodies ( ia-2a , gada ) in the type 1 diabetic case subjects was tested using plasma stored in aliquots at 80c . autoantibodies to gad and ia-2 were measured in the department of clinical sciences at north bristol , university of bristol , between 2006 and 2007 , using a radioimmunoassay ( 21,22 ) . in the 2007 proficiency evaluation of the diabetes antibody standardization program ( 23 ) , the gada assay achieved 94% sensitivity with 96% specificity , whereas the ia-2 assay sensitivity was 70% sensitivity with 98% specificity . presence of gada and ia-2a was taken as above 14 and 6 world health organization units / ml , respectively , which corresponds to the 97.5th percentile of the distribution of these autoantibodies in 2,860 school children from oxford , u.k . we did not measure iaa since most case subjects were recruited after diagnosis and antibodies to exogenous insulin are produced . all statistical analyses were carried out in stata ( www.stata.com ) or r ( 24 ) . associations with autoantibodies were tested using logistic regression models with autoantibody status ( positive / negative ) as the outcome variable and genotype at the test locus and any covariates ( sex , duration , age - at - diagnosis ; see results ) as predictors . inclusion of the covariates accounted for their effects , such that association of genotype with autoantibody positivity was tested , rather than , for example , association of genotype with decline in autoantibody positivity ( as a result of disease duration ) . a model with and without the genotype variable haplotypes were generated using the r library haplo.stats ( available from www.r-project.org ) and analyzed by logistic regression using the posterior probabilities as weights and robust variance estimates to account for the dependency between haplotype assignments . despite being genotyped at four - digit resolution , to maximize statistical power , analyses of the classical loci were confined to two - digit resolution , except for hla - dqb1 * 0301 and hla - dqb1 * 0302 . all type 1 diabetic case subjects were recruited for the juvenile diabetes research foundation / wellcome trust diabetes and inflammation laboratory s genetic resource investigating diabetes british , type 1 diabetes collection ( www.childhood-diabetes.org.uk/grid.shtml ) . case subjects were diagnosed with type 1 diabetes under age 17 years ( mean age - at - diagnosis = 7.6 years ) and on average had diabetes for 6.8 years ( table 1 ) . the appropriate ethics committees approved the collection of all dna samples , and written consent was obtained from all individuals or parents of individuals who were too young to consent . the hla - drb1 , hla - dqb1 , hla - a , hla - b , and hla - c genes were typed at four - digit resolution using dynal reli sso assays ( invitrogen , paisley , u.k . ) . a subset of 2,960 case subjects was genotyped at hla - drb1 and hla - dqb1 using roche molecular systems sso reverse dot blot technology . as described previously , the mica - str was genotyped using pcr followed by capillary electrophoresis of the amplified product ( 18 ) ; the 2,420 snps from the type 1 diabetes genetics consortium ( t1dgc ) genome - wide association ( gwa ) study were genotyped using the illumina 550 k infinium platform ( 19 ) , and 1,687 snps from the wellcome trust case - control consortium gwa study were genotyped using the affymetrix genechip human mapping 500 k array set ( 20 ) . three hundred and twenty - eight snps that passed quality control were common to both platforms . follow - up and tag snps were genotyped blind to case - control status using the taqman 5 nuclease assay ( applied biosystems , warrington , u.k . ) according to manufacturer s protocols . presence of each of the autoantibodies ( ia-2a , gada ) in the type 1 diabetic case subjects was tested using plasma stored in aliquots at 80c . autoantibodies to gad and ia-2 were measured in the department of clinical sciences at north bristol , university of bristol , between 2006 and 2007 , using a radioimmunoassay ( 21,22 ) . in the 2007 proficiency evaluation of the diabetes antibody standardization program ( 23 ) , the gada assay achieved 94% sensitivity with 96% specificity , whereas the ia-2 assay sensitivity was 70% sensitivity with 98% specificity . presence of gada and ia-2a was taken as above 14 and 6 world health organization units / ml , respectively , which corresponds to the 97.5th percentile of the distribution of these autoantibodies in 2,860 school children from oxford , u.k . we did not measure iaa since most case subjects were recruited after diagnosis and antibodies to exogenous insulin are produced . all statistical analyses were carried out in stata ( www.stata.com ) or r ( 24 ) . associations with autoantibodies were tested using logistic regression models with autoantibody status ( positive / negative ) as the outcome variable and genotype at the test locus and any covariates ( sex , duration , age - at - diagnosis ; see results ) as predictors . inclusion of the covariates accounted for their effects , such that association of genotype with autoantibody positivity was tested , rather than , for example , association of genotype with decline in autoantibody positivity ( as a result of disease duration ) . a model with and without the genotype variable haplotypes were generated using the r library haplo.stats ( available from www.r-project.org ) and analyzed by logistic regression using the posterior probabilities as weights and robust variance estimates to account for the dependency between haplotype assignments . despite being genotyped at four - digit resolution , to maximize statistical power , analyses of the classical loci were confined to two - digit resolution , except for hla - dqb1 * 0301 and hla - dqb1 * 0302 . gada were measured in 2,530 type 1 diabetic case subjects , 1,274 ( 50.4% ) of whom were found to have levels of gada above the 97.5th percentile for the reference population ( research design and methods ) and were defined as gada positive . ia-2a were measured in 2,521 type 1 diabetic case subjects , 1,475 ( 58.5% ) of whom had levels above the 97.5th percentile for the reference population ( research design and methods ) and were defined as ia-2a positive . of the 2,520 case subjects tested for both ia-2a and gada , 788 ( 31.3% ) were positive for both autoantibodies and 1,958 ( 77.7% ) were positive for at least one of ia-2a and gada . as expected , gada positivity and ia-2a positivity were both found to be correlated with a shorter duration of diabetes ( p = 9.80 10 and 8.65 10 , respectively , having accounted for age - at - diagnosis effects ; table 1 ) . in addition , gada positivity and ia-2a positivity were also correlated with a later age - at - diagnosis ( p = 3.72 10 and 7.22 10 , respectively , having accounted for duration of diabetes ; supplementary data ) . more females were gada positive than males ( p = 2.91 10 ; table 1 ) . hence , age - at - diagnosis and duration of disease were included as covariates in the statistical models testing for association at ia-2a and gada ; in addition , sex was included for gada . in total 3,779 snps between 25 and 33 mb on chromosome 6p21 containing the extended mhc region , and the hla - dqb1 , hla - drb1 , hla - b , hla - c , hla - a , and mica(str ) genes were analyzed for association with gada . in type 1 diabetes , the strongest association signal was with the hla class ii region ( 10,11 ) , which was also found to have the strongest signal for association with gada positivity ( fig . the strongest association with gada positivity was with hla - dqb1 ( p = 9.00 10 ) , but hla - drb1 was also convincingly associated ( p = 3.10 10 ; table 2 ) . indeed , the two snps most associated with gada positivity ( rs502055 , located at 32.579 mb between hla - drb1 and hla - dqa1 , p = 6.53 10 ; and rs2187668 , located at 32.606 mb in intron 1 of hla - dqa1 , p = 1.28 10 ; fig . 1a ) were in linkage disequilibrium ( ld ) with the type 1 diabetes susceptible allele , hla - dqb1 * 02 ( r > 0.69 ) and were in strong ld with the hla - drb1 * 03 type 1 diabetes susceptible allele ( r > 0.85 ) . a : association of snps used in the t1dgc illumina experiment ( using 1,218 type 1 diabetic case subjects ) and the wtccc affymetrix experiment ( using 1,056 case subjects ) and the classical loci ( using between 2,408 and 1,275 type 1 diabetic case subjects ; table 2 ) with gada positivity . the top snps from both the wtccc and t1dgc experiments are in linkage disequilibrium with hla - drb1 * 03 ( r = 0.85 and d = 1.00 with rs502055 and r = 1.00 and d = 1.00 with rs2187668 ) . b : association of snps and genes with gada positivity conditional on hla - dqb1 genotypes . association of the hla genes with gada positivity in type 1 diabetic case subjects * the multiplicative allelic effects model was not an appropriate approximation ( p = 5.66 10 for hla - dqb1 , p = 0.0011 for hla - drb1 , p = 0.0015 for hla - a ) ; hence the p value for the genotype effects model is reported . there is very little to distinguish the effects of hla - drb1 and hla - dqb1 on gada positivity ( p = 0.002 for addition of hla - dqb1 to hla - drb1 and p = 0.04 for addition of hla - drb1 to hla - dqb1 ; table 2 ) , such that either hla - drb1 or hla - dqb1 is sufficient to model the association , but both are not required . this contrasts with type 1 diabetes in which both genes are independently associated with disease and add to the association of each other ( p < 3 10 ; supplementary data and supplementary tables 1 and 2 ) . we examined which specific class ii alleles and genotypes were involved in the gada association and whether these were consistent with the type 1 diabetes associations . despite having full four - digit hla typing , owing to the rarity of many four - digit alleles , the hla loci were analyzed at two - digit resolution , except at hla - dqb1 for the hla - dqb1 * 0301 and * 0302 alleles , which are known to have opposite effects in type 1 diabetes ( odds ratio [ or ] [ 95% ci ] = 4.21 [ 3.465.13 ] and 0.44 [ 0.360.54 ] , respectively ; data not shown ) . at hla - dqb1 , the 02/02 genotype was most associated with gada positivity ( or [ 95% ci ] = 2.32 [ 1.713.13 ] ; table 3 ) , whereas at hla - drb1 , as expected owing to ld , the 03/03 genotype was most associated with gada positivity ( or [ 95% ci ] = 2.60 [ 1.863.65 ] ; table 4 ) . these findings , and that hla - drb1 * 03 was convincingly associated with gada positivity ( p = 4.80 10 ; supplementary table 4 ) , unexpectedly , the highly type 1 diabetes susceptible hla - dqb1 * 02/0302 genotype ( supplementary data and supplementary table 1 ) had a comparable frequency in gada positives ( 0.38 ) and gada negatives ( 0.40 ; table 3 ) . similarly , the most type 1 diabetes associated genotype at hla - drb1 , hla - drb1 * 03/04 ( supplementary tables 2 and 3 ) , was not associated with gada ( or = 1.23 [ 0.901.68 ] using 04/04 as reference [ table 4 ] ) . there was also no suggestion of an hla - drb1 * 03/04 synergic effect with gada ( p = 0.19 , compared with p < 7 10 , in type 1 diabetes ; supplementary data ) . both the hla - dqb1 * 0302/0302 genotype and the hla - drb1 * 04/04 genotype are highly susceptible for type 1 diabetes ( supplementary tables 1 and 2 ) , but these genotypes were not associated with gada positivity ( tables 3 and 4 ) . this is different from type 1 diabetes in which all hla - dqb1 * 0302/x and hla - drb1 * 04/x genotypes confer susceptibility ( supplementary tables 1 and 2 ) . hla - dqb1 * 0302 and hla - drb1 * 04 alleles were only ( negatively ) associated with gada positivity when they were with specific type 1 diabetes low risk alleles ( * 06 or * 04 at hla - dqb1 and * 01 or * 13 at hla - drb1 ; tables 3 and 4 ) , which contrasts other reports ( 13,14 ) . association of hla - dqb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . or are reported with 95% ci using the most common , neutrally associated , genotype hla - dqb1 * 02/0302 as reference and also the less common neutral genotype hla - dqb1 * 0302/0302 as reference . association of hla - drb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were calculated accounting for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . hla - drb1 * 0403 and * 0407 alleles have opposite effects in type 1 diabetes to the remaining hla - dbr1 * 04 alleles ; however , they are of such low frequency in the case subjects that an extra subtype was not warranted and so are included with the hla - drb1 * 04 allele . the apparent association of certain hla - drb1 genotypes following conditioning on hla - dqb1 are likely to be attributable to effects outside of the class ii region ( see main text ) and all have very large 95% cis reflecting uncertainty in the model . haplotypes of hla-dqb1.hla-drb1 were also tested for association with gada ( supplementary table 5 ) and reflected the unconditional associations observed ( tables 3 and 4 ) . the strongest association with gada positivity was with the hla - dqb1 * 02.hla - drb1 * 03/hla - dqb1 * 02.hla - drb1 * 03 homozygous genotype ( or [ 95% ci ] = 2.62 [ 1.873.67 ] ; supplementary table 5 ) ; hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 05.hla - drb1 * 01 , hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 0402.hla - drb1 * 08 , and hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 06.hla - drb1 * 13 were negatively associated with gada positivity . in contrast , hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 05.hla - drb1 * 01 and hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 0402.hla - drb1 * 08 were associated with a significant increase in risk of type 1 diabetes ( supplementary table 1 ) . hla - b*18 is associated with increased risk of type 1 diabetes , whereas hla - b*08 is not ( 10,11 ) . however , although hla - b*08.hla - drb1 * 03.hla - dqb1 * 02 could well be driving the association of hla - drb1 * 03 and hla - dqb1 * 02 with gada positivity , the effect could not strictly be distinguished from hla - b*18.hla - drb1 * 03.hla - dqb1 * 02 or hla - b*x.hla - drb1 * 03.hla - dqb1 * 02 since the 95% cis overlapped ( supplementary table 6 ) . the extended dr3 haplotype , hla - a*01.hla - b*08.hla - drb1 * 03 , was also associated with gada positivity ( p = 1.24 10 ; or = 1.75 [ 1.402.19 ] ) . the association with gada positivity extended into the class i region , and included hla - b , hla - c , hla - a , and mica ( table 2 and fig . given the strong ld in the region , these associations were likely to be attributable to the hla class ii effects . hla - dqb1 was the most associated locus with gada and so was used to test whether there were additional associations that were outside and independent of the hla class ii region . having conditioned on hla - dqb1 , there remained some evidence of association around mica ( p = 1.08 10 ) including two snps , rs9266722 and rs4713462 , both at 31.35 mb ( p = 2.84 10 and 9.66 10 ; fig . 1b ) , which were in ld with each other ( d = 0.98 ; r = 0.36 ) . the genotyping of rs9266722 was checked and extended to the full collection with gada measures ( an additional 1,124 samples ) to maximize power . the genotyping was 99.90% consistent , and the association of rs9266722 remained in the 2,320 samples tested for association with gada conditional on hla - dqb1 ( p = 4.84 10 ) . the snp , rs9266722 , was in ld with hla - b*15 ( r = 0.58 ; d = 0.93 ) and the mica - str*a5 allele ( r = 0.48 ; d = 0.79 ) . however , the association with mica did not remain once rs9266722 , which lies in the pseudo - gene hla - s located between hla - b and mica , was in the model ( p = 0.33 ) . even the mica - str*a5 allele alone did not add to the association of rs9266722 ( p = 0.16 ) . conversely , rs9266722 did not add convincingly to the association of the mica - str*a5 ( p = 0.017 ) . likewise , the effects of hla - b and rs9266722 could not be distinguished ; hla - b did not improve the association of hla - dqb1 and rs9266722 ( p = 0.04 ; table 2 ) and rs9266722 did not improve the association of hla - dqb1 and hla - b ( p = 0.13 ) . at hla - a , although no alleles were in ld with rs9266722 ( r > 0.04 ) , the effect of rs9266722 could not be distinguished from hla - a ( p = 0.0045 for the addition of hla - a to a model including hla - dqb1 and rs9266722 ; p = 0.0039 for the addition of rs9266722 to a model including hla - dqb1 and hla - a ) . more samples , therefore , are needed to decipher the association with gada positivity in the class i region . once the effect of hla - dqb1 was accounted for , as well as the association discussed in the class i region , evidence for an additional association with gada positivity was found with the intronic snp , rs926850 in fam65b at 25.0 mb ( p = 3.15 10 ; fig . 1b ) . to increase power , this snp was genotyped in an additional 1,101 case subjects and further tested for association with gada . the evidence did not improve , becoming relatively unconvincing ( p = 0.0067 ) , and , hence , associations with gada were confined to the hla class ii region and a second independent effect in the class i region . convincing evidence of the association with ia-2a was obtained in both the hla class i and ii regions ( p < 3 10 ; fig . hla - drb1 had the strongest evidence for association with ia-2a positivity ( p = 1.94 10 ; fig . however , in contrast with the association with type 1 diabetes , there was no evidence of deviation from a multiplicative allelic effects model ( p > 0.2 for the class i and ii genes ) . 2a ) , which was genotyped in the wtccc experiment ( and by the t1dgc , p = 5.9 10 ) , was also in the class ii region , between the pseudo - genes mtc03p1 and hla - dqb3 . it was in strong ld with hla - drb1 * 03 ( r = 0.72 ; d = 1.00 ) . a : association of snps used in the t1dgc illumina experiment ( using 1,215 type 1 diabetic case subjects ) and the wtccc affymetrix experiment ( using 1,051 case subjects ) with ia-2a and the classical loci ( using between 2,399 and 1,268 type 1 diabetic case subjects ) . the most associated snp , rs9275572 , is in ld with hla - drb1 * 03 ( r = 0.72 , d = 1.00 ) . b : association of snps and classical genes with ia-2a , conditional on hla - drb1 alleles . the most associated snp , rs9258750 , is in ld with the hla - a*24 allele ( r = 0.55 , d = 0.99 ) . association of the hla genes with ia-2a positivity in type 1 diabetic case subjects * physical positions are from grch37 ( www.t1dbase.org ) . the hla - drb1 * 03 allele ( and the hla - dqb1 * 02 allele ) was negatively associated with ia-2a positivity ( or [ 95% ci ] = 0.44 [ 0.340.58 ] ) for hla - drb1 * 03 using hla - drb1 * 0404 as reference ( table 6 ) . in contrast , hla - drb1 * 0401 ( and its associated hla - dqb1 allele , hla - dqb1 * 0302 ) were strongly associated with ia-2a positivity ( or = 1.72 [ 1.342.20 ] ; table 6 ) , a major similarity to type 1 diabetes susceptibility ( supplementary table 2 ) . interestingly , because the effects of hla - drb1 * 0401 and hla - drb1 * 03 were in opposite directions , the most susceptible type 1 diabetes genotype , hla - drb1 * 03/04 , was not associated with ia-2a , having a frequency of 0.331 in the ia-2a positive case subjects and 0.356 in ia-2a negatives ( or = 1.07 [ 0.741.54 ] ) using as a reference the non - hla - drb1 * 03 , non - hla - drb1 * 04 homozygous genotype , which had a frequency of 0.064 in ia-2a positives and negatives ( supplementary table 7 ) . however , unlike another report of only 618 samples ( 17 ) , we found that the type 1 diabetes protective allele , hla - drb1 * 07 ( supplementary table 2 ) , was negatively correlated with ia-2a ( or = 0.65 [ 0.450.93 ] ; table 6 ) . the highly type 1 diabetes protective allele , hla - dqb1 * 0301 , however , hla - drb1 and hla - dqb1 allelic associations with ia-2a positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates , duration of disease , and age - at - diagnosis , whereas the listed allele frequencies were not . rare : alleles 10 , 12 , 14 , 15 , 16 , and 0403 had a frequency of less than 0.5% and so were combined . na , not available ( the 04 alleles were combined , except 0404 , which was neutral , and 0401 , which was associated ) . in addition to the evidence of association in the class ii region , the hla class i region also showed evidence of association with ia-2a positivity ( fig . the snp , rs915668 near hla - a , was the most associated in that region ( p = 5.71 10 ) . however , these class i associations with ia-2a positivity could be attributable to ld with hla class ii alleles , and , hence , we tested the class i variants conditional on class ii to ensure they are independent of the primary class ii effect . a model including the hla - drb1 alleles was only improved by hla - a ( p = 6.13 10 ; table 5 ) and snps in the region adjacent to hla - a ( fig . no evidence of additional effects at the other genes and snps tested was obtained ( fig . the class ii independent effect in the class i region with ia-2a was largely attributable to hla - a*24 ( p = 1.21 10 for the association of hla - a*24 alone conditional on hla - drb1 ) . despite being associated with susceptibility to type 1 diabetes ( 10 ) , hla - a*24 was negatively associated with ia-2a positivity ( or [ 95% ci ] = 0.44 [ 0.340.57 ] ) using all other non - hla - a*24 alleles at hla - a as reference . strikingly , haplotype analyses showed that hla - a*24 significantly reduced the positive ia-2a association of hla - drb1 * 04 from ( or [ 95% ci ] = 2.02 [ 1.492.73 ] ) for the hla - a*02.hla - drb1 * 04 haplotype to a neutral effect for the hla - a*24.hla - drb1 * 04 haplotype ( or = 0.94 [ 0.631.40 ] ; supplementary table 8) . although hla - drb1 * 03 was negatively associated with ia-2a ( table 6 ) , the association was significantly stronger in the presence of hla - a*24 on the same haplotype ( or = 0.55 [ 0.390.77 ] hla - a*02.hla - drb1 * 03 compared with or = 0.25 [ 0.160.38 ] for hla - a*24.hla - drb1 * 03 ; supplementary table 8) . note , however , that hla - drb1 * 03 remained negatively associated with ia-2a positivity even after conditioning on hla - a*24 ( or = 0.35 [ 0.260.49 ] using hla - drb1 * 0404 as reference ; table 6 ) . the hla - a*01.hla - b*08.hla - drb1 * 03 extended haplotype was also negatively associated with ia-2a positivity ( or [ 95% ci ] = 0.59 [ 0.440.78 ] ) , and again presence of hla - a*24 increased this association ( or = 0.26 [ 0.130.52 ] ; supplementary table 9 ) . the negative association of hla - drb1 * 03 was , as expected given the lack of independent association at hla - b , unaltered whether it was on the hla - b*08.hla - drb1 * 03 haplotype or the hla - b*18.hla - drb1 * 03 haplotype ( or = 0.29 and 0.34 , respectively ; supplementary table 10 ) . gada were measured in 2,530 type 1 diabetic case subjects , 1,274 ( 50.4% ) of whom were found to have levels of gada above the 97.5th percentile for the reference population ( research design and methods ) and were defined as gada positive . ia-2a were measured in 2,521 type 1 diabetic case subjects , 1,475 ( 58.5% ) of whom had levels above the 97.5th percentile for the reference population ( research design and methods ) and were defined as ia-2a positive . of the 2,520 case subjects tested for both ia-2a and gada , 788 ( 31.3% ) were positive for both autoantibodies and 1,958 ( 77.7% ) were positive for at least one of ia-2a and gada . as expected , gada positivity and ia-2a positivity were both found to be correlated with a shorter duration of diabetes ( p = 9.80 10 and 8.65 10 , respectively , having accounted for age - at - diagnosis effects ; table 1 ) . in addition , gada positivity and ia-2a positivity were also correlated with a later age - at - diagnosis ( p = 3.72 10 and 7.22 10 , respectively , having accounted for duration of diabetes ; supplementary data ) . more females were gada positive than males ( p = 2.91 10 ; table 1 ) . hence , age - at - diagnosis and duration of disease were included as covariates in the statistical models testing for association at ia-2a and gada ; in addition , sex was included for gada . in total 3,779 snps between 25 and 33 mb on chromosome 6p21 containing the extended mhc region , and the hla - dqb1 , hla - drb1 , hla - b , hla - c , hla - a , and mica(str ) genes were analyzed for association with gada . in type 1 diabetes , the strongest association signal was with the hla class ii region ( 10,11 ) , which was also found to have the strongest signal for association with gada positivity ( fig . the strongest association with gada positivity was with hla - dqb1 ( p = 9.00 10 ) , but hla - drb1 was also convincingly associated ( p = 3.10 10 ; table 2 ) . indeed , the two snps most associated with gada positivity ( rs502055 , located at 32.579 mb between hla - drb1 and hla - dqa1 , p = 6.53 10 ; and rs2187668 , located at 32.606 mb in intron 1 of hla - dqa1 , p = 1.28 10 ; fig . 1a ) were in linkage disequilibrium ( ld ) with the type 1 diabetes susceptible allele , hla - dqb1 * 02 ( r > 0.69 ) and were in strong ld with the hla - drb1 * 03 type 1 diabetes susceptible allele ( r > 0.85 ) . a : association of snps used in the t1dgc illumina experiment ( using 1,218 type 1 diabetic case subjects ) and the wtccc affymetrix experiment ( using 1,056 case subjects ) and the classical loci ( using between 2,408 and 1,275 type 1 diabetic case subjects ; table 2 ) with gada positivity . the top snps from both the wtccc and t1dgc experiments are in linkage disequilibrium with hla - drb1 * 03 ( r = 0.85 and d = 1.00 with rs502055 and r = 1.00 and d = 1.00 with rs2187668 ) . b : association of snps and genes with gada positivity conditional on hla - dqb1 genotypes . association of the hla genes with gada positivity in type 1 diabetic case subjects * the multiplicative allelic effects model was not an appropriate approximation ( p = 5.66 10 for hla - dqb1 , p = 0.0011 for hla - drb1 , p = 0.0015 for hla - a ) ; hence the p value for the genotype effects model is reported . there is very little to distinguish the effects of hla - drb1 and hla - dqb1 on gada positivity ( p = 0.002 for addition of hla - dqb1 to hla - drb1 and p = 0.04 for addition of hla - drb1 to hla - dqb1 ; table 2 ) , such that either hla - drb1 or hla - dqb1 is sufficient to model the association , but both are not required . this contrasts with type 1 diabetes in which both genes are independently associated with disease and add to the association of each other ( p < 3 10 ; supplementary data and supplementary tables 1 and 2 ) . we examined which specific class ii alleles and genotypes were involved in the gada association and whether these were consistent with the type 1 diabetes associations . despite having full four - digit hla typing , owing to the rarity of many four - digit alleles , the hla loci were analyzed at two - digit resolution , except at hla - dqb1 for the hla - dqb1 * 0301 and * 0302 alleles , which are known to have opposite effects in type 1 diabetes ( odds ratio [ or ] [ 95% ci ] = 4.21 [ 3.465.13 ] and 0.44 [ 0.360.54 ] , respectively ; data not shown ) . at hla - dqb1 , the 02/02 genotype was most associated with gada positivity ( or [ 95% ci ] = 2.32 [ 1.713.13 ] ; table 3 ) , whereas at hla - drb1 , as expected owing to ld , the 03/03 genotype was most associated with gada positivity ( or [ 95% ci ] = 2.60 [ 1.863.65 ] ; table 4 ) . these findings , and that hla - drb1 * 03 was convincingly associated with gada positivity ( p = 4.80 10 ; supplementary table 4 ) , agree with previous reports in the literature ( 13,14 ) . unexpectedly , the highly type 1 diabetes susceptible hla - dqb1 * 02/0302 genotype ( supplementary data and supplementary table 1 ) had a comparable frequency in gada positives ( 0.38 ) and gada negatives ( 0.40 ; table 3 ) . similarly , the most type 1 diabetes associated genotype at hla - drb1 , hla - drb1 * 03/04 ( supplementary tables 2 and 3 ) , was not associated with gada ( or = 1.23 [ 0.901.68 ] using 04/04 as reference [ table 4 ] ) . there was also no suggestion of an hla - drb1 * 03/04 synergic effect with gada ( p = 0.19 , compared with p < 7 10 , in type 1 diabetes ; supplementary data ) . both the hla - dqb1 * 0302/0302 genotype and the hla - drb1 * 04/04 genotype are highly susceptible for type 1 diabetes ( supplementary tables 1 and 2 ) , but these genotypes were not associated with gada positivity ( tables 3 and 4 ) . this is different from type 1 diabetes in which all hla - dqb1 * 0302/x and hla - drb1 * 04/x genotypes confer susceptibility ( supplementary tables 1 and 2 ) . hla - dqb1 * 0302 and hla - drb1 * 04 alleles were only ( negatively ) associated with gada positivity when they were with specific type 1 diabetes low risk alleles ( * 06 or * 04 at hla - dqb1 and * 01 or * 13 at hla - drb1 ; tables 3 and 4 ) , which contrasts other reports ( 13,14 ) . association of hla - dqb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . or are reported with 95% ci using the most common , neutrally associated , genotype hla - dqb1 * 02/0302 as reference and also the less common neutral genotype hla - dqb1 * 0302/0302 as reference . association of hla - drb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were calculated accounting for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . hla - drb1 * 0403 and * 0407 alleles have opposite effects in type 1 diabetes to the remaining hla - dbr1 * 04 alleles ; however , they are of such low frequency in the case subjects that an extra subtype was not warranted and so are included with the hla - drb1 * 04 allele . the apparent association of certain hla - drb1 genotypes following conditioning on hla - dqb1 are likely to be attributable to effects outside of the class ii region ( see main text ) and all have very large 95% cis reflecting uncertainty in the model . haplotypes of hla-dqb1.hla-drb1 were also tested for association with gada ( supplementary table 5 ) and reflected the unconditional associations observed ( tables 3 and 4 ) . the strongest association with gada positivity was with the hla - dqb1 * 02.hla - drb1 * 03/hla - dqb1 * 02.hla - drb1 * 03 homozygous genotype ( or [ 95% ci ] = 2.62 [ 1.873.67 ] ; supplementary table 5 ) ; hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 05.hla - drb1 * 01 , hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 0402.hla - drb1 * 08 , and hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 06.hla - drb1 * 13 were negatively associated with gada positivity . in contrast , hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 05.hla - drb1 * 01 and hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 0402.hla - drb1 * 08 were associated with a significant increase in risk of type 1 diabetes ( supplementary table 1 ) . hla - b*18 is associated with increased risk of type 1 diabetes , whereas hla - b*08 is not ( 10,11 ) . however , although hla - b*08.hla - drb1 * 03.hla - dqb1 * 02 could well be driving the association of hla - drb1 * 03 and hla - dqb1 * 02 with gada positivity , the effect could not strictly be distinguished from hla - b*18.hla - drb1 * 03.hla - dqb1 * 02 or hla - b*x.hla - drb1 * 03.hla - dqb1 * 02 since the 95% cis overlapped ( supplementary table 6 ) . the extended dr3 haplotype , hla - a*01.hla - b*08.hla - drb1 * 03 , was also associated with gada positivity ( p = 1.24 10 ; or = 1.75 [ 1.402.19 ] ) . the association with gada positivity extended into the class i region , and included hla - b , hla - c , hla - a , and mica ( table 2 and fig . 1a ) . given the strong ld in the region , these associations were likely to be attributable to the hla class ii effects . hla - dqb1 was the most associated locus with gada and so was used to test whether there were additional associations that were outside and independent of the hla class ii region . having conditioned on hla - dqb1 , there remained some evidence of association around mica ( p = 1.08 10 ) including two snps , rs9266722 and rs4713462 , both at 31.35 mb ( p = 2.84 10 and 9.66 10 ; fig . 1b ) , which were in ld with each other ( d = 0.98 ; r = 0.36 ) . the genotyping of rs9266722 was checked and extended to the full collection with gada measures ( an additional 1,124 samples ) to maximize power . the genotyping was 99.90% consistent , and the association of rs9266722 remained in the 2,320 samples tested for association with gada conditional on hla - dqb1 ( p = 4.84 10 ) . the snp , rs9266722 , was in ld with hla - b*15 ( r = 0.58 ; d = 0.93 ) and the mica - str*a5 allele ( r = 0.48 ; d = 0.79 ) . however , the association with mica did not remain once rs9266722 , which lies in the pseudo - gene hla - s located between hla - b and mica , was in the model ( p = 0.33 ) . even the mica - str*a5 allele alone did not add to the association of rs9266722 ( p = 0.16 ) . conversely , rs9266722 did not add convincingly to the association of the mica - str*a5 ( p = 0.017 ) . likewise , the effects of hla - b and rs9266722 could not be distinguished ; hla - b did not improve the association of hla - dqb1 and rs9266722 ( p = 0.04 ; table 2 ) and rs9266722 did not improve the association of hla - dqb1 and hla - b ( p = 0.13 ) . at hla - a , although no alleles were in ld with rs9266722 ( r > 0.04 ) , the effect of rs9266722 could not be distinguished from hla - a ( p = 0.0045 for the addition of hla - a to a model including hla - dqb1 and rs9266722 ; p = 0.0039 for the addition of rs9266722 to a model including hla - dqb1 and hla - a ) . more samples , therefore , are needed to decipher the association with gada positivity in the class i region . once the effect of hla - dqb1 was accounted for , as well as the association discussed in the class i region , evidence for an additional association with gada positivity was found with the intronic snp , rs926850 in fam65b at 25.0 mb ( p = 3.15 10 ; fig . 1b ) . to increase power , this snp was genotyped in an additional 1,101 case subjects and further tested for association with gada . the evidence did not improve , becoming relatively unconvincing ( p = 0.0067 ) , and , hence , associations with gada were confined to the hla class ii region and a second independent effect in the class i region . convincing evidence of the association with ia-2a was obtained in both the hla class i and ii regions ( p < 3 10 ; fig . hla - drb1 had the strongest evidence for association with ia-2a positivity ( p = 1.94 10 ; fig . however , in contrast with the association with type 1 diabetes , there was no evidence of deviation from a multiplicative allelic effects model ( p > 0.2 for the class i and ii genes ) . 2a ) , which was genotyped in the wtccc experiment ( and by the t1dgc , p = 5.9 10 ) , was also in the class ii region , between the pseudo - genes mtc03p1 and hla - dqb3 . it was in strong ld with hla - drb1 * 03 ( r = 0.72 ; d = 1.00 ) . a : association of snps used in the t1dgc illumina experiment ( using 1,215 type 1 diabetic case subjects ) and the wtccc affymetrix experiment ( using 1,051 case subjects ) with ia-2a and the classical loci ( using between 2,399 and 1,268 type 1 diabetic case subjects ) . the most associated snp , rs9275572 , is in ld with hla - drb1 * 03 ( r = 0.72 , d = 1.00 ) . b : association of snps and classical genes with ia-2a , conditional on hla - drb1 alleles . the most associated snp , rs9258750 , is in ld with the hla - a*24 allele ( r = 0.55 , d = 0.99 ) . association of the hla genes with ia-2a positivity in type 1 diabetic case subjects * physical positions are from grch37 ( www.t1dbase.org ) . the hla - drb1 * 03 allele ( and the hla - dqb1 * 02 allele ) was negatively associated with ia-2a positivity ( or [ 95% ci ] = 0.44 [ 0.340.58 ] ) for hla - drb1 * 03 using hla - drb1 * 0404 as reference ( table 6 ) . in contrast , hla - drb1 * 0401 ( and its associated hla - dqb1 allele , hla - dqb1 * 0302 ) were strongly associated with ia-2a positivity ( or = 1.72 [ 1.342.20 ] ; table 6 ) , a major similarity to type 1 diabetes susceptibility ( supplementary table 2 ) . interestingly , because the effects of hla - drb1 * 0401 and hla - drb1 * 03 were in opposite directions , the most susceptible type 1 diabetes genotype , hla - drb1 * 03/04 , was not associated with ia-2a , having a frequency of 0.331 in the ia-2a positive case subjects and 0.356 in ia-2a negatives ( or = 1.07 [ 0.741.54 ] ) using as a reference the non - hla - drb1 * 03 , non - hla - drb1 * 04 homozygous genotype , which had a frequency of 0.064 in ia-2a positives and negatives ( supplementary table 7 ) . however , unlike another report of only 618 samples ( 17 ) , we found that the type 1 diabetes protective allele , hla - drb1 * 07 ( supplementary table 2 ) , was negatively correlated with ia-2a ( or = 0.65 [ 0.450.93 ] ; table 6 ) . the highly type 1 diabetes protective allele , hla - dqb1 * 0301 , however , hla - drb1 and hla - dqb1 allelic associations with ia-2a positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates , duration of disease , and age - at - diagnosis , whereas the listed allele frequencies were not . rare : alleles 10 , 12 , 14 , 15 , 16 , and 0403 had a frequency of less than 0.5% and so were combined . na , not available ( the 04 alleles were combined , except 0404 , which was neutral , and 0401 , which was associated ) . in addition to the evidence of association in the class ii region , the hla class i region also showed evidence of association with ia-2a positivity ( fig . the snp , rs915668 near hla - a , was the most associated in that region ( p = 5.71 10 ) . however , these class i associations with ia-2a positivity could be attributable to ld with hla class ii alleles , and , hence , we tested the class i variants conditional on class ii to ensure they are independent of the primary class ii effect . a model including the hla - drb1 alleles was only improved by hla - a ( p = 6.13 10 ; table 5 ) and snps in the region adjacent to hla - a ( fig . no evidence of additional effects at the other genes and snps tested was obtained ( fig . the class ii independent effect in the class i region with ia-2a was largely attributable to hla - a*24 ( p = 1.21 10 for the association of hla - a*24 alone conditional on hla - drb1 ) . despite being associated with susceptibility to type 1 diabetes ( 10 ) , hla - a*24 was negatively associated with ia-2a positivity ( or [ 95% ci ] = 0.44 [ 0.340.57 ] ) using all other non - hla - a*24 alleles at hla - a as reference . strikingly , haplotype analyses showed that hla - a*24 significantly reduced the positive ia-2a association of hla - drb1 * 04 from ( or [ 95% ci ] = 2.02 [ 1.492.73 ] ) for the hla - a*02.hla - drb1 * 04 haplotype to a neutral effect for the hla - a*24.hla - drb1 * 04 haplotype ( or = 0.94 [ 0.631.40 ] ; supplementary table 8) . although hla - drb1 * 03 was negatively associated with ia-2a ( table 6 ) , the association was significantly stronger in the presence of hla - a*24 on the same haplotype ( or = 0.55 [ 0.390.77 ] hla - a*02.hla - drb1 * 03 compared with or = 0.25 [ 0.160.38 ] for hla - a*24.hla - drb1 * 03 ; supplementary table 8) . note , however , that hla - drb1 * 03 remained negatively associated with ia-2a positivity even after conditioning on hla - a*24 ( or = 0.35 [ 0.260.49 ] using hla - drb1 * 0404 as reference ; table 6 ) . the hla - a*01.hla - b*08.hla - drb1 * 03 extended haplotype was also negatively associated with ia-2a positivity ( or [ 95% ci ] = 0.59 [ 0.440.78 ] ) , and again presence of hla - a*24 increased this association ( or = 0.26 [ 0.130.52 ] ; supplementary table 9 ) . the negative association of hla - drb1 * 03 was , as expected given the lack of independent association at hla - b , unaltered whether it was on the hla - b*08.hla - drb1 * 03 haplotype or the hla - b*18.hla - drb1 * 03 haplotype ( or = 0.29 and 0.34 , respectively ; supplementary table 10 ) . because levels of autoantibodies reduce after diagnosis of disease ( 26 ) , there will be an increased false - negative rate for ia-2a and gada compared with at time of diagnosis . a faster decline in gada and/or ia-2a positivity in the younger onset case subjects , which we had limited power to detect , may be possible . however , we have adjusted for diabetes duration in our analyses , so the genetic associations we report were not attributable to decline in autoantibody levels ( supplementary data ) . although the false - negative rate reduces the power of our study to detect genetic associations , it does not affect the significant associations obtained . we can not comment on genetic associations with autoantibody positivity prediagnosis since we only measured autoantibodies at a single time point at or after diagnosis . however , we were interested in the genetic association of these autoantibodies among case subjects , not in using them for prediction of type 1 diabetes onset ; hence this will not affect our study since the samples were positive at the time of measurement . indeed , our findings are highly consistent with other studies of ia-2a and gada ( 1316 ) , and 30% of our case subjects were sampled within 2 years of diagnosis . our data show that although both gada and ia-2a are very strongly associated with type 1 diabetes , and risk of all three localizes to the mhc region , the alleles and genotypes involved differ between autoantibodies and type 1 diabetes . our major novel finding is that even though the hla - drb1 * 03/04 ( and the hla - dqb1 * 02/0302 ) genotype is highly predisposing for type 1 diabetes , it has very little effect on positivity for gada or ia-2a , a finding that is also reflected in the 434 case subjects recruited within 1 year of diagnosis ( frequency of hla - drb1 * 03/04 was 0.28 on gada positives , 0.26 in gada negatives , 0.33 in ia-2a positives , 0.35 in ia-2a negatives ) . furthermore , hla - drb1 * 03/04 was not associated with decline in gada or ia-2a positivity with disease duration ( supplementary data ) . hla - drb1 * 03/04 has its strongest effects in children with the youngest age - at - diagnosis . in contrast , in our type 1 diabetic case subjects , both positivity for ia-2a and gada were associated with an older age - at - diagnosis ( table 1 ) such that case subjects with the youngest age - at - diagnosis were less likely to be autoantibody positive . this agrees with other studies that also found gada positivity was associated with an older age - at - diagnosis ( 13,27 ) . one possibility is that perhaps hla - drb1 * 03/04 recognizes a yet undiscovered , highly diabetogenic autoantigen , which accelerates disease progression . this leads to a younger age - at - diagnosis , with gada and ia-2a produced as a result of disease pathogenesis , but not having caused disease . it is believed that hla - drb1 * 03/04 genotype susceptibility is attributable to a hla dq transdimer molecule ( 28 ) , and it presumably binds certain preproinsulin peptides , including from the signal peptide sequence ( 29,30 ) . the hla - drb1 * 03/04 genotype susceptibility is directed toward a potent , highly pathogenic antipreproinsulin response . ( 14 ) have also shown that hla - dqb1 * 02/0302 is associated with iaa positivity . iaa has also been shown to be more common in young onset type 1 diabetic case subjects and is often the first autoantibody to appear before age 3 years ( 8) . functional masking has been observed for hla class i molecules in a mouse model of multiple sclerosis in which hla - a3 and hla - a2 have opposite functional effects on autoreactive cd8 + t cells ( 31 ) . so , another explanation for the lack of hla - drb1 * 03/04 association with ia-2a is that there is functional antigen competition between the hla - drb1 * 03 and hla - drb1 * 04 associated haplotypes , with one favoring one autoantigen response and the other competing or suppressing that response . together these alleles maintain an equilibrium that manifests as no association of hla - drb1 * 03/04 with autoantibody positivity . our results do not support a simple model in which hla - drb1 * 03/04 drives early and rapid -cell destruction , effectively lowering the amount of gad and ia-2 antigens available to the autoimmune response , since a negative association of this genotype would be expected . the positive association of the type 1 diabetes protective allele , hla - dqb1 * 0301 , with ia-2a positivity was also unexpected . however , in contrast with hla - drb1 * 03/04 , which is associated with a younger age - at - diagnosis , both hla - dqb1 * 0301 and ia-2a positivity ( table 1 ) were associated with an older age - at - diagnosis ( p = 4.48 10 and 7.22 10 , respectively ) . the frequency of hla - dqb1 * 0301 doubled from 0.04 in type 1 diabetes under 4 years to 0.08 in over 11 years . the inverse associations of the classical alleles with type 1 diabetes compared with ia-2a and gada are further illustrated by our results . hla - drb1 * 03 is associated with risk of type 1 diabetes , and also of gada positivity , but is negatively associated with ia-2a positivity . hla - drb1 * 04 is highly predisposing for type 1 diabetes , is only associated with gada ( negatively ) in the presence of hla - drb1 * 01 or * 13 , and is positively associated with ia-2a . both hla - a*24 and hla - b*39 have been shown to independently confer susceptibility to type 1 diabetes ( 10,11 ) . yet hla - a*24 was negatively associated with ia-2a positivity ( in agreement with another report ) , whereas hla - b*39 was not associated with either gada or ia-2a independently of the class ii effects . consequently , as suggested previously ( 32,33 ) , autoantibody positivity does not necessarily reflect what is occurring at the t - cell level . overall , given that there are these strong differences in the genetics of type 1 diabetes and of ia-2a and gada , these data suggest that gada and ia-2a may not be the first consequence of the autoimmune attack against -cells ( 34 ) , and their exact role in pathogenesis remains to be established .

objectivea major feature of type 1 diabetes is the appearance of islet autoantibodies before diagnosis . however , although the genetics of type 1 diabetes is advanced , the genetics of islet autoantibodies needs further investigation . the primary susceptibility loci in type 1 diabetes , the hla class i and ii genes , are believed to determine the specificity and magnitude of the autoimmune response to islet antigens . we investigated the association of glutamic acid decarboxylase autoantibodies ( gada ) and insulinoma - associated antigen-2 autoantibodies ( ia-2a ) with the hla region.research design and methodsassociations of gada and ia-2a with hla - drb1 , hla - dqb1 , hla - b , hla - c , hla - a , mica , and 3,779 single nucleotide polymorphisms ( snps ) were analyzed in 2,531 childhood - onset case subjects ( median time since diagnosis 5 years ) . all analyses were adjusted for age - at - diagnosis and duration of diabetes.resultsgada and ia-2a were associated with an older age - at - diagnosis ( p < 1019 ) . for gada , the primary association was with hla - dqb1 ( p = 9.00 1018 ) , with evidence of a second independent effect in the hla class i region with snp , rs9266722 ( p = 2.84 106 ) . hla - drb1 had the strongest association with ia-2a ( p = 1.94 1041 ) , with hla - a*24 adding to the association , albeit negatively ( p = 1.21 1010 ) . there was no evidence of association of either ia-2a or gada with the highly type 1 diabetes predisposing genotype , hla - drb1 * 03/04.conclusionsdespite genetic association of type 1 diabetes and the islet autoantibodies localizing to the same hla class ii genes , hla - drb1 and hla - dqb1 , the effects of the class ii alleles and genotypes involved are quite different . therefore , the presence of autoantibodies is unlikely to be causal , and their role in pathogenesis remains to be established .

RESEARCH DESIGN AND METHODS Subjects. Genotyping. Autoantibodies. Statistical analyses. RESULTS Autoantibodies. GADA associations. IA-2A associations. DISCUSSION Supplementary Material

distribution of phenotypes in type 1 diabetic case subjects overall and for each autoantibody subgroup the hla - drb1 , hla - dqb1 , hla - a , hla - b , and hla - c genes were typed at four - digit resolution using dynal reli sso assays ( invitrogen , paisley , u.k . ) the hla - drb1 , hla - dqb1 , hla - a , hla - b , and hla - c genes were typed at four - digit resolution using dynal reli sso assays ( invitrogen , paisley , u.k . ) as expected , gada positivity and ia-2a positivity were both found to be correlated with a shorter duration of diabetes ( p = 9.80 10 and 8.65 10 , respectively , having accounted for age - at - diagnosis effects ; table 1 ) . in total 3,779 snps between 25 and 33 mb on chromosome 6p21 containing the extended mhc region , and the hla - dqb1 , hla - drb1 , hla - b , hla - c , hla - a , and mica(str ) genes were analyzed for association with gada . in type 1 diabetes , the strongest association signal was with the hla class ii region ( 10,11 ) , which was also found to have the strongest signal for association with gada positivity ( fig . the strongest association with gada positivity was with hla - dqb1 ( p = 9.00 10 ) , but hla - drb1 was also convincingly associated ( p = 3.10 10 ; table 2 ) . 1a ) were in linkage disequilibrium ( ld ) with the type 1 diabetes susceptible allele , hla - dqb1 * 02 ( r > 0.69 ) and were in strong ld with the hla - drb1 * 03 type 1 diabetes susceptible allele ( r > 0.85 ) . association of the hla genes with gada positivity in type 1 diabetic case subjects * the multiplicative allelic effects model was not an appropriate approximation ( p = 5.66 10 for hla - dqb1 , p = 0.0011 for hla - drb1 , p = 0.0015 for hla - a ) ; hence the p value for the genotype effects model is reported . there is very little to distinguish the effects of hla - drb1 and hla - dqb1 on gada positivity ( p = 0.002 for addition of hla - dqb1 to hla - drb1 and p = 0.04 for addition of hla - drb1 to hla - dqb1 ; table 2 ) , such that either hla - drb1 or hla - dqb1 is sufficient to model the association , but both are not required . these findings , and that hla - drb1 * 03 was convincingly associated with gada positivity ( p = 4.80 10 ; supplementary table 4 ) , unexpectedly , the highly type 1 diabetes susceptible hla - dqb1 * 02/0302 genotype ( supplementary data and supplementary table 1 ) had a comparable frequency in gada positives ( 0.38 ) and gada negatives ( 0.40 ; table 3 ) . similarly , the most type 1 diabetes associated genotype at hla - drb1 , hla - drb1 * 03/04 ( supplementary tables 2 and 3 ) , was not associated with gada ( or = 1.23 [ 0.901.68 ] using 04/04 as reference [ table 4 ] ) . association of hla - dqb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . association of hla - drb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were calculated accounting for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . hla - drb1 * 0403 and * 0407 alleles have opposite effects in type 1 diabetes to the remaining hla - dbr1 * 04 alleles ; however , they are of such low frequency in the case subjects that an extra subtype was not warranted and so are included with the hla - drb1 * 04 allele . the strongest association with gada positivity was with the hla - dqb1 * 02.hla - drb1 * 03/hla - dqb1 * 02.hla - drb1 * 03 homozygous genotype ( or [ 95% ci ] = 2.62 [ 1.873.67 ] ; supplementary table 5 ) ; hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 05.hla - drb1 * 01 , hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 0402.hla - drb1 * 08 , and hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 06.hla - drb1 * 13 were negatively associated with gada positivity . the association with gada positivity extended into the class i region , and included hla - b , hla - c , hla - a , and mica ( table 2 and fig . likewise , the effects of hla - b and rs9266722 could not be distinguished ; hla - b did not improve the association of hla - dqb1 and rs9266722 ( p = 0.04 ; table 2 ) and rs9266722 did not improve the association of hla - dqb1 and hla - b ( p = 0.13 ) . once the effect of hla - dqb1 was accounted for , as well as the association discussed in the class i region , evidence for an additional association with gada positivity was found with the intronic snp , rs926850 in fam65b at 25.0 mb ( p = 3.15 10 ; fig . the evidence did not improve , becoming relatively unconvincing ( p = 0.0067 ) , and , hence , associations with gada were confined to the hla class ii region and a second independent effect in the class i region . convincing evidence of the association with ia-2a was obtained in both the hla class i and ii regions ( p < 3 10 ; fig . however , in contrast with the association with type 1 diabetes , there was no evidence of deviation from a multiplicative allelic effects model ( p > 0.2 for the class i and ii genes ) . in contrast , hla - drb1 * 0401 ( and its associated hla - dqb1 allele , hla - dqb1 * 0302 ) were strongly associated with ia-2a positivity ( or = 1.72 [ 1.342.20 ] ; table 6 ) , a major similarity to type 1 diabetes susceptibility ( supplementary table 2 ) . interestingly , because the effects of hla - drb1 * 0401 and hla - drb1 * 03 were in opposite directions , the most susceptible type 1 diabetes genotype , hla - drb1 * 03/04 , was not associated with ia-2a , having a frequency of 0.331 in the ia-2a positive case subjects and 0.356 in ia-2a negatives ( or = 1.07 [ 0.741.54 ] ) using as a reference the non - hla - drb1 * 03 , non - hla - drb1 * 04 homozygous genotype , which had a frequency of 0.064 in ia-2a positives and negatives ( supplementary table 7 ) . the highly type 1 diabetes protective allele , hla - dqb1 * 0301 , however , hla - drb1 and hla - dqb1 allelic associations with ia-2a positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates , duration of disease , and age - at - diagnosis , whereas the listed allele frequencies were not . in addition to the evidence of association in the class ii region , the hla class i region also showed evidence of association with ia-2a positivity ( fig . the class ii independent effect in the class i region with ia-2a was largely attributable to hla - a*24 ( p = 1.21 10 for the association of hla - a*24 alone conditional on hla - drb1 ) . although hla - drb1 * 03 was negatively associated with ia-2a ( table 6 ) , the association was significantly stronger in the presence of hla - a*24 on the same haplotype ( or = 0.55 [ 0.390.77 ] hla - a*02.hla - drb1 * 03 compared with or = 0.25 [ 0.160.38 ] for hla - a*24.hla - drb1 * 03 ; supplementary table 8) . as expected , gada positivity and ia-2a positivity were both found to be correlated with a shorter duration of diabetes ( p = 9.80 10 and 8.65 10 , respectively , having accounted for age - at - diagnosis effects ; table 1 ) . in total 3,779 snps between 25 and 33 mb on chromosome 6p21 containing the extended mhc region , and the hla - dqb1 , hla - drb1 , hla - b , hla - c , hla - a , and mica(str ) genes were analyzed for association with gada . in type 1 diabetes , the strongest association signal was with the hla class ii region ( 10,11 ) , which was also found to have the strongest signal for association with gada positivity ( fig . the strongest association with gada positivity was with hla - dqb1 ( p = 9.00 10 ) , but hla - drb1 was also convincingly associated ( p = 3.10 10 ; table 2 ) . 1a ) were in linkage disequilibrium ( ld ) with the type 1 diabetes susceptible allele , hla - dqb1 * 02 ( r > 0.69 ) and were in strong ld with the hla - drb1 * 03 type 1 diabetes susceptible allele ( r > 0.85 ) . association of the hla genes with gada positivity in type 1 diabetic case subjects * the multiplicative allelic effects model was not an appropriate approximation ( p = 5.66 10 for hla - dqb1 , p = 0.0011 for hla - drb1 , p = 0.0015 for hla - a ) ; hence the p value for the genotype effects model is reported . there is very little to distinguish the effects of hla - drb1 and hla - dqb1 on gada positivity ( p = 0.002 for addition of hla - dqb1 to hla - drb1 and p = 0.04 for addition of hla - drb1 to hla - dqb1 ; table 2 ) , such that either hla - drb1 or hla - dqb1 is sufficient to model the association , but both are not required . similarly , the most type 1 diabetes associated genotype at hla - drb1 , hla - drb1 * 03/04 ( supplementary tables 2 and 3 ) , was not associated with gada ( or = 1.23 [ 0.901.68 ] using 04/04 as reference [ table 4 ] ) . association of hla - dqb1 genotypes with gada positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates : sex , duration of disease , and age - at - diagnosis , whereas genotype frequencies were not . the strongest association with gada positivity was with the hla - dqb1 * 02.hla - drb1 * 03/hla - dqb1 * 02.hla - drb1 * 03 homozygous genotype ( or [ 95% ci ] = 2.62 [ 1.873.67 ] ; supplementary table 5 ) ; hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 05.hla - drb1 * 01 , hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 0402.hla - drb1 * 08 , and hla - dqb1 * 0302.hla - drb1 * 04/hla - dqb1 * 06.hla - drb1 * 13 were negatively associated with gada positivity . the association with gada positivity extended into the class i region , and included hla - b , hla - c , hla - a , and mica ( table 2 and fig . likewise , the effects of hla - b and rs9266722 could not be distinguished ; hla - b did not improve the association of hla - dqb1 and rs9266722 ( p = 0.04 ; table 2 ) and rs9266722 did not improve the association of hla - dqb1 and hla - b ( p = 0.13 ) . once the effect of hla - dqb1 was accounted for , as well as the association discussed in the class i region , evidence for an additional association with gada positivity was found with the intronic snp , rs926850 in fam65b at 25.0 mb ( p = 3.15 10 ; fig . the evidence did not improve , becoming relatively unconvincing ( p = 0.0067 ) , and , hence , associations with gada were confined to the hla class ii region and a second independent effect in the class i region . convincing evidence of the association with ia-2a was obtained in both the hla class i and ii regions ( p < 3 10 ; fig . however , in contrast with the association with type 1 diabetes , there was no evidence of deviation from a multiplicative allelic effects model ( p > 0.2 for the class i and ii genes ) . in contrast , hla - drb1 * 0401 ( and its associated hla - dqb1 allele , hla - dqb1 * 0302 ) were strongly associated with ia-2a positivity ( or = 1.72 [ 1.342.20 ] ; table 6 ) , a major similarity to type 1 diabetes susceptibility ( supplementary table 2 ) . interestingly , because the effects of hla - drb1 * 0401 and hla - drb1 * 03 were in opposite directions , the most susceptible type 1 diabetes genotype , hla - drb1 * 03/04 , was not associated with ia-2a , having a frequency of 0.331 in the ia-2a positive case subjects and 0.356 in ia-2a negatives ( or = 1.07 [ 0.741.54 ] ) using as a reference the non - hla - drb1 * 03 , non - hla - drb1 * 04 homozygous genotype , which had a frequency of 0.064 in ia-2a positives and negatives ( supplementary table 7 ) . the highly type 1 diabetes protective allele , hla - dqb1 * 0301 , however , hla - drb1 and hla - dqb1 allelic associations with ia-2a positivity in type 1 diabetic case subjects effect estimates were adjusted for the known covariates , duration of disease , and age - at - diagnosis , whereas the listed allele frequencies were not . in addition to the evidence of association in the class ii region , the hla class i region also showed evidence of association with ia-2a positivity ( fig . the class ii independent effect in the class i region with ia-2a was largely attributable to hla - a*24 ( p = 1.21 10 for the association of hla - a*24 alone conditional on hla - drb1 ) . although hla - drb1 * 03 was negatively associated with ia-2a ( table 6 ) , the association was significantly stronger in the presence of hla - a*24 on the same haplotype ( or = 0.55 [ 0.390.77 ] hla - a*02.hla - drb1 * 03 compared with or = 0.25 [ 0.160.38 ] for hla - a*24.hla - drb1 * 03 ; supplementary table 8) . our data show that although both gada and ia-2a are very strongly associated with type 1 diabetes , and risk of all three localizes to the mhc region , the alleles and genotypes involved differ between autoantibodies and type 1 diabetes . our major novel finding is that even though the hla - drb1 * 03/04 ( and the hla - dqb1 * 02/0302 ) genotype is highly predisposing for type 1 diabetes , it has very little effect on positivity for gada or ia-2a , a finding that is also reflected in the 434 case subjects recruited within 1 year of diagnosis ( frequency of hla - drb1 * 03/04 was 0.28 on gada positives , 0.26 in gada negatives , 0.33 in ia-2a positives , 0.35 in ia-2a negatives ) . in contrast , in our type 1 diabetic case subjects , both positivity for ia-2a and gada were associated with an older age - at - diagnosis ( table 1 ) such that case subjects with the youngest age - at - diagnosis were less likely to be autoantibody positive . however , in contrast with hla - drb1 * 03/04 , which is associated with a younger age - at - diagnosis , both hla - dqb1 * 0301 and ia-2a positivity ( table 1 ) were associated with an older age - at - diagnosis ( p = 4.48 10 and 7.22 10 , respectively ) . hla - drb1 * 04 is highly predisposing for type 1 diabetes , is only associated with gada ( negatively ) in the presence of hla - drb1 * 01 or * 13 , and is positively associated with ia-2a . overall , given that there are these strong differences in the genetics of type 1 diabetes and of ia-2a and gada , these data suggest that gada and ia-2a may not be the first consequence of the autoimmune attack against -cells ( 34 ) , and their exact role in pathogenesis remains to be established .

gastric acid secretion is regulated by paracrine , endocrine , and neural stimuli [ 17 ] . this involves translocating / collating essential proteins to appropriate cytoplasmic and membrane sites , vesicle trafficking , membrane docking , fusion and fission [ 810 ] , modulation of mitochondrial activities , and the coordinated regulation of ion transport proteins , as well as coupling of cytoskeletal proteins to the canalicular membrane via intermediate linking . equitable and sustainable energy and ion transport , coordinated in all parts of the cell , is needed to maintain cell homeostasis and successful acid secretion , ultimately affecting and defining parietal cell morphology [ 1113 ] . ultrastructurally , there is long - standing historical precedence for equating secretory activity with the relative volume densities ( vds ) of canalicular and apical membranes ( cams ) and tubulovesicular membranes ( tv ) . the modulation of these membranes has served as an indicator of quiescent and active secretion , which was established early - on with rigorous morphometry [ 1 , 3 , 8 , 1419 ] . a paucity of tv was equated with hyperstimulation of parietal cell , and inhibition of acid secretion with a cytoplasm containing mostly tubulovesicles . similar quantitative evaluations of membranes and other organelles in genetically engineered achlorhydric mouse models are much less common [ 2023 ] , and since results from these models are inconsistent , the debate continues over the relationship between tv and cam . the purpose of this study was to correlate parietal cell ultrastructure with the reported level of acid secretion in several mouse models of achlorhydria , in order to define a more reliable phenotype . brief descriptions of the normo- , hypo- , and achlorhydric mouse models used in this study are as follows : the ae2 cl / hco3 exchanger ( anion exchanger 2 , slc4a2 ) is expressed in most cell types at low levels but is highly expressed on the basolateral membranes of parietal cells , [ 11 , 24 , 25 ] where it mediates chloride and bicarbonate exchange required for hcl secretion and participates in the maintenance of cell volume and intracellular ph [ 26 , 27 ] . the loss of ae2 produced achlorhydria but did not have a major effect on parietal cell viability . the vd of secretory canaliculi and of tv was severely reduced , basolateral membrane infoldings were increased , and the gastric gland lumens were filled with electron dense debris . the gastric h , k , atpase alpha subunit ( ghka , atp4a ) is abundantly expressed in parietal cells and its loss produces achlorhydria . the alpha subunit contains the atp and cation binding sites required for the export of h from the apical microvilli into the gastric gland lumen . ghka appears to interact directly with cytoskeletal proteins and , as such , may actively participate in the modulation of tv and cam during stimulation . numerous and severe ultrastructural abnormalities including dilated canalicular spaces , abnormal microvilli , absence of and/or replacement of tv with round vesicles , abnormally large mitochondria with whorled cristi , the accumulation of parietal cell glycogen , and epithelial metaplasia accompanied the loss of ghka . the nhe2 na / h exchanger ( slc9a2 ) has not yet been localized within the parietal cell . although it may be localized to basolateral membranes , it is expressed on apical membranes in other cell types [ 31 , 32 ] . nhe2/ mice are hypochlorhydric initially , the amount of gastric acid measured in stomach progressively declines , and parietal cell viability is very much decreased . parietal cell ultrastructure showed mostly normal looking cam and tv and mitochondria . the nhe4 na / h exchanger ( slc9a4 ) , required for acid secretion , is also abundant in parietal cells and is expressed on basolateral membranes [ 12 , 33 ] . nhe4/ mice are severely hypochlorhydric , have compromised parietal cell development or viability , and show a greatly reduced vd of both cam and tv . cams are normal - looking but the cytoplasm just adjacent to the microvilli is mostly devoid of mitochondria and vesicles of any type ( a site where both are usually present in wt ) . cams almost exclusively appeared as shallow invaginations from the apical membrane , rather than deep canalicular nidi . morphology of the double null mice ( nhe2/ae2/ and nhe2/ ghka/ ) has not been reported in detail previously , but parietal cells appeared to default to the characteristics of the ae2/ and ghka/ mice , respectively , with some uniqueness in each . normochlorhydric knockout ( ko ) mouse strains used as controls included those for the nhe1 ( slc9a1 ) and nhe3 ( slc9a3 ) na / hexchangers , the nkcc1 ( slc12a2 ) na - k-2cl cotransporter , and the pmca4 ( atp2b4 ) plasma membrane ca2-atpase . these ko mice , and their parietal cells have also been described previously , and were determined in this study , by morphometry , not to be significantly different from parietal cells in wild - type ( wt ) mice [ 3437 ] . our aim was to identify parietal cell phenotypes which predictably reflected the level of acid secretion reported for the ph of gastric contents . it was concluded that of the numerous morphometric parameters that we measured , the vd of mitochondria ( alone ) , the vd of all secretory membranes ( together ) , and the vd of basolateral membranes / vd of mitochondria were good predictors of parietal cell activity . the latter two were less influenced by the structural defects in the ko mice which compromised modulation of tv and cam . wt mice plus 4 strains of normochlorhydric mice ( nhe1/ , nhe3/ , nkcc1/ , pmca4/ ) were used as controls . two hypochlorhydric / achlorhydric mouse lines ( nhe2/ and nhe4/ ) and 4 strains of achlorhydric mice ( single knockouts , ae2/ , ghka/ , and both nhe2/ae2/ and nhe2/ ghka/ double knockouts ) were used experimentally . all mice were housed in humidity and temperature controlled rooms , on a 12-hour light / dark cycle , with access to standard mouse chow and water ad libitum , except when fasted . ae2/ mice and their matched wt pairs were usually under three weeks old , because of early mortality in the former . all other mice varied from 3 weeks to over 1 year old at euthanasia , with approximately equal numbers of males and females in those pairs . animal protocols were approved by the university of cincinnati institutional animal care and use committee and animal handlers were trained in an american association of assessment and accreditation of laboratory animal care facility . acid secretion , in wt and nhe2/ mice only , was inhibited by fasting , omeprazole treatment ( 200 moles / kg orally two times per day for 2 weeks ) or , vagotomy . fasting included withholding food from mice housed in wire cages with no bedding for 12 hours prior to euthanasia . paired wt and nhe2/ mice were surgically vagotomized ( charles river laboratory ) when animals were approximately 5 months of age . hyperchlorhydria was induced in wt mice using histamine ( sigma ) , at a subcutaneous dose of 2 g / gram body weight in phosphate buffered saline and animals were euthanized after either 15 or 20 minutes . the stomach was dissected immediately , opened along the greater curvature , rinsed free of food in phosphate buffered saline ( pbs ) , and fixed by immersion in 2% paraformaldehyde/2.5% glutaraldehyde in pbs or in 4% paraformaldehyde in pbs for at least 24 hours . all tissue blocks were postfixed in 1% osmium tetroxide in either millonig 's buffer or pbs for 2 hours , dehydrated stepwise in increasing concentrations of ethanol , two changes of propylene oxide , and in one change each of 1 : 1 and 1 : 3 propylene oxide : spurrr ( electron microscopy sciences , hatfield pa ) . tissues were left overnight in fresh 100% resin and embedded in beemr capsules ( electron microscopy sciences , hatfield pa ) the following day in fresh resin with tissue oriented so that epithelium could be cut perpendicularly to the plane of the basement membrane . sections were cut at 1.5 to 2 microns thick and stained with toluidine blue for light microscopy . tissue was also preserved in formalin , dehydrated in ethanol and xylene , blocked in paraffin , sectioned at 5 microns , and stained with hematoxylin and eosin ( h&e ) . thin ( gold ) plastic sections were stained with uranyl acetate and lead citrate for transmission electron microscopy . the number of ko , double - ko , and wt mice per group varied from 2 to 46 . the number ( n ) of mice in any given analysis was included within the bar graphs . the 4 normochlorhydric ko genotypes and 4 normochlorhydric heterozygous mice from among these genotypes were pooled with wt mice , since no significant differences were found in morphometry compared to wt . electron micrographs of parietal cells for morphometry were taken as they were sequentially encountered at the microscope , avoiding operator bias in the selection of individual cells . parietal cells in the mucus pit region of the gastric epithelium were excluded ; however , the parietal cells included in the dataset represented a summary of those from the gastric gland neck and basal regions . intersections of the grid which fell over the following cytoplasmic and nuclear events were counted : ( 1 ) basal and lateral membranes ( blm ) , ( 2 ) round vesicles ( rv ) , ( 3 ) tubulovesicles ( tv ) ( distinctly oval or tubular or irregularly shaped ) , ( 4 ) canalicular and apical microvillar membranes ( cam ) , ( 5 ) apical membrane without characteristic microvilli ( as in the case of the ghka/ parietal cells ) , ( 6 ) mitochondria , ( 7 ) lamellar and dense bodies , ( 8) nucleoplasm , and ( 9 ) glycogen . volume densities were calculated by dividing the number of points lying over a given structure by the total number of points counted over that parietal cell [ 29 , 30 ] . data were analyzed using sas v8.0 , or 9.1 ( sas institute , cary nc ) , and sigma plot 2000 . means and standard errors of the means were determined by genotype , gender , treatment and age using the general linear model . for data shown in bar graphs , n = an individual animal . images for final figures were scanned from prints , contrast enhanced , and occasionally dodged , burned , or the dust and scratches filter applied , when needed , with photoshop 7 ( adobe.com ) . parietal cells in the normochlorhydric ko mice ( nhe1/ , nhe3/ , nkcc1/ and pmca4/ ) were not morphometrically different from wt mice , and these data were pooled ( hereafter referred to collectively as wt ) . generally , untreated wt parietal cell morphology and morphometry agreed very well with historical data ( figures 1 and 2 ) . this was also true for those wt mice in which gastric acid secretion was inhibited by vagotomy or by omeprazole [ 15 , 17 , 18 ] ( figure 1 ) . eighty - five to 90 percent of the cytoplasmic membranes , that is , ( tv + cam)/total membranes were directly related to acid secretion in wt mice . data were ordered according to the number of ko genotypes which achieved a statistically significant difference from wt for each morphometric parameter ( the number of achlorhydric genotypes significantly different from wt / the total number of achlorhydric genotypes tested ) : microvillar length and width ( 2/6 ) , vd of tv ( 3/6 ) , vd of basolateral membranes ( 4/6 ) , vd of lamellar and dense bodies ( 4/6 ) , vd of canaliculi ( 4/6 ) , the vd of all secretory membranes ( 5/6 ) , and the vd of mitochondria ( 5/6 ) ( figure 2 ) . the vd of the nucleus showed no statistically significant changes among all groups ( wt , normochlorhydric or achlorhydric ) but data were put aside as incomplete because ( 1 ) the vd of nuclear profiles appearing in micrographs was highly variable and ( 2 ) roles for cell volume regulation are predicted for ae2 , nhe2 , and nhe4 . the vd of secretory membranes , grouped as tv plus cam , was significantly reduced in four of the six achlorhydric ko groups only . in the ghka/ genotype , canalicular nidi and vesicles were present but were round and contained within themselves smaller vesicles and thus were not typical of tv . this underscores the grey areas in interpretation as to what constituted cam or tv . several achlorhydric genotypes showed an absence of tvs or a decrease in tvs leading to a high ratio of cam to tv , characteristics usually associated with stimulated acid secretion ( figures 2 and 3 ) . we found that only the hypochlorhydric genotype , nhe2/ , under both untreated and inhibited conditions , showed qualitatively similar responses in secretory membranes compared to wt , though quantitatively different ( figure 3 ) . ratios of volume densities of various membranes and organelles were also used to see whether secretory membranes modulated reciprocally , since the latter has been suggested in several reports [ 15 , 18 ] , and to determine whether ratios would be of value for defining a stable histological phenotype of achlorhydria . data were ranked again , according to how many achlorhydric genotypes showed significant differences in the ratios of volume densities , compared to untreated wt mice ( table 1 ) . the vd of total secretory membrane / vd of mitochondria was a fair predictor of gastric acid ph , but not as good at predicting overall secretory activity in the knockout models as other parameters , because abnormal morphology ( particularly in ghka/ mice ) produced misleading counts of cam and tv . the following genotypes had very few vesicles ( whether round or tubular ) present in the cytoplasm : ae2/ , nhe2/ ae2/ , and nhe4/ ( figure 3 ) . our data also show that significant fluctuations ( reductions or increases ) in tv , cam , and blm occurred while the vd of total secretory membranes remained nearly unchanged ( figure 2 ) . secretory membranes in wt mice were more or less equitably exchanged after vagotomy and omeprazole treatments . while some reports have demonstrated almost equal gains and losses within the whole secretory membrane compartment [ 17 , 3842 ] , this was not the case with 5 of the 6 achlorhydric kos . while equal gains and losses support the generally accepted hypothesis about modulation of membranes , it is implicit in this approach that all membranes be capable of modulating and those membranes to be included be named . the sum of all secretory membranes ( tv + cam + blm ) showed a trend to increase in wt animals in which acid secretion was inhibited ( 26.8 1.1 versus 31.8 2.5 p = .07 ) , showing inequitable exchange , due in large part to a relatively greater increase in tv . in the nhe2/ null mice where secretion was inhibited by vagotomy or omeprazole , no change in total vd of secretory membranes occurred ( 22.84 2.3 versus 22.1 2.5 p = 0.8 ) , and changes a decrease in the ratio of cam to tv in both wt and nhe2/ mice was significant during inhibition however , when each was compared to their respective untreated control ( wt , p = .0187 ; nhe2/ , p = .023 ) . each was also accompanied by an increased vd of basolateral membrane ( significant only in wt , p < .0001 ) . nhe2/ mice were unique among the ko genotypes in this study because organelles changed in parallel ( but not on a par ) with wt mice . the vd of total secretory membranes was reduced , often severely , in the achlorhydric mice in this study , with p - values ranging between.058 and < .0001 for the 6 genotypes . in nhe2/ mice , where the reduction in secretory membranes was not as dramatic as the others kos ( p = .058 ) , frequently the canalicular lumens were grossly dilated , accompanied by evidence of impending parietal cell death and debris ( figure 3 and ) . the reduction in vd of total secretory membranes , particularly in the nhe4/ , nhe2/ ae2/ , and ghka/ mice , resulted mostly from the loss of tv . inhibition of acid secretion , in our hands , produced an unbalanced membrane exchange in wt and nhe2/ parietal cells ( figure 2 ) . apical membranes with microvilli of an appropriate length and width , however , were clearly present in all ko genotypes except for the ghka/ mice , where they were significantly shorter than in wt . basolateral membranes were increased in all of the kos but did not increase sufficiently to compensate for the loss of other secretory membranes , resulting in unbalanced modulation . tv were almost always visible in wt parietal cells , representing around 8% of the cell cytoplasm ( vd = 7.68 0.96 , n = 47 ) ( figure 1 ) under normal conditions and were greatly increased when acid secretion was inhibited by vagotomy or omeprazole ( 13.99 2.73 n = 7 , p = .010 to untreated wt ) . fasting and stimulation produced variable morphometric outcomes , especially in the vd of tv in wt parietal cells , which varied from 0% to 100% of the secretory membranes in any given cell profile . this required the assessment of large numbers of micrographs of parietal cells , from many animals , to obtain representative data . when the morphology of tv was compromised , or when tvs were absent , gastric contents were less acidic . unlike tvs , microvilli maintained a fairly constant morphology ( ghka/ parietal cells excepted ) . thus , while the tv in some models did not reflect acid secretion , the morphology of tv gave important clues as to the causes of failed secretion , and highlighted the relatively unchanging appearance of cam as well as mitochondria . canalicular nidi were still present in the cytoplasm , and seemingly normal microvilli were seen on the apical membrane , but the cytoplasm just adjacent to the canaliculi was most often devoid of organelles and vesicles ( whether round or tubular ) . similarly , in ae2/ mice , tvs were reduced ( vd 2.67 0.79 n = 3 , p = .033 to wt)(figure 3 ) but some round vesicles were observed . even more dramatically , the nhe2/ae2/ and nhe2/ghka/ parietal cells had almost no vesicles at all ( figure 3 ) . wt and nhe2/ mice had no paucity of canaliculi and incipient canalicular nidi were frequently close to the basal portion of the parietal cells . no comment can be made in this study as to whether those nidi were separate from , or connected to , the infoldings of the apical membrane since 3d reconstruction from serial sections was not performed . strikingly however , canaliculi in the cytoplasm of ae2/ , nhe4/ , and the nhe2/ ae2/ mice were infrequent compared to wt ( figure 3 ) , and invaginations were nearly always seen in close proximity to the apical membrane . especially in the nhe2/ ae2/ in addition , glandular lumens were often filled with electron dense debris that was ultrastructurally similar to secretory granules from nearby zymogen cells ( figure 4(d ) ) , which mimicked the single ko of ae2 . failure to generate deep cytoplasmic canalicular nidi , which become , or are already , part of the branched secretory cam , was apparent in 3 of the 6 ko genotypes . in contrast , the other three genotypes ( ghka/ , nhe2/ , and the nhe2/ ghka/ double ko ) showed sporadic , but often massive dilation of their canaliculi , with canalicular nidi seemingly deep in the cytoplasm and separate from the apical membrane . microvilli in wt mice were approximately 1 m long 0.2 m wide , undulated modestly in thickness along their lengths , and were slightly bulbous at the ends . nhe2/ parietal cells had normal microvillar morphology but they were significantly longer than in wt parietal cells ( wt , 1.17 0.037 by 0.26 0.012 microns , n = 40 ; nhe2/ , 2.04 0.65 by 0.35 0.1 microns , n = 13 , p = .012 ) . both microvillar length and width tended to decrease during inhibition of acid secretion ( vagotomy and omeprazole , combined data ) in wt and nhe2/ mice , but this did not reach statistical significance . in opportune cross sections actin filaments within cam were apparent , and most often arranged in a circle , close to the plasmalemma , but occasionally were found clustered centrally as a core bundle . no quantification of the ratio of centrally to peripherally located actin was performed in the current study , but it was obvious , subjectively , that wt parietal cells had considerably more microvilli with peripherally located actin than with centrally bundled actin , while in the achlorhydric kos the latter arrangement was much more frequent . ( 1 ) the hypochlorhydric nhe2/ mice had actin located peripherally in the microvilli , again underscoring that the parietal cells in this ko must have some functional acid secretion , and ( 2 ) in the ghka/ mice , where parietal cells always showed centrally bundled actin , microvilli were significantly shorter than in wt mice . the increase in vd of the basolateral membrane was significant in 4 of the 6 achlorhydric genotypes , and elevated in the other two ( figures 3 and 4 ) . in addition to the vd of the blm being increased with achlorhydria , the normally closely aligned intercellular interdigitations give way to broader infoldings , that extended further into the cytoplasm than in wt cells ( figure 5 ) . ( this is a rarely reported observation . ) in nhe4/ and ae2/ mice , and in the nhe2/ ae2/ double ko especially , interdigitations were broader ( figure 5 ) . the vd of basolateral membranes alone was about equivalent to the vd of canaliculi in predicting the level of acid secretion . however , when the vd of blm was expressed as a ratio with the vd of mitochondria , the p values were significant for 6/6 of the achlorhydric genotypes , thus making this ratio one of the better markers derived from morphometry . the vd of mitochondria provided a reasonably good measure of acid secretory competence , being significantly reduced in 5 of 6 achlorhydric models compared to wt , reflecting decreased secretory activity . vd of mitochondria was about 30% in wt parietal cells and was significantly reduced in parietal cells of nhe2/ mice ( untreated ) , compared to wt . inhibition of acid secretion with omeprazole or vagotomy did not decrease mitochondrial vd further ( figure 2 ) . though mitochondrial vd was reduced in the hypo- and achlorhydria phenotypes , mitochondria still appeared to be of normal size and shape , and cristi were unremarkable , and were overall quite similar to wt , except for the ghka ko . ghka/ and nhe2/ ghka/ double ko mice showed some very large mitochondria with whorled cristi in young animals ( figure 4 ) , a phenotype which disappeared as the animals aged . the position of mitochondria within wt parietal cells is characteristically closely juxtaposed to tv and cam , and indeed , areas of tv and cam are intimately surrounded by mitochondria . several ko genotypes however , in nhe4/ , ae2/ and nhe2/ ae2/ parietal cells , mitochondria were positioned at a distance from cam , rarely coming in close proximity to those membranes . subjectively , the void zone was inversely related to the amount of tv and vesicular membranes present in the cytoplasm , and thus inversely related to the acid secretion . inclusion bodies , or phagolysosomes , the morphology of which was divided into two phenotypes in this study ( dense granular and lamellar ) , were significantly increased in 4/6 achlorhydric genotypes . dense granular inclusions were almost exclusively found in the ghka/ mice ( ghka/ and nhe2/ ghka/ ) , appeared to be large , and composed of both dark and lucent rounded granules , lacking much in the way of myelin - like components . the remaining achlorhydric genotypes displayed myelin or lamellar - type inclusions with some granular components also . the highest vd of lamellar inclusions was found in the nhe2/ ae2/ double ko mice ( p < .0001 to wt ) ( figure 3 ) . ae2/ and nhe4/ mice also showed a preponderance of lamellar type bodies , rather than dense granular inclusions ( figure 4 ) . although the two different types of cytoplasmic inclusions , dense granular and lamellar , were combined into a single category for statistical purposes , they were very different in structure , and likely resulted from quite different mechanisms . in nhe2/ single ko mice , parietal cell death was elevated and cell debris containing large lamellar inclusions was common in the gastric gland lumens . the vd of lamellar inclusions in nhe2/ mice decreased upon inhibition of acid secretion by vagotomy or omeprazole ( nhe2/ untreated 1.31 0.31 , n = 13 , versus nhe2/ inhibited 0.76 0.37 , n = 9 , p = .073 ) , suggesting that they may result from faulty modulation of tv and cam . interestingly , where the tvs were decreased because of loss of ghka in the double null mice , the vd of lamellar inclusions diminished , but in the nhe2/ ae2/ null mice , it increased above that of the nhe2/ single ko ( figure 3 ) . gastric acidity in wt mice parallels the relative volume densities of canalicular and apical membranes ( cam ) and tubulovesicular membranes ( tv ) in the parietal cell cytoplasm in a predictable manner . although the relationship between the level of acid secretion and the relative amounts of cam and tv has been useful as a reference point for histology , it is not without inconsistencies . in addition , methods of long - term inhibition ( omeprazole and vagotomy ) have many undesirable secondary effects , such as hypergastrinemia and changes in morphology . just as problematic is the fact that adjacent parietal cells can vary widely in the vd of tvs . our data from our normal parietal cells supported the occurrence of diminished vd of tv with stimulation and a cytoplasm containing mostly tvs and only a few cam after inhibition of acid secretion . during examination of the various mouse models of hypochlorhydria and achlorhydria it became clear that using the ratio of cam to tv was not an unambiguous indicator of activity . our ultrastructural quantification of cam and tv and other membranes and organelles in mice which were normochlorhydric , compared with 6 mouse models with defective acid secretion , demonstrated that the vd of tv was more variable than other organelles when predicting secretory competence . in fact , the vd of basolateral membranes , and mitochondria , and the ratio of these two parameters , paralleled the gastric ph data from published studies far better . the most critical event in disrupting the ratio of cam to tv appeared to involve the interruption of vesicle trafficking between tv and cam . this was alluded to in the ae2/ and nhe4/ single kos [ 11 , 12 , 30 ] and the nhe2/ ae2/ double ko , but comparisons clearly suggest that with significant decreases in vd of tv , there is little acid secretion . tv appeared to vary morphologically ( and morphometrically ) independently from cam , since even in an almost total absence of both round vesicles and tv , the presence of an ultrastructurally normal - looking canalicular and apical membrane microvillar morphology can persist . this raises the question of whether minute amounts of acid are being secreted from these ostensibly normal looking microvilli . shape ( whether round or tubular ) and location with respect to mitochondria and canaliculi were subject to viewer judgement , and physical factors such as buffering and fixation greatly influenced the appearance of tv [ 15 , 38 ] . consistently applied criteria for tubular and vesicular shape were used in this study . if a vesicle appeared to be round rigid , infrequent , and not located in proximity to cam , then it was not counted as tv . in one of the few papers which quantified parietal cells in achlorhydric mice ( these lacked ezrin ) a significant increase in tv and apical membranes in parietal cells was shown , but in images from that study , apical membrane microvilli looked disorganized and were reduced in amount compared to their wt controls , and what was identified as a vesicular component resembled the rounded vesicles in ghka/ parietal cells ( figure 4 ) in our study . our data suggest that a common hindrance to acid secretion is the loss of membrane modulation , vesicle fusion or fission events , or other factors , leading to a block in the formation of tv . the morphology of tv and other membranes is important clues as to the causes of failed secretion , but the relatively unchanging appearance of cam and mitochondria may be better equated with overall parietal cell acid secretion . emerging studies demonstrate the importance of proteins such as the f - actin- and clathrin - binding protein huntingtin interacting protein 1 related ( hip1r ) in vesicular trafficking for parietal cell acid secretion . in addition , the gastric morphogen sonic hedgehog ( shh ) is also involved in activation of parietal cell function possibly via membrane modulation . in contrast to the ezrin and ghka/ mouse models [ 22 , 28 , 29 ] both the hip1r and the parietal cell - specific shh null animals lack well - defined tubulovesicles and exhibit expanded canalicular membranes . neither of the latter mouse models respond to histamine stimulation , perhaps with important implications for other models of secretion . failure to generate deep separate cytoplasmic canalicular nidi ( which become , or are already , part of the branched secretory cam ) was apparent in 3 of the 6 ko genotypes . in contrast , the other three genotypes ( ghka/ , nhe2/ , and the nhe2/ ghka/ double kos ) showed sporadic , but often massive dilation of their canaliculi , with canalicular nidi seemingly deep in the cytoplasm and separate from the apical membrane . the ezrin ko mouse showed cam which seemed to be derived from short invaginations from the apical membrane , but not from deeper nidi in the cytoplasm . common between the ezrin ko and the ghka/ was that both apparently failed to connect actin filaments to the secretory membranes [ 20 , 28 ] . reports reveal that the loss of proteins critical for acid secretion sometimes results in dramatic ultrastructural consequences , as was seen in parietal cells of mice lacking ghka [ 13 , 28 , 29 ] . in other models , less dramatic ultrastructural changes were found , such as those reported for the nhe2/ mouse , where we also observed that both cam and tv appeared to modulate appropriately . it is important to point out that in the nhe2/ mice the loss of acid secretion appeared to be due primarily to diminished parietal cell viability , reduced parietal cell numbers , and increased base secretion . parietal cells in young nhe2/ mice can actually show highly developed cam and numerous tv and overall , present with a fairly normal morphology . f - actin and ezrin regulate movement of vesicles and organelles through the cytoplasm , are essential for modulation of parietal cell membranes in gastric acid secretion [ 7 , 20 , 45 ] , and also interact with some major transport proteins , including the gastric h , k - atpase ( ghka ) [ 28 , 29 ] . as surmised from the ghka ko mouse , microvillar ultrastructure requires the presence of ghka and the linking of actin to the microvillar membrane , and is also necessary for the modulation of cam to tv and vice versa . it has been reported that the distribution of ezrin is at the interface of tv and cam and that it participates in membrane - vesicle fusion and movement events [ 46 , 47 ] , regardless of the controversy over whether tv and cam are distinct , continuous , or both , and in what manner they are recycled and recruited [ 8 , 42 , 48 ] . whether secretory membranes in wt mice were equitably exchanged after vagotomy and omeprazole treatments our data show that significant fluctuations ( reductions or increases ) in tv , cam , and blm occurred while the vd of total secretory membranes remained nearly unchanged . according to ito and schofield , during stimulation of acid secretion with insulin , microvillar membranes increased from 12% to 20% , lateral and basal cell membranes decreased from 6% to 5% , and there was a simultaneous reduction in tubulovesicular membranes from a vd of about 24% in untreated , to around 4% in stimulated cells . helander also implied an even exchange of membranes ( in this case tv and cam ) during stimulation of acid secretion . other studies have demonstrated almost equal gains and losses within the whole secretory membrane compartment [ 17 , 38 , 39 ] . the inhibition of acid secretion , in our experiments , produced an unbalanced membrane exchange in both wt and nhe2/ parietal cells and also was of a lesser magnitude than what others reported . while equal gains and losses support a generally accepted hypothesis , it is implicit in this approach that all membranes must first be able to modulate . even then , it comprises only a few percent of the total membrane vd in parietal cells and this increase would appear to be insufficient to compensate for the large fluctuations in the vd of tv and cam . it is clear , of course , that basal and lateral membranes form a separate compartment from that of secretory membranes ( tv and cam ) , and too that combining them into one category is an oversimplification . the microenvironment that basal portions of the plasmalemma abut has a completely different milieu than lateral membanes . evidence in support of the involvement of basolateral membrane processes in acid secretion comes from the required activation of cl / hco3 and na / h exchangers on the basolateral membrane [ 11 , 12 , 49 ] . these ion exchangers are critical components of the overall system of basolateral transporters needed to support ongoing hcl and kcl secretion across canalicular membranes . somewhat counterintuitively , however , in this study we found a significant increase in vd of basolateral membranes when acid secretion was diminished . the increased basolateral infoldings observed in achlorhydric phenotypes may be due to a physical effect : a compensation for changes in cell shape accompanying the greatly reduced canalicular space , which normally extends deep into the parietal cell cytoplasm during stimulated secretion . the activation of secretion might result in fewer infoldings as the cell expands and the infoldings are drawn into the surface plasmalemma . however , when reduced acid secretion was accompanied by increased canalicular space , vd of basal and lateral membranes was still increased ( as was seen in the nhe2/ and ghka parietal cells ) . basolateral membranes are also critical sites of ligand binding ( acetylcholine , gastrin , histamine ) for regulation of acid secretion , and it is possible that the vd of basolateral membranes is regulated separately from the vd of tv and cam . the vd of mitochondria was about 30% in the wt parietal cells in this study , which was very close to reports in the literature [ 11 , 12 , 15 , 18 , 29 , 30 , 36 , 38 , 39 , 50 , 51 ] . obtained a mitochondrial vd of 40% , but this was after excluding nuclei and canalicular space from the analysis and was therefore higher than the values for our wt and achlorhydric mice . mitochondrial vd appeared not to fluctuate after stimulation or inhibition of secretion in the short term [ 15 , 38 ] but it did decrease under conditions of chronic inhibition ( wt , vagotomy , and omeprazole ) as shown in the current study . mitochondrial vd was significantly reduced in untreated nhe2/ mice , and inhibition with omeprazole and vagotomy did not decrease it further ( figure 2 ) . vd of mitochondria was an improved indicator of achlorhydria , perhaps posing better as an overall indicator of cell activity , reflecting principally , acid secretion . mitochondria within wt parietal cells were typically closely juxtaposed to tv and cam , and indeed , areas of tv and cam and mitochondria were intimately associated in wt and in nhe2/ parietal cells , and in ghka/ parietal cells to some degree . uncharacteristically however , and especially prominent in the nhe4/ ko , but also in ae2/ and nhe2/ ae2/ double ko parietal cells , mitochondria were positioned quite a distance from cam , rarely coming in close proximity to those membranes . . showed ae2/ parietal cells which clearly pointed to a decreased vd of mitochondria and a greater separation between mitochondria , tv , and cam . subjectively , the void zone is inversely related to the amount of tv and vesicular membranes present in the cytoplasm and inversely related to measured acid secretion . it is possible , perhaps , that pentalamellar bodies were coincident in a few instances with lamellar bodies , or that they may fall into the same membrane recycling cascade as lamellar bodies . sawaguchi mentioned that dense autophagolysosomes were heavily invested with the gastric h , k atpase . what is interesting is that lamellar inclusions were common in all genotypes except those where ghka was lacking . in summary , using various knockout mouse models in which acid secretion was impaired and experimentally induced inhibition of acid secretion it was determined that the ratio of cam to tv was a good indicator of acid secretion in parietal cells which have functional membrane trafficking potential . nhe2/ mice had the capacity to modulate secretory membranes and showed a parallel response to wt parietal cells in morphometry and morphology , though attenuated . however , it was more common for defects in membrane trafficking to produce atypical ratios of tv to cam . it appears that the vd of mitochondria and vd of basolateral membranes and the ratio of the two , are measures which can be applied both to wt and achlorhydric models that reflect the level of gastric acid secretion .

acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking . histologically , consensus defines acid secretion as the ratio of the volume density ( vd ) of canalicular and apical membranes ( cams ) to tubulovesicular ( tv ) membranes , a value which varies widely under normal conditions . examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion . vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion . we confirmed that cam to tv ratio fairly represented secretory activity in untreated and secretion - inhibited wild - type ( wt ) mice and in nhe2/ mice as well , though the response was significantly attenuated in the latter . however , high cam to tv ratios wrongly posed as active acid secretion in ae2/ , ghka/ , and nhe4/ mice . achlorhydric genotypes also had a significantly higher vd of basolateral membrane than wt mice , and reduced vd of mitochondria and canaliculi . the vd of mitochondria , and ratio of the vd of basolateral membranes / vd of mitochondria were preferred predictors of the level of acid secretion . alterations in acid secretion , then , cause significant changes not only in the vd of secretory membranes but also in mitochondria and basolateral membranes .

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

ultrastructurally , there is long - standing historical precedence for equating secretory activity with the relative volume densities ( vds ) of canalicular and apical membranes ( cams ) and tubulovesicular membranes ( tv ) . the purpose of this study was to correlate parietal cell ultrastructure with the reported level of acid secretion in several mouse models of achlorhydria , in order to define a more reliable phenotype . brief descriptions of the normo- , hypo- , and achlorhydric mouse models used in this study are as follows : the ae2 cl / hco3 exchanger ( anion exchanger 2 , slc4a2 ) is expressed in most cell types at low levels but is highly expressed on the basolateral membranes of parietal cells , [ 11 , 24 , 25 ] where it mediates chloride and bicarbonate exchange required for hcl secretion and participates in the maintenance of cell volume and intracellular ph [ 26 , 27 ] . the vd of secretory canaliculi and of tv was severely reduced , basolateral membrane infoldings were increased , and the gastric gland lumens were filled with electron dense debris . the nhe4 na / h exchanger ( slc9a4 ) , required for acid secretion , is also abundant in parietal cells and is expressed on basolateral membranes [ 12 , 33 ] . nhe4/ mice are severely hypochlorhydric , have compromised parietal cell development or viability , and show a greatly reduced vd of both cam and tv . cams are normal - looking but the cytoplasm just adjacent to the microvilli is mostly devoid of mitochondria and vesicles of any type ( a site where both are usually present in wt ) . these ko mice , and their parietal cells have also been described previously , and were determined in this study , by morphometry , not to be significantly different from parietal cells in wild - type ( wt ) mice [ 3437 ] . our aim was to identify parietal cell phenotypes which predictably reflected the level of acid secretion reported for the ph of gastric contents . it was concluded that of the numerous morphometric parameters that we measured , the vd of mitochondria ( alone ) , the vd of all secretory membranes ( together ) , and the vd of basolateral membranes / vd of mitochondria were good predictors of parietal cell activity . two hypochlorhydric / achlorhydric mouse lines ( nhe2/ and nhe4/ ) and 4 strains of achlorhydric mice ( single knockouts , ae2/ , ghka/ , and both nhe2/ae2/ and nhe2/ ghka/ double knockouts ) were used experimentally . parietal cells in the mucus pit region of the gastric epithelium were excluded ; however , the parietal cells included in the dataset represented a summary of those from the gastric gland neck and basal regions . intersections of the grid which fell over the following cytoplasmic and nuclear events were counted : ( 1 ) basal and lateral membranes ( blm ) , ( 2 ) round vesicles ( rv ) , ( 3 ) tubulovesicles ( tv ) ( distinctly oval or tubular or irregularly shaped ) , ( 4 ) canalicular and apical microvillar membranes ( cam ) , ( 5 ) apical membrane without characteristic microvilli ( as in the case of the ghka/ parietal cells ) , ( 6 ) mitochondria , ( 7 ) lamellar and dense bodies , ( 8) nucleoplasm , and ( 9 ) glycogen . parietal cells in the normochlorhydric ko mice ( nhe1/ , nhe3/ , nkcc1/ and pmca4/ ) were not morphometrically different from wt mice , and these data were pooled ( hereafter referred to collectively as wt ) . eighty - five to 90 percent of the cytoplasmic membranes , that is , ( tv + cam)/total membranes were directly related to acid secretion in wt mice . data were ordered according to the number of ko genotypes which achieved a statistically significant difference from wt for each morphometric parameter ( the number of achlorhydric genotypes significantly different from wt / the total number of achlorhydric genotypes tested ) : microvillar length and width ( 2/6 ) , vd of tv ( 3/6 ) , vd of basolateral membranes ( 4/6 ) , vd of lamellar and dense bodies ( 4/6 ) , vd of canaliculi ( 4/6 ) , the vd of all secretory membranes ( 5/6 ) , and the vd of mitochondria ( 5/6 ) ( figure 2 ) . the vd of the nucleus showed no statistically significant changes among all groups ( wt , normochlorhydric or achlorhydric ) but data were put aside as incomplete because ( 1 ) the vd of nuclear profiles appearing in micrographs was highly variable and ( 2 ) roles for cell volume regulation are predicted for ae2 , nhe2 , and nhe4 . the vd of secretory membranes , grouped as tv plus cam , was significantly reduced in four of the six achlorhydric ko groups only . several achlorhydric genotypes showed an absence of tvs or a decrease in tvs leading to a high ratio of cam to tv , characteristics usually associated with stimulated acid secretion ( figures 2 and 3 ) . ratios of volume densities of various membranes and organelles were also used to see whether secretory membranes modulated reciprocally , since the latter has been suggested in several reports [ 15 , 18 ] , and to determine whether ratios would be of value for defining a stable histological phenotype of achlorhydria . the vd of total secretory membrane / vd of mitochondria was a fair predictor of gastric acid ph , but not as good at predicting overall secretory activity in the knockout models as other parameters , because abnormal morphology ( particularly in ghka/ mice ) produced misleading counts of cam and tv . the following genotypes had very few vesicles ( whether round or tubular ) present in the cytoplasm : ae2/ , nhe2/ ae2/ , and nhe4/ ( figure 3 ) . our data also show that significant fluctuations ( reductions or increases ) in tv , cam , and blm occurred while the vd of total secretory membranes remained nearly unchanged ( figure 2 ) . the sum of all secretory membranes ( tv + cam + blm ) showed a trend to increase in wt animals in which acid secretion was inhibited ( 26.8 1.1 versus 31.8 2.5 p = .07 ) , showing inequitable exchange , due in large part to a relatively greater increase in tv . in the nhe2/ null mice where secretion was inhibited by vagotomy or omeprazole , no change in total vd of secretory membranes occurred ( 22.84 2.3 versus 22.1 2.5 p = 0.8 ) , and changes a decrease in the ratio of cam to tv in both wt and nhe2/ mice was significant during inhibition however , when each was compared to their respective untreated control ( wt , p = .0187 ; nhe2/ , p = .023 ) . each was also accompanied by an increased vd of basolateral membrane ( significant only in wt , p < .0001 ) . the vd of total secretory membranes was reduced , often severely , in the achlorhydric mice in this study , with p - values ranging between.058 and < .0001 for the 6 genotypes . in nhe2/ mice , where the reduction in secretory membranes was not as dramatic as the others kos ( p = .058 ) , frequently the canalicular lumens were grossly dilated , accompanied by evidence of impending parietal cell death and debris ( figure 3 and ) . the reduction in vd of total secretory membranes , particularly in the nhe4/ , nhe2/ ae2/ , and ghka/ mice , resulted mostly from the loss of tv . inhibition of acid secretion , in our hands , produced an unbalanced membrane exchange in wt and nhe2/ parietal cells ( figure 2 ) . basolateral membranes were increased in all of the kos but did not increase sufficiently to compensate for the loss of other secretory membranes , resulting in unbalanced modulation . tv were almost always visible in wt parietal cells , representing around 8% of the cell cytoplasm ( vd = 7.68 0.96 , n = 47 ) ( figure 1 ) under normal conditions and were greatly increased when acid secretion was inhibited by vagotomy or omeprazole ( 13.99 2.73 n = 7 , p = .010 to untreated wt ) . fasting and stimulation produced variable morphometric outcomes , especially in the vd of tv in wt parietal cells , which varied from 0% to 100% of the secretory membranes in any given cell profile . thus , while the tv in some models did not reflect acid secretion , the morphology of tv gave important clues as to the causes of failed secretion , and highlighted the relatively unchanging appearance of cam as well as mitochondria . strikingly however , canaliculi in the cytoplasm of ae2/ , nhe4/ , and the nhe2/ ae2/ mice were infrequent compared to wt ( figure 3 ) , and invaginations were nearly always seen in close proximity to the apical membrane . both microvillar length and width tended to decrease during inhibition of acid secretion ( vagotomy and omeprazole , combined data ) in wt and nhe2/ mice , but this did not reach statistical significance . no quantification of the ratio of centrally to peripherally located actin was performed in the current study , but it was obvious , subjectively , that wt parietal cells had considerably more microvilli with peripherally located actin than with centrally bundled actin , while in the achlorhydric kos the latter arrangement was much more frequent . ( 1 ) the hypochlorhydric nhe2/ mice had actin located peripherally in the microvilli , again underscoring that the parietal cells in this ko must have some functional acid secretion , and ( 2 ) in the ghka/ mice , where parietal cells always showed centrally bundled actin , microvilli were significantly shorter than in wt mice . the increase in vd of the basolateral membrane was significant in 4 of the 6 achlorhydric genotypes , and elevated in the other two ( figures 3 and 4 ) . in nhe4/ and ae2/ mice , and in the nhe2/ ae2/ double ko especially , interdigitations were broader ( figure 5 ) . the vd of basolateral membranes alone was about equivalent to the vd of canaliculi in predicting the level of acid secretion . however , when the vd of blm was expressed as a ratio with the vd of mitochondria , the p values were significant for 6/6 of the achlorhydric genotypes , thus making this ratio one of the better markers derived from morphometry . the vd of mitochondria provided a reasonably good measure of acid secretory competence , being significantly reduced in 5 of 6 achlorhydric models compared to wt , reflecting decreased secretory activity . vd of mitochondria was about 30% in wt parietal cells and was significantly reduced in parietal cells of nhe2/ mice ( untreated ) , compared to wt . the position of mitochondria within wt parietal cells is characteristically closely juxtaposed to tv and cam , and indeed , areas of tv and cam are intimately surrounded by mitochondria . the vd of lamellar inclusions in nhe2/ mice decreased upon inhibition of acid secretion by vagotomy or omeprazole ( nhe2/ untreated 1.31 0.31 , n = 13 , versus nhe2/ inhibited 0.76 0.37 , n = 9 , p = .073 ) , suggesting that they may result from faulty modulation of tv and cam . interestingly , where the tvs were decreased because of loss of ghka in the double null mice , the vd of lamellar inclusions diminished , but in the nhe2/ ae2/ null mice , it increased above that of the nhe2/ single ko ( figure 3 ) . gastric acidity in wt mice parallels the relative volume densities of canalicular and apical membranes ( cam ) and tubulovesicular membranes ( tv ) in the parietal cell cytoplasm in a predictable manner . although the relationship between the level of acid secretion and the relative amounts of cam and tv has been useful as a reference point for histology , it is not without inconsistencies . just as problematic is the fact that adjacent parietal cells can vary widely in the vd of tvs . our data from our normal parietal cells supported the occurrence of diminished vd of tv with stimulation and a cytoplasm containing mostly tvs and only a few cam after inhibition of acid secretion . during examination of the various mouse models of hypochlorhydria and achlorhydria it became clear that using the ratio of cam to tv was not an unambiguous indicator of activity . our ultrastructural quantification of cam and tv and other membranes and organelles in mice which were normochlorhydric , compared with 6 mouse models with defective acid secretion , demonstrated that the vd of tv was more variable than other organelles when predicting secretory competence . in fact , the vd of basolateral membranes , and mitochondria , and the ratio of these two parameters , paralleled the gastric ph data from published studies far better . the most critical event in disrupting the ratio of cam to tv appeared to involve the interruption of vesicle trafficking between tv and cam . this was alluded to in the ae2/ and nhe4/ single kos [ 11 , 12 , 30 ] and the nhe2/ ae2/ double ko , but comparisons clearly suggest that with significant decreases in vd of tv , there is little acid secretion . in one of the few papers which quantified parietal cells in achlorhydric mice ( these lacked ezrin ) a significant increase in tv and apical membranes in parietal cells was shown , but in images from that study , apical membrane microvilli looked disorganized and were reduced in amount compared to their wt controls , and what was identified as a vesicular component resembled the rounded vesicles in ghka/ parietal cells ( figure 4 ) in our study . reports reveal that the loss of proteins critical for acid secretion sometimes results in dramatic ultrastructural consequences , as was seen in parietal cells of mice lacking ghka [ 13 , 28 , 29 ] . it is important to point out that in the nhe2/ mice the loss of acid secretion appeared to be due primarily to diminished parietal cell viability , reduced parietal cell numbers , and increased base secretion . whether secretory membranes in wt mice were equitably exchanged after vagotomy and omeprazole treatments our data show that significant fluctuations ( reductions or increases ) in tv , cam , and blm occurred while the vd of total secretory membranes remained nearly unchanged . according to ito and schofield , during stimulation of acid secretion with insulin , microvillar membranes increased from 12% to 20% , lateral and basal cell membranes decreased from 6% to 5% , and there was a simultaneous reduction in tubulovesicular membranes from a vd of about 24% in untreated , to around 4% in stimulated cells . the inhibition of acid secretion , in our experiments , produced an unbalanced membrane exchange in both wt and nhe2/ parietal cells and also was of a lesser magnitude than what others reported . even then , it comprises only a few percent of the total membrane vd in parietal cells and this increase would appear to be insufficient to compensate for the large fluctuations in the vd of tv and cam . it is clear , of course , that basal and lateral membranes form a separate compartment from that of secretory membranes ( tv and cam ) , and too that combining them into one category is an oversimplification . evidence in support of the involvement of basolateral membrane processes in acid secretion comes from the required activation of cl / hco3 and na / h exchangers on the basolateral membrane [ 11 , 12 , 49 ] . somewhat counterintuitively , however , in this study we found a significant increase in vd of basolateral membranes when acid secretion was diminished . however , when reduced acid secretion was accompanied by increased canalicular space , vd of basal and lateral membranes was still increased ( as was seen in the nhe2/ and ghka parietal cells ) . basolateral membranes are also critical sites of ligand binding ( acetylcholine , gastrin , histamine ) for regulation of acid secretion , and it is possible that the vd of basolateral membranes is regulated separately from the vd of tv and cam . the vd of mitochondria was about 30% in the wt parietal cells in this study , which was very close to reports in the literature [ 11 , 12 , 15 , 18 , 29 , 30 , 36 , 38 , 39 , 50 , 51 ] . mitochondrial vd appeared not to fluctuate after stimulation or inhibition of secretion in the short term [ 15 , 38 ] but it did decrease under conditions of chronic inhibition ( wt , vagotomy , and omeprazole ) as shown in the current study . mitochondrial vd was significantly reduced in untreated nhe2/ mice , and inhibition with omeprazole and vagotomy did not decrease it further ( figure 2 ) . mitochondria within wt parietal cells were typically closely juxtaposed to tv and cam , and indeed , areas of tv and cam and mitochondria were intimately associated in wt and in nhe2/ parietal cells , and in ghka/ parietal cells to some degree . uncharacteristically however , and especially prominent in the nhe4/ ko , but also in ae2/ and nhe2/ ae2/ double ko parietal cells , mitochondria were positioned quite a distance from cam , rarely coming in close proximity to those membranes . showed ae2/ parietal cells which clearly pointed to a decreased vd of mitochondria and a greater separation between mitochondria , tv , and cam . in summary , using various knockout mouse models in which acid secretion was impaired and experimentally induced inhibition of acid secretion it was determined that the ratio of cam to tv was a good indicator of acid secretion in parietal cells which have functional membrane trafficking potential . nhe2/ mice had the capacity to modulate secretory membranes and showed a parallel response to wt parietal cells in morphometry and morphology , though attenuated . it appears that the vd of mitochondria and vd of basolateral membranes and the ratio of the two , are measures which can be applied both to wt and achlorhydric models that reflect the level of gastric acid secretion .

premenstrual syndrome ( pms ) is a set of somatic and psychological symptoms which occur during the luteal phase of the menstrual cycle ( 1 ) . up to 85% of women of reproductive age report experiencing one or more of the symptoms of pms ( 2 ) and approximately 5% suffer from a severe form of pms called premenstrual dysphoric disorder ( pmdd ) ( 3 ) . the exact cause of pms still remains unclear and many factors are supposed to contribute to the condition ; therefore , it has been proposed that pms is a multicausal problem ( 4 ) . according to the neurobiological data on the subject , the activities of the neurotransmitter system are mainly affected by gonadal steroids . through this process , estrogens , progestins , and androgens may be indirectly implicit in the development of depression ( 5 , 6 ) . although several studies have focused on the hormone and lipid profiles of women with pms , they report findings that are controversial . for example , several studies that have assessed the connection between pms and testosterone levels , present findings that conflict ( 7 , 8) . similarly , there is also disagreement over the association between prolactin ( prl ) levels and pms ; while benedek - jaszmann and hearn - sturtevant reported higher levels of prl in women with pms ( 9 ) , backstrom and aakvaag failed to find any association ( 10 ) . there are also conflicting data on the mean concentration of cholesterol among women with and without pms ( 11 ) . by conducting epidemiological and clinical studies , many researchers have uncovered conflicting results regarding the association between estradiol ( e2 ) , follicle - stimulating hormone ( fsh ) , testosterone ( t ) , dehydroepiandrosterone ( dhea ) , and dehydroepiandrosterone sulfate ( dhea - s ) levels and depression ( 12 - 15 ) . to date , few studies have evaluated the association between hormonal and metabolic factors and pms . most of the results were restricted by their small sample size , inappropriate inclusion criteria for women with pms , and by their comparison of a limited number of variables between the two groups of women , both with and without pms ( 10 , 16 ) . as a result , in the present study we aimed to investigate the association of hormonal and metabolic factors with pms among iranian women of reproductive age . the subjects of this study were selected from among the participants in the iranian pcos prevalence study which was a community based cross - sectional study of 1026 women , aged 18 - 45 years , conducted between 2009 - 2010 ( 17 ) . the eligible women were invited to participate in a comprehensive interview and their blood pressure , anthropometric , hormonal , and metabolic measurements were documented . data were completed for all but 97 women who did not come to the clinics and 19 participants whose hormonal and metabolic profiles were unavailable . we also excluded women who were using antidepressants ( n = 34 ) , oral contraceptive pills ( n = 151 ) , or taking hormonal medication for irregular menses . furthermore , those women who were pregnant at the time of the study ( n = 43 ) and menopausal women ( n = 37 ) were also excluded . finally , after the exclusion of the women who did not meet our inclusion criteria , 656 women were enrolled in the study . the american college of obstetricians and gynecologists ( acog ) criteria were used to diagnose pms . according to these criteria , the women needed to experience at least one of each of the following affective symptoms ( depression , angry outbursts , irritability , anxiety , confusion , social withdrawal ) and one somatic symptom ( breast tenderness , abdominal bloating , headache , swelling of extremities ) during the five days before menses in order to be diagnosed as having pms . each symptom must appear in three consecutive menstrual cycles and be scored on a scale of 1 ( low intensity ) , 2 ( moderate intensity ) and 3 ( severe intensity ) . the sum of the pms score ranges from 2 to 30 . using these criteria , our study participants were categorized into two groups : women with pms ( n = 354 ) and those without pms ( n = 302 ) . all the participants underwent clinical examinations , where their body weight , height , waist and hip circumferences , and blood pressure were measured by trained staff . height and weight were also measured with the subjects wearing light clothes but without shoes , using standard apparatus . height and waist circumferences ( wc ) were measured to the nearest 0.5 cm using a measuring tape . the waist was measured midway between the lower rib margin and the iliac crest , after a gentle expiration . body mass index ( bmi ) was calculated by dividing a participant s weight in kilograms by their height in meters squared ( kg / m ) . a blood sample that was used to determine biochemical measurements was taken from each subject on the second or third day of their menstrual cycle , after 12 hours of overnight fasting . blood samples were collected in edta treated test tubes . written informed consent was obtained from all participants before study entry . 17-hydroxyprogesterone ( 17oh - p ) , total testosterone ( tt ) and androstenedione ( a4 ) were measured by enzyme immunoassay ( eia ) , ( diagnostics biochem canada inc . , ontario , canada ) . sex hormone - binding globulin ( shbg ) was measured by immunoenzymometric assay ( iema ) , ( mercodia , uppsala , sweden ) . all elisa tests were performed using a sunrise elisa reader ( tecan co. , salzburg , austria ) . luteinizing hormone ( lh ) , follicle stimulating hormone ( fsh ) , prolactin ( prl ) , and thyroid stimulating hormone ( tsh ) were measured by immunoradiometric assay ( irma ) ( izotop , budapest , hungary ) using a gamma counter ( wallac wizard , turku , finland ) . it has been shown that in women , the free androgen index ( fai ) has a good correlation with free testosterone measured by a physical separation method ( 18 ) ; therefore , in this study the fai was calculated by using the formula : the intra- and inter - assay coefficients of variation for tt were 5.6% and 6.6% ; for shbg , 1.2% and 5.7% ; for a4 , 2.2% and 3.5% ; for lh , 3% and 5.8% ; for fsh , 3.5% and 4% ; for tsh , 1.7% and 3.4% , and for prl , they were 2.1% and 4.1% , respectively . according to the acog practice guidelines for the diagnosis of pms , one or more of the disturbing affective or somatic symptoms must have occurred in the five days before menses in each of three previous menstrual cycles . metabolic syndrome , based on the joint interim statement ( jis ) definition , was considered to be the presence of any three of the following five risk factors ( 19 ) : wc 95 ( country - specific cutoff point for iranians ( 20 ) , hdl < 50 , sbp 130 or dbp 85 , tg 150 and fbs 100 . according to the sixth report of the joint national committee ( jnc - vi ) criteria , hypertension or high bp was defined as mean sbp _ 140 mmhg , mean dbp _ 90 mmhg , or applied to a person undergoing current treatment for hypertension with prescription medication ( 21 ) . based on the american diabetes association s ( ada ) definition of diabetes , participants who met the following criteria were considered to be diabetic : 1 ) using anti - diabetic drugs or with fasting blood sugar ( fbs ) of _ 7 mmol / l or 2-h plasma glucose ( 2hpg ) _ 11.1 mmol / l ; 2 ) those with 2hpg between 7.77 and 11.1 mmol / l were defined as igt , and ; 3 ) fbs between 5.6 - 6.9 mmol / l was defined as impaired fasting plasma glucose ( ifg ) ( 10 , 22 ) . based on atp ii , dyslipidemia was defined as tc _ 240 mg / dl or ldl _ 160 mg / dl or tg _ 200 mg / dl or hdl < 35 mg / dl ( 23 ) . biochemical hyperandrogenism was detected by fai and/or a4 levels above the upper 95 percentile for the 362 women studied who were not on any hormonal medication and had no clinical evidence of hyperandrogenism , anovu , or pco . specifically , the upper normal limits for total t were = 0.88 ng / ml , a4 = 2.3 ng / ml , and fai = 5.47 . continuous variables were checked for normality using the one - sample kolmogorov - smirnov test , and expressed as mean , standard deviation , and/or median ( iq : 25 - 75 ) , as appropriate . linear regression ( forward method ) was used to identify the association between pms scores and metabolic disorders ( dependent variable ) after adjustment for age and bmi . the data analysis was performed using the spss 15.0 pc package ( spss inc . , the subjects of this study were selected from among the participants in the iranian pcos prevalence study which was a community based cross - sectional study of 1026 women , aged 18 - 45 years , conducted between 2009 - 2010 ( 17 ) . the eligible women were invited to participate in a comprehensive interview and their blood pressure , anthropometric , hormonal , and metabolic measurements were documented . data were completed for all but 97 women who did not come to the clinics and 19 participants whose hormonal and metabolic profiles were unavailable . we also excluded women who were using antidepressants ( n = 34 ) , oral contraceptive pills ( n = 151 ) , or taking hormonal medication for irregular menses . furthermore , those women who were pregnant at the time of the study ( n = 43 ) and menopausal women ( n = 37 ) were also excluded . finally , after the exclusion of the women who did not meet our inclusion criteria , 656 women were enrolled in the study . the american college of obstetricians and gynecologists ( acog ) criteria were used to diagnose pms . according to these criteria , the women needed to experience at least one of each of the following affective symptoms ( depression , angry outbursts , irritability , anxiety , confusion , social withdrawal ) and one somatic symptom ( breast tenderness , abdominal bloating , headache , swelling of extremities ) during the five days before menses in order to be diagnosed as having pms . each symptom must appear in three consecutive menstrual cycles and be scored on a scale of 1 ( low intensity ) , 2 ( moderate intensity ) and 3 ( severe intensity ) . the sum of the pms score ranges from 2 to 30 . using these criteria , our study participants were categorized into two groups : women with pms ( n = 354 ) and those without pms ( n = 302 ) . all the participants underwent clinical examinations , where their body weight , height , waist and hip circumferences , and blood pressure were measured by trained staff . height and weight were also measured with the subjects wearing light clothes but without shoes , using standard apparatus . height and waist circumferences ( wc ) were measured to the nearest 0.5 cm using a measuring tape . the waist was measured midway between the lower rib margin and the iliac crest , after a gentle expiration . body mass index ( bmi ) was calculated by dividing a participant s weight in kilograms by their height in meters squared ( kg / m ) . a blood sample that was used to determine biochemical measurements was taken from each subject on the second or third day of their menstrual cycle , after 12 hours of overnight fasting . blood samples were collected in edta treated test tubes . written informed consent was obtained from all participants before study entry . 17-hydroxyprogesterone ( 17oh - p ) , total testosterone ( tt ) and androstenedione ( a4 ) were measured by enzyme immunoassay ( eia ) , ( diagnostics biochem canada inc . , ontario , canada ) . sex hormone - binding globulin ( shbg ) was measured by immunoenzymometric assay ( iema ) , ( mercodia , uppsala , sweden ) . all elisa tests were performed using a sunrise elisa reader ( tecan co. , salzburg , austria ) . luteinizing hormone ( lh ) , follicle stimulating hormone ( fsh ) , prolactin ( prl ) , and thyroid stimulating hormone ( tsh ) were measured by immunoradiometric assay ( irma ) ( izotop , budapest , hungary ) using a gamma counter ( wallac wizard , turku , finland ) . it has been shown that in women , the free androgen index ( fai ) has a good correlation with free testosterone measured by a physical separation method ( 18 ) ; therefore , in this study the fai was calculated by using the formula : the intra- and inter - assay coefficients of variation for tt were 5.6% and 6.6% ; for shbg , 1.2% and 5.7% ; for a4 , 2.2% and 3.5% ; for lh , 3% and 5.8% ; for fsh , 3.5% and 4% ; for tsh , 1.7% and 3.4% , and for prl , they were 2.1% and 4.1% , respectively . according to the acog practice guidelines for the diagnosis of pms , one or more of the disturbing affective or somatic symptoms must have occurred in the five days before menses in each of three previous menstrual cycles . metabolic syndrome , based on the joint interim statement ( jis ) definition , was considered to be the presence of any three of the following five risk factors ( 19 ) : wc 95 ( country - specific cutoff point for iranians ( 20 ) , hdl < 50 , sbp 130 or dbp 85 , tg 150 and fbs 100 . according to the sixth report of the joint national committee ( jnc - vi ) criteria , hypertension or high bp was defined as mean sbp _ 140 mmhg , mean dbp _ 90 mmhg , or applied to a person undergoing current treatment for hypertension with prescription medication ( 21 ) . based on the american diabetes association s ( ada ) definition of diabetes , participants who met the following criteria were considered to be diabetic : 1 ) using anti - diabetic drugs or with fasting blood sugar ( fbs ) of _ 7 mmol / l or 2-h plasma glucose ( 2hpg ) _ 11.1 mmol / l ; 2 ) those with 2hpg between 7.77 and 11.1 mmol / l were defined as igt , and ; 3 ) fbs between 5.6 - 6.9 mmol / l was defined as impaired fasting plasma glucose ( ifg ) ( 10 , 22 ) . based on atp ii , dyslipidemia was defined as tc _ 240 mg / dl or ldl _ 160 mg / dl or tg _ 200 mg / dl or hdl < 35 mg / dl ( 23 ) . biochemical hyperandrogenism was detected by fai and/or a4 levels above the upper 95 percentile for the 362 women studied who were not on any hormonal medication and had no clinical evidence of hyperandrogenism , anovu , or pco . specifically , the upper normal limits for total t were = 0.88 ng / ml , a4 = 2.3 ng / ml , and fai = 5.47 . continuous variables were checked for normality using the one - sample kolmogorov - smirnov test , and expressed as mean , standard deviation , and/or median ( iq : 25 - 75 ) , as appropriate . correlations between hormone concentrations and premenstrual syndrome scores were checked using the pearson correlation . linear regression ( forward method ) was used to identify the association between pms scores and metabolic disorders ( dependent variable ) after adjustment for age and bmi . the data analysis was performed using the spss 15.0 pc package ( spss inc . , the demographic characteristics of the women who did not complete the questionnaire or those without hormonal measurements available did not significantly differ from those who completed the study procedure ( data has not been presented ) . according to our findings , the education levels in women with pms were significantly higher than those without the condition . table 1 demonstrates some of the demographic , reproductive , and anthropometric characteristics of the studied women , as divided into two groups , i.e. women with pms and those without it . the values are expressed as mean sd except income , marital and job statuses that are presented as % . comparison carried out using t - tests or tests for continuous and categorical variables , respectively . table 2 shows a comparison of the hormonal , metabolic , and lipid profiles of women with and without pms . our results suggest that there was a significant increase in prl and tg among women with pms , whereas tes , hdl and 17oh - p were significantly decreased when compared to women without pms . mean prl and tes for women with pms were 18.5 and 0.58 , respectively , whereas for the controls these values were 16.3 and 0.67 , respectively ( p < 0.05 ) . the mean serum levels of tg , hdl and 17oh - p for women with and without pms were 147.3 , 44.2 , and 1.8 versus 128.2 , 46.3 , and 2 , respectively . using ancova , we compared all the above variables between the two groups in order to adjust for age and bmi ; the results of the t - test analysis were not significantly different . the results of the pearson correlation analysis showed that t4 , prl , tes , and 17oh - p had significant correlations with pms scores among the affected women ( p < 0.05 ) . other variants , including serum levels of tsh , lh , shbg , hdl , tg , insulin , fbs , ldl , chl , systolic bp , diastolic bp , lh / fsh , fai , and a4 were not significantly correlated with pms . after adjusting for age and bmi , using partial correlation after adjusting for age and bmi , a linear regression analysis demonstrated a significant association between pms scores and the prevalence of metabolic syndrome ( p = 0.033 ) . according to our findings , for every one unit increase in pms score there was a 12% increase in the probability of having metabolic syndrome . using a linear regression analysis we found a non - significant association between pms scores and other metabolic disorders , such as diabetes , hyperandrogenism , dyslipidemia , hypertension , and hypothyroidism ( table 3 ) . abbreviations : bmi , body mass index ; ci , confidence interval ; pms , premenstrual syndrome . in the present study , we assessed a wide range of hormones and metabolites in order to establish their associations with pms . the association found between lower tt levels and pms in the present study contradicts the findings of bloch et al.s study ( 7 ) which indicated significantly lower tt and free t levels in women with pms . however , eriksson et al . ( 16 ) and backstrom and aakvaag ( 10 ) discussed the contrary . it has been found that low testosterone in women can cause a number of physical and emotional symptoms , including depression , loss of sexual desire , and declining libido ( 24 - 26 ) . despite there being a limited number of studies concerning the effects of testosterone on mood , the results show that testosterone treatment per se , or with estrogen , improves mood in women ( 27 ) . we observed significantly lower 17-ohp levels among women with pms which are findings that contradict those of eriksson et al . ( 16 ) who assessed women with pms during the luteal phase and reported higher 17-ohp levels when compared to age - matched controls ; these differences may be explained by the difference in the age of the subjects and the effects of age on hormone levels . the present study demonstrated that the serum prl level is significantly higher in women with pms , which is a finding that is in line with previous studies ( 9 , 28 ) . by contrast , another study ( 10 ) reported that the mean plasma prolactin level among 15 women affected with pms was not significantly different from that of the 17 women in the control group ; it appears that because of the small sample size , the researchers found no association between prl and pms . prolactin plays an indirect role in premenstrual syndrome and may cause renal retention of water , sodium , and potassium , and it interacts with lithium . prolactin can also interact with ovarian hormones to cause symptoms of depression , anxiety , or irritable hostility ( 29 ) . the data report higher levels of total cholesterol in women with pms with no significant alteration in tg and hdl ( 11 ) ; conversely , our data showed higher tg and lower hdl levels . to our knowledge , this is the first study examining the association of metabolic disorders in iranian women with pms . several studies have demonstrated that depression is significantly associated with metabolic syndrome ; for example , raikkonen et al . ( 30 ) also suggest that there is an association between metabolic syndrome and higher rates of depression . since depression is one of the main criteria of diagnosing pms , it can be speculated that pms and metabolic syndrome are associated . according to the data produced by this study , there was no significant association between insulin levels and pms , which is a consonant result with that of zarei et al . eriksson et al . ( 16 ) found no significant difference between pms cases and controls in shbg levels , which was also consistent with our findings . one of the strengths of our study was the large cross - sectional community based sample that it used . since most similar studies have focused on the small number of variants that contribute to pms disorders , we were able to investigate a broad range of anthropometric parameters and hormone levels in order to determine the independent association between endogenous hormones and metabolic parameters in multivariate analyses . however , a potential limitation that must be recognized is that we used homa - ir as a surrogate marker for assessing insulin resistance ( ir ) ; in spite of the good correlation between homa - ir and gold standard clamp methods , it might be inaccurate in women with pcos . moreover , we did not measure free testosterone due to our inability to access a proper method for its measurement . however , we calculated the fai using equation 1 it has been shown that fai correlates with free testosterone as measured by the physical separation method in women , which is the same as calculated free testosterone ( 17 ) . in conclusion , we found a significant association between pms scores and the prevalence of metabolic syndrome . moreover , we observed higher levels of prl and lower levels of tes , ohp-17 , tg , and hdl among women with pms , when compared to women without the syndrome . further studies are needed to confirm and validate the relationships between lipid profile abnormalities and metabolic disorders with pms .

backgroundpremenstrual syndrome ( pms ) is reported by up to 85% of women of reproductive age . although several studies have focused on the hormone and lipid profiles of females with pms , the results are controversial.objectivesthis study was designed to investigate the association of hormonal and metabolic factors with pms among iranian women of reproductive age.materials and methodsthis study was a community based cross - sectional study . anthropometric measurements , biochemical parameters , and metabolic disorders were compared between 354 women with pms and 302 healthy controls selected from among 1126 women of reproductive age who participated in the iranian pcos prevalence study . p values < 0.05 were considered significant.resultsprolactin ( prl ) and triglycerides ( tg ) were significantly elevated in women with pms , whereas their testosterone ( tes ) , high density lipoprotein ( hdl ) and 17-hydroxyprogesterone ( 17-ohp ) levels were significantly less than they were in women without the syndrome ( p < 0.05 ) . after adjusting for age and body mass index ( bmi ) , linear regression analysis demonstrated that for every one unit increase in pms score there was 12% rise in the probability of having metabolic syndrome ( p = 0.033).conclusionsthere was a significant association between pms scores and the prevalence of metabolic syndrome . further studies are needed to confirm and validate the relationships between lipid profile abnormalities and metabolic disorders with pms .

1. Background 2. Objectives 3. Materials and Methods 3.1. Subjects 3.2. Laboratory Measurements 3.3. Definition 3.4. Statistical Analysis 4. Results 5. Discussion

premenstrual syndrome ( pms ) is a set of somatic and psychological symptoms which occur during the luteal phase of the menstrual cycle ( 1 ) . up to 85% of women of reproductive age report experiencing one or more of the symptoms of pms ( 2 ) and approximately 5% suffer from a severe form of pms called premenstrual dysphoric disorder ( pmdd ) ( 3 ) . according to the neurobiological data on the subject , the activities of the neurotransmitter system are mainly affected by gonadal steroids . through this process , estrogens , progestins , and androgens may be indirectly implicit in the development of depression ( 5 , 6 ) . although several studies have focused on the hormone and lipid profiles of women with pms , they report findings that are controversial . for example , several studies that have assessed the connection between pms and testosterone levels , present findings that conflict ( 7 , 8) . similarly , there is also disagreement over the association between prolactin ( prl ) levels and pms ; while benedek - jaszmann and hearn - sturtevant reported higher levels of prl in women with pms ( 9 ) , backstrom and aakvaag failed to find any association ( 10 ) . there are also conflicting data on the mean concentration of cholesterol among women with and without pms ( 11 ) . by conducting epidemiological and clinical studies , many researchers have uncovered conflicting results regarding the association between estradiol ( e2 ) , follicle - stimulating hormone ( fsh ) , testosterone ( t ) , dehydroepiandrosterone ( dhea ) , and dehydroepiandrosterone sulfate ( dhea - s ) levels and depression ( 12 - 15 ) . to date , few studies have evaluated the association between hormonal and metabolic factors and pms . most of the results were restricted by their small sample size , inappropriate inclusion criteria for women with pms , and by their comparison of a limited number of variables between the two groups of women , both with and without pms ( 10 , 16 ) . as a result , in the present study we aimed to investigate the association of hormonal and metabolic factors with pms among iranian women of reproductive age . the subjects of this study were selected from among the participants in the iranian pcos prevalence study which was a community based cross - sectional study of 1026 women , aged 18 - 45 years , conducted between 2009 - 2010 ( 17 ) . the eligible women were invited to participate in a comprehensive interview and their blood pressure , anthropometric , hormonal , and metabolic measurements were documented . data were completed for all but 97 women who did not come to the clinics and 19 participants whose hormonal and metabolic profiles were unavailable . we also excluded women who were using antidepressants ( n = 34 ) , oral contraceptive pills ( n = 151 ) , or taking hormonal medication for irregular menses . furthermore , those women who were pregnant at the time of the study ( n = 43 ) and menopausal women ( n = 37 ) were also excluded . finally , after the exclusion of the women who did not meet our inclusion criteria , 656 women were enrolled in the study . according to these criteria , the women needed to experience at least one of each of the following affective symptoms ( depression , angry outbursts , irritability , anxiety , confusion , social withdrawal ) and one somatic symptom ( breast tenderness , abdominal bloating , headache , swelling of extremities ) during the five days before menses in order to be diagnosed as having pms . each symptom must appear in three consecutive menstrual cycles and be scored on a scale of 1 ( low intensity ) , 2 ( moderate intensity ) and 3 ( severe intensity ) . the sum of the pms score ranges from 2 to 30 . using these criteria , our study participants were categorized into two groups : women with pms ( n = 354 ) and those without pms ( n = 302 ) . height and waist circumferences ( wc ) were measured to the nearest 0.5 cm using a measuring tape . the waist was measured midway between the lower rib margin and the iliac crest , after a gentle expiration . body mass index ( bmi ) was calculated by dividing a participant s weight in kilograms by their height in meters squared ( kg / m ) . a blood sample that was used to determine biochemical measurements was taken from each subject on the second or third day of their menstrual cycle , after 12 hours of overnight fasting . 17-hydroxyprogesterone ( 17oh - p ) , total testosterone ( tt ) and androstenedione ( a4 ) were measured by enzyme immunoassay ( eia ) , ( diagnostics biochem canada inc . sex hormone - binding globulin ( shbg ) was measured by immunoenzymometric assay ( iema ) , ( mercodia , uppsala , sweden ) . luteinizing hormone ( lh ) , follicle stimulating hormone ( fsh ) , prolactin ( prl ) , and thyroid stimulating hormone ( tsh ) were measured by immunoradiometric assay ( irma ) ( izotop , budapest , hungary ) using a gamma counter ( wallac wizard , turku , finland ) . it has been shown that in women , the free androgen index ( fai ) has a good correlation with free testosterone measured by a physical separation method ( 18 ) ; therefore , in this study the fai was calculated by using the formula : the intra- and inter - assay coefficients of variation for tt were 5.6% and 6.6% ; for shbg , 1.2% and 5.7% ; for a4 , 2.2% and 3.5% ; for lh , 3% and 5.8% ; for fsh , 3.5% and 4% ; for tsh , 1.7% and 3.4% , and for prl , they were 2.1% and 4.1% , respectively . according to the acog practice guidelines for the diagnosis of pms , one or more of the disturbing affective or somatic symptoms must have occurred in the five days before menses in each of three previous menstrual cycles . metabolic syndrome , based on the joint interim statement ( jis ) definition , was considered to be the presence of any three of the following five risk factors ( 19 ) : wc 95 ( country - specific cutoff point for iranians ( 20 ) , hdl < 50 , sbp 130 or dbp 85 , tg 150 and fbs 100 . based on the american diabetes association s ( ada ) definition of diabetes , participants who met the following criteria were considered to be diabetic : 1 ) using anti - diabetic drugs or with fasting blood sugar ( fbs ) of _ 7 mmol / l or 2-h plasma glucose ( 2hpg ) _ 11.1 mmol / l ; 2 ) those with 2hpg between 7.77 and 11.1 mmol / l were defined as igt , and ; 3 ) fbs between 5.6 - 6.9 mmol / l was defined as impaired fasting plasma glucose ( ifg ) ( 10 , 22 ) . specifically , the upper normal limits for total t were = 0.88 ng / ml , a4 = 2.3 ng / ml , and fai = 5.47 . continuous variables were checked for normality using the one - sample kolmogorov - smirnov test , and expressed as mean , standard deviation , and/or median ( iq : 25 - 75 ) , as appropriate . linear regression ( forward method ) was used to identify the association between pms scores and metabolic disorders ( dependent variable ) after adjustment for age and bmi . , the subjects of this study were selected from among the participants in the iranian pcos prevalence study which was a community based cross - sectional study of 1026 women , aged 18 - 45 years , conducted between 2009 - 2010 ( 17 ) . the eligible women were invited to participate in a comprehensive interview and their blood pressure , anthropometric , hormonal , and metabolic measurements were documented . data were completed for all but 97 women who did not come to the clinics and 19 participants whose hormonal and metabolic profiles were unavailable . we also excluded women who were using antidepressants ( n = 34 ) , oral contraceptive pills ( n = 151 ) , or taking hormonal medication for irregular menses . furthermore , those women who were pregnant at the time of the study ( n = 43 ) and menopausal women ( n = 37 ) were also excluded . according to these criteria , the women needed to experience at least one of each of the following affective symptoms ( depression , angry outbursts , irritability , anxiety , confusion , social withdrawal ) and one somatic symptom ( breast tenderness , abdominal bloating , headache , swelling of extremities ) during the five days before menses in order to be diagnosed as having pms . each symptom must appear in three consecutive menstrual cycles and be scored on a scale of 1 ( low intensity ) , 2 ( moderate intensity ) and 3 ( severe intensity ) . the sum of the pms score ranges from 2 to 30 . using these criteria , our study participants were categorized into two groups : women with pms ( n = 354 ) and those without pms ( n = 302 ) . all the participants underwent clinical examinations , where their body weight , height , waist and hip circumferences , and blood pressure were measured by trained staff . height and waist circumferences ( wc ) were measured to the nearest 0.5 cm using a measuring tape . the waist was measured midway between the lower rib margin and the iliac crest , after a gentle expiration . body mass index ( bmi ) was calculated by dividing a participant s weight in kilograms by their height in meters squared ( kg / m ) . a blood sample that was used to determine biochemical measurements was taken from each subject on the second or third day of their menstrual cycle , after 12 hours of overnight fasting . 17-hydroxyprogesterone ( 17oh - p ) , total testosterone ( tt ) and androstenedione ( a4 ) were measured by enzyme immunoassay ( eia ) , ( diagnostics biochem canada inc . sex hormone - binding globulin ( shbg ) was measured by immunoenzymometric assay ( iema ) , ( mercodia , uppsala , sweden ) . luteinizing hormone ( lh ) , follicle stimulating hormone ( fsh ) , prolactin ( prl ) , and thyroid stimulating hormone ( tsh ) were measured by immunoradiometric assay ( irma ) ( izotop , budapest , hungary ) using a gamma counter ( wallac wizard , turku , finland ) . it has been shown that in women , the free androgen index ( fai ) has a good correlation with free testosterone measured by a physical separation method ( 18 ) ; therefore , in this study the fai was calculated by using the formula : the intra- and inter - assay coefficients of variation for tt were 5.6% and 6.6% ; for shbg , 1.2% and 5.7% ; for a4 , 2.2% and 3.5% ; for lh , 3% and 5.8% ; for fsh , 3.5% and 4% ; for tsh , 1.7% and 3.4% , and for prl , they were 2.1% and 4.1% , respectively . according to the acog practice guidelines for the diagnosis of pms , one or more of the disturbing affective or somatic symptoms must have occurred in the five days before menses in each of three previous menstrual cycles . metabolic syndrome , based on the joint interim statement ( jis ) definition , was considered to be the presence of any three of the following five risk factors ( 19 ) : wc 95 ( country - specific cutoff point for iranians ( 20 ) , hdl < 50 , sbp 130 or dbp 85 , tg 150 and fbs 100 . based on the american diabetes association s ( ada ) definition of diabetes , participants who met the following criteria were considered to be diabetic : 1 ) using anti - diabetic drugs or with fasting blood sugar ( fbs ) of _ 7 mmol / l or 2-h plasma glucose ( 2hpg ) _ 11.1 mmol / l ; 2 ) those with 2hpg between 7.77 and 11.1 mmol / l were defined as igt , and ; 3 ) fbs between 5.6 - 6.9 mmol / l was defined as impaired fasting plasma glucose ( ifg ) ( 10 , 22 ) . specifically , the upper normal limits for total t were = 0.88 ng / ml , a4 = 2.3 ng / ml , and fai = 5.47 . continuous variables were checked for normality using the one - sample kolmogorov - smirnov test , and expressed as mean , standard deviation , and/or median ( iq : 25 - 75 ) , as appropriate . linear regression ( forward method ) was used to identify the association between pms scores and metabolic disorders ( dependent variable ) after adjustment for age and bmi . according to our findings , the education levels in women with pms were significantly higher than those without the condition . table 1 demonstrates some of the demographic , reproductive , and anthropometric characteristics of the studied women , as divided into two groups , i.e. women with pms and those without it . table 2 shows a comparison of the hormonal , metabolic , and lipid profiles of women with and without pms . our results suggest that there was a significant increase in prl and tg among women with pms , whereas tes , hdl and 17oh - p were significantly decreased when compared to women without pms . mean prl and tes for women with pms were 18.5 and 0.58 , respectively , whereas for the controls these values were 16.3 and 0.67 , respectively ( p < 0.05 ) . the mean serum levels of tg , hdl and 17oh - p for women with and without pms were 147.3 , 44.2 , and 1.8 versus 128.2 , 46.3 , and 2 , respectively . using ancova , we compared all the above variables between the two groups in order to adjust for age and bmi ; the results of the t - test analysis were not significantly different . the results of the pearson correlation analysis showed that t4 , prl , tes , and 17oh - p had significant correlations with pms scores among the affected women ( p < 0.05 ) . other variants , including serum levels of tsh , lh , shbg , hdl , tg , insulin , fbs , ldl , chl , systolic bp , diastolic bp , lh / fsh , fai , and a4 were not significantly correlated with pms . after adjusting for age and bmi , using partial correlation after adjusting for age and bmi , a linear regression analysis demonstrated a significant association between pms scores and the prevalence of metabolic syndrome ( p = 0.033 ) . according to our findings , for every one unit increase in pms score there was a 12% increase in the probability of having metabolic syndrome . using a linear regression analysis we found a non - significant association between pms scores and other metabolic disorders , such as diabetes , hyperandrogenism , dyslipidemia , hypertension , and hypothyroidism ( table 3 ) . abbreviations : bmi , body mass index ; ci , confidence interval ; pms , premenstrual syndrome . in the present study , we assessed a wide range of hormones and metabolites in order to establish their associations with pms . the association found between lower tt levels and pms in the present study contradicts the findings of bloch et al.s study ( 7 ) which indicated significantly lower tt and free t levels in women with pms . ( 16 ) and backstrom and aakvaag ( 10 ) discussed the contrary . it has been found that low testosterone in women can cause a number of physical and emotional symptoms , including depression , loss of sexual desire , and declining libido ( 24 - 26 ) . despite there being a limited number of studies concerning the effects of testosterone on mood , the results show that testosterone treatment per se , or with estrogen , improves mood in women ( 27 ) . we observed significantly lower 17-ohp levels among women with pms which are findings that contradict those of eriksson et al . ( 16 ) who assessed women with pms during the luteal phase and reported higher 17-ohp levels when compared to age - matched controls ; these differences may be explained by the difference in the age of the subjects and the effects of age on hormone levels . the present study demonstrated that the serum prl level is significantly higher in women with pms , which is a finding that is in line with previous studies ( 9 , 28 ) . by contrast , another study ( 10 ) reported that the mean plasma prolactin level among 15 women affected with pms was not significantly different from that of the 17 women in the control group ; it appears that because of the small sample size , the researchers found no association between prl and pms . prolactin plays an indirect role in premenstrual syndrome and may cause renal retention of water , sodium , and potassium , and it interacts with lithium . the data report higher levels of total cholesterol in women with pms with no significant alteration in tg and hdl ( 11 ) ; conversely , our data showed higher tg and lower hdl levels . to our knowledge , this is the first study examining the association of metabolic disorders in iranian women with pms . several studies have demonstrated that depression is significantly associated with metabolic syndrome ; for example , raikkonen et al . ( 30 ) also suggest that there is an association between metabolic syndrome and higher rates of depression . since depression is one of the main criteria of diagnosing pms , it can be speculated that pms and metabolic syndrome are associated . according to the data produced by this study , there was no significant association between insulin levels and pms , which is a consonant result with that of zarei et al . ( 16 ) found no significant difference between pms cases and controls in shbg levels , which was also consistent with our findings . one of the strengths of our study was the large cross - sectional community based sample that it used . since most similar studies have focused on the small number of variants that contribute to pms disorders , we were able to investigate a broad range of anthropometric parameters and hormone levels in order to determine the independent association between endogenous hormones and metabolic parameters in multivariate analyses . however , a potential limitation that must be recognized is that we used homa - ir as a surrogate marker for assessing insulin resistance ( ir ) ; in spite of the good correlation between homa - ir and gold standard clamp methods , it might be inaccurate in women with pcos . however , we calculated the fai using equation 1 it has been shown that fai correlates with free testosterone as measured by the physical separation method in women , which is the same as calculated free testosterone ( 17 ) . in conclusion , we found a significant association between pms scores and the prevalence of metabolic syndrome . moreover , we observed higher levels of prl and lower levels of tes , ohp-17 , tg , and hdl among women with pms , when compared to women without the syndrome . further studies are needed to confirm and validate the relationships between lipid profile abnormalities and metabolic disorders with pms .

hospital - acquired pneumonia ( hap ) is defined as a low respiratory tract infection , which develops 48 h after hospital admission in a patient without infection at admission . hap currently ranks second among nosocomial infections and accounts for 25% of the infections in intensive care units . hap has a significant impact on the financial burden of health care , and new cases drive the increasing emergence of pathogens with multi- or pan - antibiotic resistance . therefore , identifying the infectious etiology in different settings is the key step for mitigating or obviating the severe infection in time . early identification of specific pathogens could significantly improve the morbidity and mortality of hap , and lower the cost of treatment as well . so far , the most common method used to implement hap etiology is still tied to sputum culture . in addition to sputum culture 's relatively low sensitivity and the difficulty with which it identifies atypical pathogens , the time required to obtain results always leads to empirical antibiotic therapies rather than target therapies for patients with hap . an rapid innovative method for etiology identification of hap , the quantitative loop - mediated isothermal amplification ( qlamp ) , has already been used in the diagnosis of virus , fungus , parasite , and tuberculosis infections and is now commercially available . it is a novel assay that focuses on the genetics of pathogens based on rapid nucleic acid amplification method . therefore , this technique has two important advantages such as rapid diagnosis and high sensitivity . in addition , qlamp is also a high specific assay , which could detect different bacteria with quantified copies . since the excellent timeliness and accuracy of qlamp for etiological diagnosis to the lower respiratory tract infection has been confirmed by our group , we initiated a pilot , prospective , and interventional study to investigate the value of qlamp to guide target antibiotics therapies in a small group of patients with hap . patients with suspected hap from august 2011 to march 2014 at peking university people 's hospital ( beijing , china ) were recruited in the study , which were approved by the ethical committee of peking university people 's hospital ( no . these patients were initially diagnosed as suspected cases of hap occurring more than 48 h after admission and were not incubated at the time of admission , having typical characteristics of pneumonia , which were firmly inferred from chest x - rays and the following criteria : [ i ] at least one of the following : ( 1 ) fever ( > 38.5c ) , ( 2 ) leukopenia ( peripheral white blood cell count [ wbc ] < 4.0 10/l ) or leukocytosis ( wbc > 10.0 10/l ) , and ( 3 ) for adults 70 years old or older , mental status changes with no other recognized cause ; [ ii ] at least two of the following : ( 1 ) new - onset of purulent sputum , or change in character of sputum , or increased respiratory secretion , or increased suctioning requirements , ( 2 ) new - onset or worsening cough , or dyspnea , or tachycardia , ( 3 ) rales or bronchial breath sounds , and ( 4 ) worsening gas exchange ( pao2/fio2 240 ) , increased oxygen requirements , or increased ventilation demand . patients with noninfectious diseases , viral infection , fungal infection , or tuberculosis were subsequently excluded from the study . once patients were enrolled , lower respiratory secretion samples were collected on the 1 day for both routine culture and qlamp assays , of which the results were reported to the clinicians . data of each patient were also collected from the medical records , with particular attention to clinical manifestations and treatment strategies before and after the qlamp results reporting . to determine the final diagnosis and assess the treatment response for each patient , 2 independent pulmonologists blinded to qlamp results reviewed all available medical records ( including patient history , physical examination , and results of laboratory tests , including blood routine examination , biochemical indicators , plasma electrolytes , blood gas analysis , and chest radiograph ) pertaining to the patient from the time of hap presentation to discharge / death . after liquefied in an equal volume of 10% naoh , dna specimen of each sample was isolated using the universal kit for bacterial dna extraction ( capitalbio corporation , china ) . the specimens were then prepared for qlamp using a set of specific primers for streptococcus pneumonia ( sp ) , staphylococcus aureus ( sa ) , escherichia coli ( ec ) , klebsiella pneumonia ( kp ) , pseudomonas aeruginosa ( pa ) , acinetobacter baumannii ( ab ) , stenotrophomonas maltophilia ( sm ) , haemophilus influenzae ( hi ) , legionella pneumophila ( lp ) , mycoplasma pneumonia ( mp ) , and chlamydophila pneumoniae ( cp ) . qlamp primer system of each species of pathogen is composed of six primers recognizing eight distinct regions on the target dna , termed a forward outer primer ( f3 ) , a backward internal primer ( bip ) ( b3 ) , a forward internal primer ( fip ) , a bip , and loop primers ( lf and lb ) . eight - pathogen primer sequences are used same as we did before , including sp , sa , ec , kp , pa , ab , sm , and hi . those for atypical pathogens were redesigned as shown in table 1 , and both their sensitivity and specificity were ensured by quantified dna isolated in 27 bacterial species as we did before . primers for atypical pathogens used in this study fip : forward internal primer ; bip : backward internal primer ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumonia . the reaction was performed at 65c for 45 min in a 25-l reaction mixture consisting of 1.6 mol / l each of fip and bip , 0.2 mol / l each of f3 and b3 , 0.4 mol / l each of lf and lb , 8 u of the bst dna polymerase large fragment ( new england biolabs inc . , beverly , mass . , usa ) , 0.4 mmol / l dntp , 0.1 mmol / l dutp , 0.8 mol / l betaine , 6 mmol / l mgso4 , 0.5 mg / ml bsa , 0.6 evagreen ( biotium , inc . , ca , usa ) , 0.1 u / ml uracil - dna glycosylase ( fermentas inc . , md , usa ) , 20 mmol / l tris - hcl ( ph 8.8 at 25c ) , 10 mmol / l kci , 10 mmol / l ( nh4)2 so4 , 0.1% triton x-100 , and 2 l template dna or pcr grade h2 o as negative control , and then heated at 80c for 5 min to terminate the reaction . all amplifications were performed with an rt - cycler real - time fluorescence quantitative pcr instrument ( capitalbio corporation , beijing , china ) . the titer was quantified according to the standard curves obtained from prequantified dna templates as described previously . biochip technology was introduced in january 2013 at peking university people 's hospital , and the reaction was performed on a microfluidic device after then . the qlamp tests and routine cultures were conducted by two experienced technicians awareness of the sample identities in two separated laboratories of peking university people 's hospital . we constructed a contingency table and used mcnemar test to evaluate the congruence of qlamp and culture results . the differences between patients with or without treatment strategies adjustment based on qlamp results were tested with fisher 's exact test or t test . baseline data of these patients were expressed as mean standard deviation ( sd ) for normally distributed values . all analyses were performed with the use of spss statistics software , version 19.0 ( ibm , usa ) . a p < 0.05 is considered statistically significant difference . patients with suspected hap from august 2011 to march 2014 at peking university people 's hospital ( beijing , china ) were recruited in the study , which were approved by the ethical committee of peking university people 's hospital ( no . these patients were initially diagnosed as suspected cases of hap occurring more than 48 h after admission and were not incubated at the time of admission , having typical characteristics of pneumonia , which were firmly inferred from chest x - rays and the following criteria : [ i ] at least one of the following : ( 1 ) fever ( > 38.5c ) , ( 2 ) leukopenia ( peripheral white blood cell count [ wbc ] < 4.0 10/l ) or leukocytosis ( wbc > 10.0 10/l ) , and ( 3 ) for adults 70 years old or older , mental status changes with no other recognized cause ; [ ii ] at least two of the following : ( 1 ) new - onset of purulent sputum , or change in character of sputum , or increased respiratory secretion , or increased suctioning requirements , ( 2 ) new - onset or worsening cough , or dyspnea , or tachycardia , ( 3 ) rales or bronchial breath sounds , and ( 4 ) worsening gas exchange ( pao2/fio2 240 ) , increased oxygen requirements , or increased ventilation demand . patients with noninfectious diseases , viral infection , fungal infection , or tuberculosis were subsequently excluded from the study . once patients were enrolled , lower respiratory secretion samples were collected on the 1 day for both routine culture and qlamp assays , of which the results were reported to the clinicians . data of each patient were also collected from the medical records , with particular attention to clinical manifestations and treatment strategies before and after the qlamp results reporting . to determine the final diagnosis and assess the treatment response for each patient , 2 independent pulmonologists blinded to qlamp results reviewed all available medical records ( including patient history , physical examination , and results of laboratory tests , including blood routine examination , biochemical indicators , plasma electrolytes , blood gas analysis , and chest radiograph ) pertaining to the patient from the time of hap presentation to discharge / death . after liquefied in an equal volume of 10% naoh , dna specimen of each sample was isolated using the universal kit for bacterial dna extraction ( capitalbio corporation , china ) . the specimens were then prepared for qlamp using a set of specific primers for streptococcus pneumonia ( sp ) , staphylococcus aureus ( sa ) , escherichia coli ( ec ) , klebsiella pneumonia ( kp ) , pseudomonas aeruginosa ( pa ) , acinetobacter baumannii ( ab ) , stenotrophomonas maltophilia ( sm ) , haemophilus influenzae ( hi ) , legionella pneumophila ( lp ) , mycoplasma pneumonia ( mp ) , and chlamydophila pneumoniae ( cp ) . qlamp primer system of each species of pathogen is composed of six primers recognizing eight distinct regions on the target dna , termed a forward outer primer ( f3 ) , a backward internal primer ( bip ) ( b3 ) , a forward internal primer ( fip ) , a bip , and loop primers ( lf and lb ) . eight - pathogen primer sequences are used same as we did before , including sp , sa , ec , kp , pa , ab , sm , and hi . those for atypical pathogens were redesigned as shown in table 1 , and both their sensitivity and specificity were ensured by quantified dna isolated in 27 bacterial species as we did before . primers for atypical pathogens used in this study fip : forward internal primer ; bip : backward internal primer ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumonia . the reaction was performed at 65c for 45 min in a 25-l reaction mixture consisting of 1.6 mol / l each of fip and bip , 0.2 mol / l each of f3 and b3 , 0.4 mol / l each of lf and lb , 8 u of the bst dna polymerase large fragment ( new england biolabs inc . , usa ) , 0.4 mmol / l dntp , 0.1 mmol / l dutp , 0.8 mol / l betaine , 6 mmol / l mgso4 , 0.5 mg / ml bsa , 0.6 evagreen ( biotium , inc . , ca , usa ) , 0.1 u / ml uracil - dna glycosylase ( fermentas inc . , md , usa ) , 20 mmol / l tris - hcl ( ph 8.8 at 25c ) , 10 mmol / l kci , 10 mmol / l ( nh4)2 so4 , 0.1% triton x-100 , and 2 l template dna or pcr grade h2 o as negative control , and then heated at 80c for 5 min to terminate the reaction . all amplifications were performed with an rt - cycler real - time fluorescence quantitative pcr instrument ( capitalbio corporation , beijing , china ) . the titer was quantified according to the standard curves obtained from prequantified dna templates as described previously . biochip technology was introduced in january 2013 at peking university people 's hospital , and the reaction was performed on a microfluidic device after then . the qlamp tests and routine cultures were conducted by two experienced technicians awareness of the sample identities in two separated laboratories of peking university people 's hospital . we constructed a contingency table and used mcnemar test to evaluate the congruence of qlamp and culture results . the differences between patients with or without treatment strategies adjustment based on qlamp results were tested with fisher 's exact test or t test . baseline data of these patients were expressed as mean standard deviation ( sd ) for normally distributed values . all analyses were performed with the use of spss statistics software , version 19.0 ( ibm , usa ) . a p < 0.05 is considered statistically significant difference . totally , 76 samples were recruited from 110 eligible samples overall in our study [ figure 1 ] . none of the samples were collected from the same onset of hap . as shown in table 2 , there were 70 samples with qlamp results greater than 10 copies / ml , 23 samples with qlamp results between 10 and 10 copies / ml , and 16 samples with qlamp results below 10 copies / ml . study profile . for each eligible patient , we collected lower respiratory secretion samples on the 1 day for routine culture and quantitative loop - mediated isothermal amplification tests and reported the results to the clinicians . we also collected the clinical data and treatment strategies before and after reporting the quantitative loop - mediated isothermal amplification results . qlamp results of specimen from patients with hospital - acquired pneumonia data are presented as number , unless otherwise indicated . qlamp : quantitative loop - mediated isothermal amplification ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumoniae ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia ; hi : haemophilus influenza . the concordance rates of the two assays for detecting sa , ec , pa , kp , sm , sp , and ab are 90.79% , 98.68% , 89.47% , 93.42% , 93.42% , 100.00% , and 77.63% , respectively [ table 3 ] . we also evaluated the difference between qlamp and culture results by mcnemar test , in which no significant difference was found ( p > 0.05 ) [ table 3 and supplementary tables 17 ] . the qlamp results of 4 samples for hi , lp , or mp were positive , while the culture results for these specimens were negative probably because of their low detectable rates in culture . we then studied the clinical data of these 4 samples and found that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . no cp positive results were reported in the 76 samples either by qlamp or culture assay . congruence of qlamp and culture results in patients with hospital - acquired pneumonia qlamp : quantitative loop - mediated isothermal amplification ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for escherichia coli * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for pseudomonas aeruginosa * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for klebsiella pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for stenotrophomonas maltophilia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for streptococcus pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for acinetobacter baumannii * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification a total of 44 qlamp - positive samples were identified in the study . eight samples were subsequently excluded from analysis because of discharge or death within 3 days after admission . the final analysis group of 36 patients consisted of 19 with treatment established or adjusted to target antibiotics therapies according to qlamp results ( pathogen target - driven therapy group ) and 17 without treatment strategies adjustment whose treatment strategy was inconsistent with qlamp results ( empirical therapy group ) . demographic and clinical characteristics of the two groups demographic and clinical characteristics of the two groups are shown in table 4 . there is no significant difference ( p > 0.05 ) in the characteristics of the patients between the two groups , including gender , age , complications , clinical manifestation ( temperature , cough , sputum , and rales ) , blood routine examination , biochemical indicators , blood gas analysis , blood coagulation index , and chest radiograph infiltration . demographic and clinical characteristics of patients in the two groups data are presented as mean sd unless otherwise indicated . : data not applicable ; sd : standard deviation ; wbc : white blood cell ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; alb : albumin ; cre : creatinine ; bun : blood urea nitrogen ; pt : prothrombin time ; aptt : activated partial thromboplastin time ; ne : neutrophil count . patients taking an advantage for clinical condition with pathogen target - driven therapy there are 16 patients from the pathogen target - driven therapy group with clinical condition improvement 3 days after qlamp results reported , while there is only 1 from the empirical therapy group . the remission rate is 84.2% in the group with pathogen target - driven therapy , and 5.9% in the group with empirical therapy . the differences in the remission rates between these two groups evaluated by fisher 's exact tests are statistically significant ( p = 2 10 ) . daily mean temperature of the group with pathogen target - driven therapy shows a more obvious tendency of improvement than the group with empirical therapy [ figure 2 ] . on the 1 day when samples were collected , the mean temperature of target - driven therapy group and empirical therapy group were 37.95c and 37.68c , respectively , while the mean temperature of those two groups changed to 37.42c and 38.02c 3 days later . the decrease of daily mean temperature was 0.53c in the group with pathogen target - driven therapy while the decrease in the group with empirical therapy was 0.34c . similarly , decrease of total wbc number in group with pathogen target - driven therapy is more significant than the group with empirical therapy ( 2.15 10/l with pathogen target - driven therapy vs. 0.70 10/l with empirical therapy ) [ figure 3 ] . the body temperature of the group with pathogen target - driven therapy decreased while the group with empirical therapy had no significant improvement in body temperature . the wbc count of the group with pathogen target - driven therapy decreased while the group with empirical therapy fluctuated in wbc count . totally , 76 samples were recruited from 110 eligible samples overall in our study [ figure 1 ] . none of the samples were collected from the same onset of hap . as shown in table 2 , there were 70 samples with qlamp results greater than 10 copies / ml , 23 samples with qlamp results between 10 and 10 copies / ml , and 16 samples with qlamp results below 10 copies / ml . study profile . for each eligible patient , we collected lower respiratory secretion samples on the 1 day for routine culture and quantitative loop - mediated isothermal amplification tests and reported the results to the clinicians . we also collected the clinical data and treatment strategies before and after reporting the quantitative loop - mediated isothermal amplification results . qlamp results of specimen from patients with hospital - acquired pneumonia data are presented as number , unless otherwise indicated . qlamp : quantitative loop - mediated isothermal amplification ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumoniae ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia ; hi : haemophilus influenza . the concordance rates of the two assays for detecting sa , ec , pa , kp , sm , sp , and ab are 90.79% , 98.68% , 89.47% , 93.42% , 93.42% , 100.00% , and 77.63% , respectively [ table 3 ] . we also evaluated the difference between qlamp and culture results by mcnemar test , in which no significant difference was found ( p > 0.05 ) [ table 3 and supplementary tables 17 ] . the qlamp results of 4 samples for hi , lp , or mp were positive , while the culture results for these specimens were negative probably because of their low detectable rates in culture . we then studied the clinical data of these 4 samples and found that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . no cp positive results were reported in the 76 samples either by qlamp or culture assay . congruence of qlamp and culture results in patients with hospital - acquired pneumonia qlamp : quantitative loop - mediated isothermal amplification ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for escherichia coli * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for pseudomonas aeruginosa * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for klebsiella pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for stenotrophomonas maltophilia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for streptococcus pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for acinetobacter baumannii * * data are presented as no . eight samples were subsequently excluded from analysis because of discharge or death within 3 days after admission . the final analysis group of 36 patients consisted of 19 with treatment established or adjusted to target antibiotics therapies according to qlamp results ( pathogen target - driven therapy group ) and 17 without treatment strategies adjustment whose treatment strategy was inconsistent with qlamp results ( empirical therapy group ) . demographic and clinical characteristics of the two groups demographic and clinical characteristics of the two groups are shown in table 4 . there is no significant difference ( p > 0.05 ) in the characteristics of the patients between the two groups , including gender , age , complications , clinical manifestation ( temperature , cough , sputum , and rales ) , blood routine examination , biochemical indicators , blood gas analysis , blood coagulation index , and chest radiograph infiltration . demographic and clinical characteristics of patients in the two groups data are presented as mean sd unless otherwise indicated . : data not applicable ; sd : standard deviation ; wbc : white blood cell ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; alb : albumin ; cre : creatinine ; bun : blood urea nitrogen ; pt : prothrombin time ; aptt : activated partial thromboplastin time ; ne : neutrophil count . patients taking an advantage for clinical condition with pathogen target - driven therapy there are 16 patients from the pathogen target - driven therapy group with clinical condition improvement 3 days after qlamp results reported , while there is only 1 from the empirical therapy group . the remission rate is 84.2% in the group with pathogen target - driven therapy , and 5.9% in the group with empirical therapy . the differences in the remission rates between these two groups evaluated by fisher 's exact tests are statistically significant ( p = 2 10 ) . daily mean temperature of the group with pathogen target - driven therapy shows a more obvious tendency of improvement than the group with empirical therapy [ figure 2 ] . on the 1 day when samples were collected , the mean temperature of target - driven therapy group and empirical therapy group were 37.95c and 37.68c , respectively , while the mean temperature of those two groups changed to 37.42c and 38.02c 3 days later . the decrease of daily mean temperature was 0.53c in the group with pathogen target - driven therapy while the decrease in the group with empirical therapy was 0.34c . similarly , decrease of total wbc number in group with pathogen target - driven therapy is more significant than the group with empirical therapy ( 2.15 10/l with pathogen target - driven therapy vs. 0.70 10/l with empirical therapy ) [ figure 3 ] . the body temperature of the group with pathogen target - driven therapy decreased while the group with empirical therapy had no significant improvement in body temperature . the wbc count of the group with pathogen target - driven therapy decreased while the group with empirical therapy fluctuated in wbc count . this is a pilot study to assess the value of qlamp in guiding early target antibiotic therapies of hap , which may have significant effects on the mortality of hap and reduce the cost . although bacterial pneumonia is a kind of curable diseases due to the advent of the antibiotics , the mortality of bacterial hap is still high , which may contribute to the delay of target antibiotics therapies according to the results of sputum culture . as a new manner of detecting the etiology of different kinds of infections , qlamp is now commercially available . with the availability of this rapid ( results are available within 12 h ) , sensitive , and specific therefore , we apply qlamp for the decision - making regarding whether we selected empirical antibiotic therapies or the target antibiotic therapies for hap patients . since we would investigate the value of qlamp steering therapies , the first important issue was whether qlamp can etiologically diagnose hap in time . as qlamp assay was much more rapid than sputum culture , the most common assay in recent clinical practice , we first focus on the congruency of the results of qlamp and sputum culture . fortunately , there was no significance between qlamp and culture results of hap patients with infections of sa , ec , pa , kp , sm , sp , and ab . in addition , qlamp can detect hi , lp , and mp , which were not detectable by culture . besides , qlamp was a candidate method which could differentiate the pathogens between colonized and infectious status . after that , we prospectively enrolled 36 patients with hap with the same baseline data to evaluate the value of qlamp steering early target therapies . among these patients , the qlamp results were all positive based on the cut - off value ( > 1.0 10 copies / ml ) which was established in our former work ( data not shown ) . we randomly adjusted the regimen of these patients with empirical therapies according to the 2005 american thoracic society / infectious diseases society of america hap guideline or target therapies based on the results of qlamp . interestingly , we found that the clinical condition was significantly improved in the group with pathogen target - driven therapy compared to the group with empirical therapies . firstly , it was performed with a small sample size , and the stochastic effects were too big to drive a definite conclusion . secondly , we did not test the infection of fungus and virus of hap , which may contribute a small number of hap infections . thirdly , we did not assess the immunological conditions and nutritional statuses of the patients in these two groups , which may influence the effects of antibiotic therapies . perhaps , we could combine qlamp and sputum drug sensitivity test to individualize the hap regimens . however , the definition of conclusion can only be driven after multi - centered , randomized , and large sample sized research . since qlamp can not test the drug sensitivity , the combination of qlamp and sputum culture is the good choice for guiding early target therapies in hap patients . in conclusion , supplementary information is linked to the online version of the paper on the chinese medical journal website . this study was supported by grants from capital medical research and development foundation ( no . 2009 - 1018 ) , special fund for health - scientific research in the public interest program ( no . 201202011 ) by ministry of health of china , national high - tech research and development program ( no . 2006aa02z4a9 ) by ministry of science and technology of china , national 12 five - year plan major scientific and technological program ( no . 2012zx10004 - 206 ) by ministry of science and technology of china . this study was supported by grants from capital medical research and development foundation ( no . 2009 - 1018 ) , special fund for health - scientific research in the public interest program ( no . 201202011 ) by ministry of health of china , national high - tech research and development program ( no . 2006aa02z4a9 ) by ministry of science and technology of china , national 12 five - year plan major scientific and technological program ( no . 2012zx10004 - 206 ) by ministry of science and technology of china .

background : it is important to achieve the definitive pathogen identification in hospital - acquired pneumonia ( hap ) , but the traditional culture results always delay the target antibiotic therapy . we assessed the method called quantitative loop - mediated isothermal amplification ( qlamp ) as a new implement for steering of the antibiotic decision - making in hap.methods:totally , 76 respiratory tract aspiration samples were prospectively collected from 60 hap patients . dna was isolated from these samples . specific dna fragments for identifying 11 pneumonia - related bacteria were amplified by qlamp assay . culture results of these patients were compared with the qlamp results . clinical data and treatment strategies were analyzed to evaluate the effects of qlamp results on clinical data . mcnemar test and fisher 's exact test were used for statistical analysis.results:the detection of staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , klebsiella pneumonia , stenotrophomonas maltophilia , streptococcus pneumonia , and acinetobacter baumannii by qlamp was consistent with sputum culture ( p > 0.05 ) . the qlamp results of 4 samples for haemophilus influenzae , legionella pneumophila , or mycoplasma pneumonia ( mp ) were inconsistent with culture results ; however , clinical data revealed that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . the improvement of clinical condition was more significant ( p < 0.001 ) in patients with pathogen target - driven therapy based on qlamp results than those with empirical therapy.conclusion:qlamp is a more promising method for detection of pathogens in an early , rapid , sensitive , and specific manner than culture .

I M Study design Procedures Statistical analysis R Quantitative loop-mediated isothermal amplification assaying outcome of specimen from hospital-acquired pneumonia patients Congruence of quantitative loop-mediated isothermal amplification and culture results Clinical benefit of quantitative loop-mediated isothermal amplification guided target therapy D Financial support and sponsorship Conflicts of interest

hospital - acquired pneumonia ( hap ) is defined as a low respiratory tract infection , which develops 48 h after hospital admission in a patient without infection at admission . in addition to sputum culture 's relatively low sensitivity and the difficulty with which it identifies atypical pathogens , the time required to obtain results always leads to empirical antibiotic therapies rather than target therapies for patients with hap . an rapid innovative method for etiology identification of hap , the quantitative loop - mediated isothermal amplification ( qlamp ) , has already been used in the diagnosis of virus , fungus , parasite , and tuberculosis infections and is now commercially available . it is a novel assay that focuses on the genetics of pathogens based on rapid nucleic acid amplification method . since the excellent timeliness and accuracy of qlamp for etiological diagnosis to the lower respiratory tract infection has been confirmed by our group , we initiated a pilot , prospective , and interventional study to investigate the value of qlamp to guide target antibiotics therapies in a small group of patients with hap . patients with suspected hap from august 2011 to march 2014 at peking university people 's hospital ( beijing , china ) were recruited in the study , which were approved by the ethical committee of peking university people 's hospital ( no . these patients were initially diagnosed as suspected cases of hap occurring more than 48 h after admission and were not incubated at the time of admission , having typical characteristics of pneumonia , which were firmly inferred from chest x - rays and the following criteria : [ i ] at least one of the following : ( 1 ) fever ( > 38.5c ) , ( 2 ) leukopenia ( peripheral white blood cell count [ wbc ] < 4.0 10/l ) or leukocytosis ( wbc > 10.0 10/l ) , and ( 3 ) for adults 70 years old or older , mental status changes with no other recognized cause ; [ ii ] at least two of the following : ( 1 ) new - onset of purulent sputum , or change in character of sputum , or increased respiratory secretion , or increased suctioning requirements , ( 2 ) new - onset or worsening cough , or dyspnea , or tachycardia , ( 3 ) rales or bronchial breath sounds , and ( 4 ) worsening gas exchange ( pao2/fio2 240 ) , increased oxygen requirements , or increased ventilation demand . once patients were enrolled , lower respiratory secretion samples were collected on the 1 day for both routine culture and qlamp assays , of which the results were reported to the clinicians . data of each patient were also collected from the medical records , with particular attention to clinical manifestations and treatment strategies before and after the qlamp results reporting . to determine the final diagnosis and assess the treatment response for each patient , 2 independent pulmonologists blinded to qlamp results reviewed all available medical records ( including patient history , physical examination , and results of laboratory tests , including blood routine examination , biochemical indicators , plasma electrolytes , blood gas analysis , and chest radiograph ) pertaining to the patient from the time of hap presentation to discharge / death . the specimens were then prepared for qlamp using a set of specific primers for streptococcus pneumonia ( sp ) , staphylococcus aureus ( sa ) , escherichia coli ( ec ) , klebsiella pneumonia ( kp ) , pseudomonas aeruginosa ( pa ) , acinetobacter baumannii ( ab ) , stenotrophomonas maltophilia ( sm ) , haemophilus influenzae ( hi ) , legionella pneumophila ( lp ) , mycoplasma pneumonia ( mp ) , and chlamydophila pneumoniae ( cp ) . we constructed a contingency table and used mcnemar test to evaluate the congruence of qlamp and culture results . the differences between patients with or without treatment strategies adjustment based on qlamp results were tested with fisher 's exact test or t test . a p < 0.05 is considered statistically significant difference . patients with suspected hap from august 2011 to march 2014 at peking university people 's hospital ( beijing , china ) were recruited in the study , which were approved by the ethical committee of peking university people 's hospital ( no . these patients were initially diagnosed as suspected cases of hap occurring more than 48 h after admission and were not incubated at the time of admission , having typical characteristics of pneumonia , which were firmly inferred from chest x - rays and the following criteria : [ i ] at least one of the following : ( 1 ) fever ( > 38.5c ) , ( 2 ) leukopenia ( peripheral white blood cell count [ wbc ] < 4.0 10/l ) or leukocytosis ( wbc > 10.0 10/l ) , and ( 3 ) for adults 70 years old or older , mental status changes with no other recognized cause ; [ ii ] at least two of the following : ( 1 ) new - onset of purulent sputum , or change in character of sputum , or increased respiratory secretion , or increased suctioning requirements , ( 2 ) new - onset or worsening cough , or dyspnea , or tachycardia , ( 3 ) rales or bronchial breath sounds , and ( 4 ) worsening gas exchange ( pao2/fio2 240 ) , increased oxygen requirements , or increased ventilation demand . once patients were enrolled , lower respiratory secretion samples were collected on the 1 day for both routine culture and qlamp assays , of which the results were reported to the clinicians . data of each patient were also collected from the medical records , with particular attention to clinical manifestations and treatment strategies before and after the qlamp results reporting . to determine the final diagnosis and assess the treatment response for each patient , 2 independent pulmonologists blinded to qlamp results reviewed all available medical records ( including patient history , physical examination , and results of laboratory tests , including blood routine examination , biochemical indicators , plasma electrolytes , blood gas analysis , and chest radiograph ) pertaining to the patient from the time of hap presentation to discharge / death . the specimens were then prepared for qlamp using a set of specific primers for streptococcus pneumonia ( sp ) , staphylococcus aureus ( sa ) , escherichia coli ( ec ) , klebsiella pneumonia ( kp ) , pseudomonas aeruginosa ( pa ) , acinetobacter baumannii ( ab ) , stenotrophomonas maltophilia ( sm ) , haemophilus influenzae ( hi ) , legionella pneumophila ( lp ) , mycoplasma pneumonia ( mp ) , and chlamydophila pneumoniae ( cp ) . we constructed a contingency table and used mcnemar test to evaluate the congruence of qlamp and culture results . the differences between patients with or without treatment strategies adjustment based on qlamp results were tested with fisher 's exact test or t test . a p < 0.05 is considered statistically significant difference . none of the samples were collected from the same onset of hap . for each eligible patient , we collected lower respiratory secretion samples on the 1 day for routine culture and quantitative loop - mediated isothermal amplification tests and reported the results to the clinicians . we also collected the clinical data and treatment strategies before and after reporting the quantitative loop - mediated isothermal amplification results . qlamp results of specimen from patients with hospital - acquired pneumonia data are presented as number , unless otherwise indicated . qlamp : quantitative loop - mediated isothermal amplification ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumoniae ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia ; hi : haemophilus influenza . we also evaluated the difference between qlamp and culture results by mcnemar test , in which no significant difference was found ( p > 0.05 ) [ table 3 and supplementary tables 17 ] . the qlamp results of 4 samples for hi , lp , or mp were positive , while the culture results for these specimens were negative probably because of their low detectable rates in culture . we then studied the clinical data of these 4 samples and found that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . no cp positive results were reported in the 76 samples either by qlamp or culture assay . congruence of qlamp and culture results in patients with hospital - acquired pneumonia qlamp : quantitative loop - mediated isothermal amplification ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for pseudomonas aeruginosa * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for klebsiella pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for stenotrophomonas maltophilia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for streptococcus pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification a total of 44 qlamp - positive samples were identified in the study . the final analysis group of 36 patients consisted of 19 with treatment established or adjusted to target antibiotics therapies according to qlamp results ( pathogen target - driven therapy group ) and 17 without treatment strategies adjustment whose treatment strategy was inconsistent with qlamp results ( empirical therapy group ) . there is no significant difference ( p > 0.05 ) in the characteristics of the patients between the two groups , including gender , age , complications , clinical manifestation ( temperature , cough , sputum , and rales ) , blood routine examination , biochemical indicators , blood gas analysis , blood coagulation index , and chest radiograph infiltration . patients taking an advantage for clinical condition with pathogen target - driven therapy there are 16 patients from the pathogen target - driven therapy group with clinical condition improvement 3 days after qlamp results reported , while there is only 1 from the empirical therapy group . the remission rate is 84.2% in the group with pathogen target - driven therapy , and 5.9% in the group with empirical therapy . the differences in the remission rates between these two groups evaluated by fisher 's exact tests are statistically significant ( p = 2 10 ) . daily mean temperature of the group with pathogen target - driven therapy shows a more obvious tendency of improvement than the group with empirical therapy [ figure 2 ] . on the 1 day when samples were collected , the mean temperature of target - driven therapy group and empirical therapy group were 37.95c and 37.68c , respectively , while the mean temperature of those two groups changed to 37.42c and 38.02c 3 days later . the decrease of daily mean temperature was 0.53c in the group with pathogen target - driven therapy while the decrease in the group with empirical therapy was 0.34c . similarly , decrease of total wbc number in group with pathogen target - driven therapy is more significant than the group with empirical therapy ( 2.15 10/l with pathogen target - driven therapy vs. 0.70 10/l with empirical therapy ) [ figure 3 ] . the body temperature of the group with pathogen target - driven therapy decreased while the group with empirical therapy had no significant improvement in body temperature . the wbc count of the group with pathogen target - driven therapy decreased while the group with empirical therapy fluctuated in wbc count . none of the samples were collected from the same onset of hap . for each eligible patient , we collected lower respiratory secretion samples on the 1 day for routine culture and quantitative loop - mediated isothermal amplification tests and reported the results to the clinicians . we also collected the clinical data and treatment strategies before and after reporting the quantitative loop - mediated isothermal amplification results . qlamp results of specimen from patients with hospital - acquired pneumonia data are presented as number , unless otherwise indicated . qlamp : quantitative loop - mediated isothermal amplification ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumoniae ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia ; hi : haemophilus influenza . we also evaluated the difference between qlamp and culture results by mcnemar test , in which no significant difference was found ( p > 0.05 ) [ table 3 and supplementary tables 17 ] . the qlamp results of 4 samples for hi , lp , or mp were positive , while the culture results for these specimens were negative probably because of their low detectable rates in culture . we then studied the clinical data of these 4 samples and found that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . no cp positive results were reported in the 76 samples either by qlamp or culture assay . congruence of qlamp and culture results in patients with hospital - acquired pneumonia qlamp : quantitative loop - mediated isothermal amplification ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for escherichia coli * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for pseudomonas aeruginosa * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for klebsiella pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for stenotrophomonas maltophilia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for streptococcus pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for acinetobacter baumannii * * data are presented as no . the final analysis group of 36 patients consisted of 19 with treatment established or adjusted to target antibiotics therapies according to qlamp results ( pathogen target - driven therapy group ) and 17 without treatment strategies adjustment whose treatment strategy was inconsistent with qlamp results ( empirical therapy group ) . there is no significant difference ( p > 0.05 ) in the characteristics of the patients between the two groups , including gender , age , complications , clinical manifestation ( temperature , cough , sputum , and rales ) , blood routine examination , biochemical indicators , blood gas analysis , blood coagulation index , and chest radiograph infiltration . patients taking an advantage for clinical condition with pathogen target - driven therapy there are 16 patients from the pathogen target - driven therapy group with clinical condition improvement 3 days after qlamp results reported , while there is only 1 from the empirical therapy group . the remission rate is 84.2% in the group with pathogen target - driven therapy , and 5.9% in the group with empirical therapy . the differences in the remission rates between these two groups evaluated by fisher 's exact tests are statistically significant ( p = 2 10 ) . daily mean temperature of the group with pathogen target - driven therapy shows a more obvious tendency of improvement than the group with empirical therapy [ figure 2 ] . on the 1 day when samples were collected , the mean temperature of target - driven therapy group and empirical therapy group were 37.95c and 37.68c , respectively , while the mean temperature of those two groups changed to 37.42c and 38.02c 3 days later . the decrease of daily mean temperature was 0.53c in the group with pathogen target - driven therapy while the decrease in the group with empirical therapy was 0.34c . similarly , decrease of total wbc number in group with pathogen target - driven therapy is more significant than the group with empirical therapy ( 2.15 10/l with pathogen target - driven therapy vs. 0.70 10/l with empirical therapy ) [ figure 3 ] . the body temperature of the group with pathogen target - driven therapy decreased while the group with empirical therapy had no significant improvement in body temperature . the wbc count of the group with pathogen target - driven therapy decreased while the group with empirical therapy fluctuated in wbc count . this is a pilot study to assess the value of qlamp in guiding early target antibiotic therapies of hap , which may have significant effects on the mortality of hap and reduce the cost . although bacterial pneumonia is a kind of curable diseases due to the advent of the antibiotics , the mortality of bacterial hap is still high , which may contribute to the delay of target antibiotics therapies according to the results of sputum culture . with the availability of this rapid ( results are available within 12 h ) , sensitive , and specific therefore , we apply qlamp for the decision - making regarding whether we selected empirical antibiotic therapies or the target antibiotic therapies for hap patients . as qlamp assay was much more rapid than sputum culture , the most common assay in recent clinical practice , we first focus on the congruency of the results of qlamp and sputum culture . fortunately , there was no significance between qlamp and culture results of hap patients with infections of sa , ec , pa , kp , sm , sp , and ab . after that , we prospectively enrolled 36 patients with hap with the same baseline data to evaluate the value of qlamp steering early target therapies . among these patients , the qlamp results were all positive based on the cut - off value ( > 1.0 10 copies / ml ) which was established in our former work ( data not shown ) . we randomly adjusted the regimen of these patients with empirical therapies according to the 2005 american thoracic society / infectious diseases society of america hap guideline or target therapies based on the results of qlamp . interestingly , we found that the clinical condition was significantly improved in the group with pathogen target - driven therapy compared to the group with empirical therapies . thirdly , we did not assess the immunological conditions and nutritional statuses of the patients in these two groups , which may influence the effects of antibiotic therapies . since qlamp can not test the drug sensitivity , the combination of qlamp and sputum culture is the good choice for guiding early target therapies in hap patients .

glioblastoma ( gbm ) is the most frequent malignant brain tumor , accounting for approximately 1215% of all intracranial neoplasms and 6075% of astrocytic tumors . in most european and north american countries , the incidence of gbm is in the range of 34 new cases per 100000 populations per year11 ) . in korea , an analysis of 5208 patients who were diagnosed histologically with primary central nervous system ( cns ) tumors in 2010 showed that gbm accounted for 5.2% of all cns tumors and 40.6% of all glial tumors915 ) . although the overall incidence rate of primary brain tumors is very low among all human cancers , gbm is a highly invasive and aggressively growing tumor that responds poorly to radiation therapy and most forms of chemotherapy . temozolomide ( tmz ) is an alkylating chemotherapeutic agent that causes dna damage by transferring alkyl groups at several sites within dna , including the o6 position of guanine . the mgmt encodes a dna repair protein that removes alkyl groups from o6-guanine , and thus plays a fundamental role in maintaining genomic integrity20 ) . epigenetic silencing by promoter methylation results in decreased mgmt expression and correlates with improved survival in patients with gbm who are treated with alkylating agents4 ) . although mgmt promoter methylation is used as a prognostic / predictive marker in patients with gbm , no consensus exists on the optimal analysis method . methylation - specific polymerase chain reaction ( pcr ) ( msp ) has been widely used , and enables cost - efficient analysis of mgmt promoter methylation . however , it is not a quantitative method and has the risk of false - positive or false - negative results , particularly when the dna quality and/or quantity are low . in the eortc 26981/22981 and ncic ce.3 trials , hegi et al.7 ) only collected data from 67% of the samples analyzed , representing 36% of cases from their archives . the success rate of msp on formalin - fixed paraffin - embedded ( ffpe ) tumor samples was highly variable and the median success rate was 75.0% ( range , 0% to 100% ) . they concluded that diagnostic mgmt testing requires sufficient and optimally preserved tumor tissue such as cryopreserved tumor specimens , thus avoiding fixation - related deterioration of tumor dna quality . an alternative technique that overcomes the problems associated with msp is pyrosequencing ( psq ) , which is based on the sequencing - by - synthesis principle and involves a simple technique for accurate and quantitative analysis of dna sequences . pcr followed by psq is a sensitive , highly reproducible , and cost - effective method for dna methylation analyses13 ) . it provides absolute quantitative information based on each interrogated cpg site , which is not possible with most other methods . additionally , unlike other methods , the assay design allows inclusion of internal controls to address inaccuracies resulting from incomplete bisulfite conversion . a group of investigators tried to compare and optimize three quantitative mgmt promoter methylation techniques and concluded that the psq assay is the most accurate and is also robust when applied to archival samples , including those of gbm14 ) . the objective of this study was to determine whether psq could be used to determine quantitatively the methylation status of the mgmt promoter as a clinical biomarker in routine practice using 2- to 15-year - old archival tissue samples of gbms . we also estimated the methylation status of the mgmt promoter using msp , which is already known to be a prognostic marker for overall survival and a predictive marker for conventional treatment of gbm patients , and verified the results obtained with psq . this study was reviewed and approved by the institutional review board of two individual institutes , and all patients or families provided written informed consent . the study set included ffpe brain tumor tissue of 129 patients from the department of pathology archives of one hospital ( collected from 19972010 ) or from another hospital ( collected from 20002012 ) , of which all were diagnosed as gbm . adjuvant conventional radiotherapy and/or chemotherapy were performed after pathologic diagnosis in most of the cases . all hematoxylin and eosin - stained slides were reviewed using world health organization classification ( 2007 ) by two pathologists without any information regarding clinical and pathologic parameters . tumors were excluded if the tissue was almost entirely necrotized or the tumor proportion of the section was < 80%14 ) . epidemiologic characteristics [ including sex , age at the time of surgery , and karnofsky performance scale ( kps ) ] , the type of postoperative treatment , therapeutic type after recurrence , duration of follow - up , and the time of death were retrospectively reviewed for each patient using medical records . the dose of irradiation and type of radiotherapy administered and the regimen and timing of chemotherapy were also investigated . radiologic evaluation of the extent of surgical resection and response to treatment was performed by two different neuroradiologists without any information on clinical and pathological parameters . extent of surgical resection was estimated from magnetic resonance imaging ( mri ) scans performed within 72 hours after surgery . subtotal resection was defined as removal of more than 90% of the gadolinium - enhancing lesion in enhanced t1-weighted mri , and gross total resection was defined as lack of detectable gadolinium - enhancing lesion . in terms of response , tumor measurement for determination of treatment response according to the macdonald criteria was based on the product of orthogonal diameters on the image with the largest gadolinium - enhancing tumor area17 ) . if multiple lesions were present , the sum of the products of individual measurable lesions was calculated . radiologic studies were performed at regular 3-monthly intervals during follow - up and when there was clinical suspicion of disease progression . genomic dna was extracted from three 10-m - thick slices of ffpe material using the qiaamp dna ffpe extraction kit and the qiacube automated dna extraction machine ( qiagen , hilden , germany ) and quantified by uv absorption ( nanodrop , thermo scientific , wilmington , de , usa ) , typically yielding a total of > 1 g of gdna per specimen . gdna ( 200 ng ) was used in the bisulfite conversion reactions , in which unmethylated cytosine was converted to uracil with the epitect bisulfite kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . briefly , dna was mixed with water , the dna was protected by the buffer and bisulfite mix , and the conversion was run on an abi 2727 thermocycler ( applied biosystems , foster city , ca , usa ) under the recommended cycle conditions . converted dna was purified and eluted in two steps in a total of 40 l buffer eb and further diluted into 20-l aliquots of 1000 cell - equivalents/l ( the cell calculations assumed 6 pg dna per diploid cell ) . msp was performed in a two - step approach using the methods of palmisano et al.16 ) . primer sets with one biotin - labeled primer were used to amplify the above bisulfite - converted dna samples . the pyromark q96 cpg mgmt kit510 ) ( ensembl i d : otthumt00000051009 ) ( qiagen , hilden , germany ) is available as a ready - to - use research kit ; this kit was able to detect the level of methylation at positions 1739 in exon 1 of the mgmt gene , which contains 5 cpgs . a cytosine not followed by a guanine , which was not methylated , served as an internal control and was programmed automatically by the psq96ma 2.1 software ( biotage , uppsala , sweden ) to verify the efficiency of bisulfite conversion . theoretically , the internal control must be zero , because all cytosines that are not followed by a guanine are converted to uracil during bisulfite conversion , and then uracil is converted to thymine after pcr . a value < 5% was considered an acceptable value for the internal control according to the manufacturer 's protocol . if the value of the internal control was > 5% , all procedures were repeated again ( from bisulfite conversion to psq ) . pcr was performed using a converted gdna equivalent of approximately 5000 cells and the pyromark pcr kit ( qiagen , hilden , germany ) . briefly , 12.5 l master mix , 2.5 l coral red , 5 pmol of each primer , 7 l of water , and 2 l sample were mixed for each reaction and run under the following thermal cycling conditions : 95 for 15 minutes , followed by 45 cycles of 30 seconds at 94 , 30 seconds at the optimized primer - specific annealing temperature , and 30 seconds at 72 , followed by a final extension for 10 minutes at 72. amplification of the correct dna product was confirmed by electrophoresis on a 2% low melting point agarose gel ( sigma - aldrich , steinheim , germany ) in tris - borate - edta buffer . a standard psq sample preparation protocol was applied5 ) . streptavidin beads ( 3 l ; ge healthcare , buckinghamshire , uk ) , 37 l pyromark binding buffer ( qiagen ) , 20 l pcr product , and 20 l water were mixed and incubated for 10 minutes on a shaking table at 1300 rpm . amplicons were separated , denatured , washed , and added to 45 l annealing buffer containing 0.33 m of psq primer using the biotage q96 vacuum workstation . primer annealing was performed by incubating the samples at 80 for 2 minutes and then cooling to room temperature prior to psq . pyrogold reagents were used for the psq reaction , and the signal was analyzed using the psq 96ma system ( biotage , uppsala , sweden ) . target cpgs were evaluated by psq96ma 2.1 instrument software ( biotage , uppsala , sweden ) , which converts the pyrograms to numerical values for peak heights and calculates the proportion of methylation at each base as a c / t ratio . methylation values 5% were considered potential background signals of questionable significance , as shown in other studies31016 ) . unmethylated ( 300 ng ; qiagen , hilden , germany ) and hypermethylated dna ( millipore , billerica , ma , usa ) were used as standard controls , and were bisulfite converted as described above . the main analyses converted individual c / t ratio data into mean values for the five cpgs analyzed in a gene segment using psq . the percentage of the mean value of the five cpgs was used to determine whether methylation affects the survival of gbm patients . receiver operating characteristic ( roc ) curve analysis was used to determine the cut - off value of mean percentage of methylation at the five cpgs for predicting the longer survival3 ) . the area under the roc curve ( auc ) was used to determine the optimal threshold of the mean percentage of the methylation at the five cpgs . sensitivity was calculated as the true positive rate ( number of true positives divided by the sum of the number of true positives and number of false negatives ) ; specificity as the true negative rate ( number of true negatives divided by the sum of the number of true negatives and number of false positives ) ; and accuracy as the sum of the number of true positives and true negatives , divided by the total number of positives and negatives . true positives mean that the methylation percentage above the cut - off value has an influence on the long survival , and true negatives mean that the methylation percentage below the cut - off value has an influence on the short survival . differences between subgroups were analyzed using the student 's t - test for normally distributed continuous values and the mann - whitney test for non - normally distributed continuous values . variables that were found to be significantly associated with survival in gbm patients by univariate analyses were subjected to multivariate analyses . in addition , several variables that were of interest to the authors and were reported to be associated with survival of gbm patients in the literature also were included in multivariate analysis . we evaluated the impact of variables on survival by comparing the overall survival curves using the log - rank test . in multivariate analysis , cox proportional hazard regression model was used to assess the independent effects of specific factors on overall survival and to define hazard ratios for significant covariates . spss software version 12.0 ( spss inc . , chicago , il , usa ) was used for statistical analysis . this study was reviewed and approved by the institutional review board of two individual institutes , and all patients or families provided written informed consent . the study set included ffpe brain tumor tissue of 129 patients from the department of pathology archives of one hospital ( collected from 19972010 ) or from another hospital ( collected from 20002012 ) , of which all were diagnosed as gbm . adjuvant conventional radiotherapy and/or chemotherapy were performed after pathologic diagnosis in most of the cases . all hematoxylin and eosin - stained slides were reviewed using world health organization classification ( 2007 ) by two pathologists without any information regarding clinical and pathologic parameters . tumors were excluded if the tissue was almost entirely necrotized or the tumor proportion of the section was < 80%14 ) . epidemiologic characteristics [ including sex , age at the time of surgery , and karnofsky performance scale ( kps ) ] , the type of postoperative treatment , therapeutic type after recurrence , duration of follow - up , and the time of death were retrospectively reviewed for each patient using medical records . the dose of irradiation and type of radiotherapy administered and the regimen and timing of chemotherapy were also investigated . radiologic evaluation of the extent of surgical resection and response to treatment was performed by two different neuroradiologists without any information on clinical and pathological parameters . extent of surgical resection was estimated from magnetic resonance imaging ( mri ) scans performed within 72 hours after surgery . subtotal resection was defined as removal of more than 90% of the gadolinium - enhancing lesion in enhanced t1-weighted mri , and gross total resection was defined as lack of detectable gadolinium - enhancing lesion . in terms of response , tumor measurement for determination of treatment response according to the macdonald criteria was based on the product of orthogonal diameters on the image with the largest gadolinium - enhancing tumor area17 ) . if multiple lesions were present , the sum of the products of individual measurable lesions was calculated . radiologic studies were performed at regular 3-monthly intervals during follow - up and when there was clinical suspicion of disease progression . genomic dna was extracted from three 10-m - thick slices of ffpe material using the qiaamp dna ffpe extraction kit and the qiacube automated dna extraction machine ( qiagen , hilden , germany ) and quantified by uv absorption ( nanodrop , thermo scientific , wilmington , de , usa ) , typically yielding a total of > 1 g of gdna per specimen . gdna ( 200 ng ) was used in the bisulfite conversion reactions , in which unmethylated cytosine was converted to uracil with the epitect bisulfite kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . briefly , dna was mixed with water , the dna was protected by the buffer and bisulfite mix , and the conversion was run on an abi 2727 thermocycler ( applied biosystems , foster city , ca , usa ) under the recommended cycle conditions . converted dna was purified and eluted in two steps in a total of 40 l buffer eb and further diluted into 20-l aliquots of 1000 cell - equivalents/l ( the cell calculations assumed 6 pg dna per diploid cell ) . msp was performed in a two - step approach using the methods of palmisano et al.16 ) . primer sets with one biotin - labeled primer were used to amplify the above bisulfite - converted dna samples . the pyromark q96 cpg mgmt kit510 ) ( ensembl i d : otthumt00000051009 ) ( qiagen , hilden , germany ) is available as a ready - to - use research kit ; this kit was able to detect the level of methylation at positions 1739 in exon 1 of the mgmt gene , which contains 5 cpgs . a cytosine not followed by a guanine , which was not methylated , served as an internal control and was programmed automatically by the psq96ma 2.1 software ( biotage , uppsala , sweden ) to verify the efficiency of bisulfite conversion . theoretically , the internal control must be zero , because all cytosines that are not followed by a guanine are converted to uracil during bisulfite conversion , and then uracil is converted to thymine after pcr . a value < 5% was considered an acceptable value for the internal control according to the manufacturer 's protocol . if the value of the internal control was > 5% , all procedures were repeated again ( from bisulfite conversion to psq ) . pcr was performed using a converted gdna equivalent of approximately 5000 cells and the pyromark pcr kit ( qiagen , hilden , germany ) . briefly , 12.5 l master mix , 2.5 l coral red , 5 pmol of each primer , 7 l of water , and 2 l sample were mixed for each reaction and run under the following thermal cycling conditions : 95 for 15 minutes , followed by 45 cycles of 30 seconds at 94 , 30 seconds at the optimized primer - specific annealing temperature , and 30 seconds at 72 , followed by a final extension for 10 minutes at 72. amplification of the correct dna product was confirmed by electrophoresis on a 2% low melting point agarose gel ( sigma - aldrich , steinheim , germany ) in tris - borate - edta buffer . streptavidin beads ( 3 l ; ge healthcare , buckinghamshire , uk ) , 37 l pyromark binding buffer ( qiagen ) , 20 l pcr product , and 20 l water were mixed and incubated for 10 minutes on a shaking table at 1300 rpm . amplicons were separated , denatured , washed , and added to 45 l annealing buffer containing 0.33 m of psq primer using the biotage q96 vacuum workstation . primer annealing was performed by incubating the samples at 80 for 2 minutes and then cooling to room temperature prior to psq . pyrogold reagents were used for the psq reaction , and the signal was analyzed using the psq 96ma system ( biotage , uppsala , sweden ) . target cpgs were evaluated by psq96ma 2.1 instrument software ( biotage , uppsala , sweden ) , which converts the pyrograms to numerical values for peak heights and calculates the proportion of methylation at each base as a c / t ratio . methylation values 5% were considered potential background signals of questionable significance , as shown in other studies31016 ) . unmethylated ( 300 ng ; qiagen , hilden , germany ) and hypermethylated dna ( millipore , billerica , ma , usa ) were used as standard controls , and were bisulfite converted as described above . the main analyses converted individual c / t ratio data into mean values for the five cpgs analyzed in a gene segment using psq . the percentage of the mean value of the five cpgs was used to determine whether methylation affects the survival of gbm patients . receiver operating characteristic ( roc ) curve analysis was used to determine the cut - off value of mean percentage of methylation at the five cpgs for predicting the longer survival3 ) . the area under the roc curve ( auc ) was used to determine the optimal threshold of the mean percentage of the methylation at the five cpgs . sensitivity was calculated as the true positive rate ( number of true positives divided by the sum of the number of true positives and number of false negatives ) ; specificity as the true negative rate ( number of true negatives divided by the sum of the number of true negatives and number of false positives ) ; and accuracy as the sum of the number of true positives and true negatives , divided by the total number of positives and negatives . true positives mean that the methylation percentage above the cut - off value has an influence on the long survival , and true negatives mean that the methylation percentage below the cut - off value has an influence on the short survival . differences between subgroups were analyzed using the student 's t - test for normally distributed continuous values and the mann - whitney test for non - normally distributed continuous values . variables that were found to be significantly associated with survival in gbm patients by univariate analyses were subjected to multivariate analyses . in addition , several variables that were of interest to the authors and were reported to be associated with survival of gbm patients in the literature also were included in multivariate analysis . we evaluated the impact of variables on survival by comparing the overall survival curves using the log - rank test . in multivariate analysis , cox proportional hazard regression model was used to assess the independent effects of specific factors on overall survival and to define hazard ratios for significant covariates . from a total of 129 gbm patients treated within the defined study period , 104 patients ( 58 male and 46 male ) were eligible for analysis ( table 1 ) . all patients were followed for at least 3 months , and the mean follow - up duration was 17.3 months ( range , 3.241.5 months ) . during follow up , 79 patients ( 76.0% ) died from gbm . seventy - four ( 71.2% ) patients could perform activities of daily life independently ( kps scores 70 ) . the most frequent major complaints at presentation were headache ( 49 cases , 47.1% ) , seizure ( 16 cases , 15.4% ) , focal neurological deficits such as motor weakness and dysphasia ( 15 cases , 14.4% ) , and altered mentation ( 14 cases , 13.5% ) . biopsy only was performed in 5 patients ( 4.8% ) , subtotal resection in 60 ( 57.7% ) , and gross total resection in 39 ( 37.5% ) . most of the patients ( 97 patients , 93.3% ) underwent active adjuvant treatment ; 13 patients ( 12.5% ) had chemotherapy alone , 33 patients ( 31.7% ) had radiotherapy alone , and 51 patients ( 49.0% ) had combination therapy with chemotherapy and radiotherapy . forty - four patients ( 42.3% ) were treated with concurrent temozolomide chemoradiotherapy as adjuvant treatment since 2005 . during follow - up , 88 patients ( 84.6% ) experienced disease progression . mean time to disease progression was 11.4 months ( range , 4.613.8 months ) . in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , mean time to progression was 12.2 months ( range , 5.715.2 months ) . after progression , only 22 patients ( 25.0% ) were able to undergo repeated surgical resection . for salvage therapy , 22 patients ( 25.0% ) underwent repeated surgery , 11 patients ( 12.5% ) were treated additionally with chemotherapy alone , 32 ( 33.4% ) with radiotherapy alone , and 16 ( 18.1% ) with both therapies . psq and msp data were obtained for all 104 samples . for determining the methylation status of mgmt promoter methylation by psq , roc curve analysis of using the mean percentage of methylation at the five cpgs to predict the longer survival showed that the auc was 0.68 . the optimal threshold of mean percentage of methylation for distinguishing between the patients with long survival and those with short survival was 9% , and this yielded a sensitivity of 53.8% [ 95% confidence interval ( ci ) 44.662.9% ] , a specificity of 66.7% ( 95% ci 52.980.5% ) , and an accuracy of 68.0% . using psq analysis , mgmt promoter methylation was detected in 43 ( 41.3% ) of the gbm samples . cases of methylated and unmethylated promoters of gbms were represented by a pyrogram ( fig . the average percentage methylation for all samples of the methylated cases over the study period of 13 years was also investigated ( table 2 ) . the average methylation percentage of all gbm samples was 14.016.8% ; the percentage for methylated cases was 39.014.7% and that for unmethylated cases was 3.21.8% ( p<0.001 ) . the percentage methylation values for each year were not significantly different ( p=0.687 ) and did not show an increasing or decreasing linear pattern according to the age of the ffpe block . in comparison , msp analysis showed that 39 samples ( 37.5% ) had a methylated mgmt promoter and 65 samples ( 62.5% ) had an unmethylated mgmt promoter . six samples ( 5.8% ) had an opposite methylation status of the mgmt promoter according to the two assays ( table 3 ) ; all 6 samples were obtained before 2003 and therefore were at least 10 years old and included one individual case for every year from 1997 to 2002 . mean overall survival ( os ) of the total 104 gbm patients was 16.9 months ( 95% ci 12.421.5 months ) and the 2-year survival rate was 25.2% . for analysis by msp , the mean os of gbm patients with an unmethylated mgmt promoter was 16.2 months ( 95% ci 11.321.1 months ) , and that of patients with a methylated mgmt promoter was 19.6 months ( 95% ci 14.824.2 months , p=0.004 ) . in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , mean os was 18.5 months ( 95% ci 12.824.5 months ) and 2-year survival rate was 26.9% . for analysis by msp , the mean os of these patients with an unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) , and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months , p=0.002 ) ( table 4 ) . according to analysis by psq , the mean os of total 104 gbm patients with unmethylated mgmt promoter was 14.1 months ( 95% ci 11.616.9 months ) and that of patients with a methylated mgmt promoter was 20.9 months ( 95% ci 17.324.4 months ) . there was a significant difference in os between the two groups ( p<0.001 ) . more detailed categorization according to the percentage of methylation showed a significant difference in os even among patients with a methylated mgmt promoter ( p=0.012 ) ; the mean os of gbm patients with a less methylated mgmt promoter ( from 939% ) was 17.8 months ( 95% ci 13.921.7 months ) , whereas that of patients with a more methylated mgmt promoter ( above 39% ) was 24.5 months ( 95% ci 19.229.8 months ) . actually , in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , the methylation status analyzed by psq was completely identical to that by msp in 44 patients who were treated with temozolomide concurrent chemoradiotherapy . therefore , there was no difference of overall survival between two groups according to the analyzing technique ; the patients with unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months ) ( table 4 ) . univariate analysis for os of all gbm patients was performed ( table 5 ) . age < 50 [ hazard ratio ( hr ) 2.912 ; p=0.042 ] , kps 70 ( hr 13.329 ; p=0.001 ) , extent of surgical resection ( p<0.05 ) , type of adjuvant therapy ( p<0.05 ) , methylation of mgmt promoter analyzed by msp ( hr 9.856 ; p=0.013 ) , and methylation of mgmt promoter analyzed by psq ( hr 18.007 ; p<0.001 ) were associated with os . however , sex ( p=0.572 ) , concurrent temozolomide chemoradiotherapy ( p=0.063 ) and method of salvage treatment ( p>0.05 ) were not associated with os . multi - factor adjustment by the cox - regression model was performed for the factors that were associated with os in univariate analysis and additional factors of interest to the authors ( such as method of salvage treatment ) ( table 6 ) . in multivariate analysis , we found that age ( < 50 years vs. 50 years ; hr 3.386 , p=0.048 ) , kps ( 70 vs. < 70 ; hr 9.417 , p=0.002 ) , extent of surgical resection ( gross total resection vs. subtotal resection , hr 8.045 ) , method of adjuvant treatment ( combination therapy vs. other treatment , hr 14.128 , p<0.001 ) , methylation status of mgmt promoter by msp ( methylated vs. unmethylated , hr 4.927 , p=0.034 ) , and methylation status of mgmt promoter by psq ( methylated vs. unmethylated , hr 9.208 , p=0.001 ) were independently associated with os . methods of salvage treatment and concurrent temozolomide chemoradiotherapy that were potentially associated with os did not have any association in multivariate analysis ( p>0.05 ) . from a total of 129 gbm patients treated within the defined study period , 104 patients ( 58 male and 46 male ) were eligible for analysis ( table 1 ) . all patients were followed for at least 3 months , and the mean follow - up duration was 17.3 months ( range , 3.241.5 months ) . during follow up , 79 patients ( 76.0% ) died from gbm . seventy - four ( 71.2% ) patients could perform activities of daily life independently ( kps scores 70 ) . the most frequent major complaints at presentation were headache ( 49 cases , 47.1% ) , seizure ( 16 cases , 15.4% ) , focal neurological deficits such as motor weakness and dysphasia ( 15 cases , 14.4% ) , and altered mentation ( 14 cases , 13.5% ) . biopsy only was performed in 5 patients ( 4.8% ) , subtotal resection in 60 ( 57.7% ) , and gross total resection in 39 ( 37.5% ) . most of the patients ( 97 patients , 93.3% ) underwent active adjuvant treatment ; 13 patients ( 12.5% ) had chemotherapy alone , 33 patients ( 31.7% ) had radiotherapy alone , and 51 patients ( 49.0% ) had combination therapy with chemotherapy and radiotherapy . forty - four patients ( 42.3% ) were treated with concurrent temozolomide chemoradiotherapy as adjuvant treatment since 2005 . during follow - up , 88 patients ( 84.6% ) experienced disease progression . mean time to disease progression was 11.4 months ( range , 4.613.8 months ) . in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , mean time to progression was 12.2 months ( range , 5.715.2 months ) . after progression , only 22 patients ( 25.0% ) were able to undergo repeated surgical resection . for salvage therapy , 22 patients ( 25.0% ) underwent repeated surgery , 11 patients ( 12.5% ) were treated additionally with chemotherapy alone , 32 ( 33.4% ) with radiotherapy alone , and 16 ( 18.1% ) with both therapies . psq and msp data were obtained for all 104 samples . for determining the methylation status of mgmt promoter methylation by psq , roc curve analysis of using the mean percentage of methylation at the five cpgs to predict the longer survival showed that the auc was 0.68 . the optimal threshold of mean percentage of methylation for distinguishing between the patients with long survival and those with short survival was 9% , and this yielded a sensitivity of 53.8% [ 95% confidence interval ( ci ) 44.662.9% ] , a specificity of 66.7% ( 95% ci 52.980.5% ) , and an accuracy of 68.0% . using psq analysis , cases of methylated and unmethylated promoters of gbms were represented by a pyrogram ( fig . the average percentage methylation for all samples of the methylated cases over the study period of 13 years was also investigated ( table 2 ) . the average methylation percentage of all gbm samples was 14.016.8% ; the percentage for methylated cases was 39.014.7% and that for unmethylated cases was 3.21.8% ( p<0.001 ) . the percentage methylation values for each year were not significantly different ( p=0.687 ) and did not show an increasing or decreasing linear pattern according to the age of the ffpe block . in comparison , msp analysis showed that 39 samples ( 37.5% ) had a methylated mgmt promoter and 65 samples ( 62.5% ) had an unmethylated mgmt promoter . six samples ( 5.8% ) had an opposite methylation status of the mgmt promoter according to the two assays ( table 3 ) ; all 6 samples were obtained before 2003 and therefore were at least 10 years old and included one individual case for every year from 1997 to 2002 . mean overall survival ( os ) of the total 104 gbm patients was 16.9 months ( 95% ci 12.421.5 months ) and the 2-year survival rate was 25.2% . for analysis by msp , the mean os of gbm patients with an unmethylated mgmt promoter was 16.2 months ( 95% ci 11.321.1 months ) , and that of patients with a methylated mgmt promoter was 19.6 months ( 95% ci 14.824.2 months , p=0.004 ) . in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , mean os was 18.5 months ( 95% ci 12.824.5 months ) and 2-year survival rate was 26.9% . for analysis by msp , the mean os of these patients with an unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) , and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months , p=0.002 ) ( table 4 ) . according to analysis by psq , the mean os of total 104 gbm patients with unmethylated mgmt promoter was 14.1 months ( 95% ci 11.616.9 months ) and that of patients with a methylated mgmt promoter was 20.9 months ( 95% ci 17.324.4 months ) . more detailed categorization according to the percentage of methylation showed a significant difference in os even among patients with a methylated mgmt promoter ( p=0.012 ) ; the mean os of gbm patients with a less methylated mgmt promoter ( from 939% ) was 17.8 months ( 95% ci 13.921.7 months ) , whereas that of patients with a more methylated mgmt promoter ( above 39% ) was 24.5 months ( 95% ci 19.229.8 months ) . actually , in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , the methylation status analyzed by psq was completely identical to that by msp in 44 patients who were treated with temozolomide concurrent chemoradiotherapy . therefore , there was no difference of overall survival between two groups according to the analyzing technique ; the patients with unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months ) ( table 4 ) . univariate analysis for os of all gbm patients was performed ( table 5 ) . age < 50 [ hazard ratio ( hr ) 2.912 ; p=0.042 ] , kps 70 ( hr 13.329 ; p=0.001 ) , extent of surgical resection ( p<0.05 ) , type of adjuvant therapy ( p<0.05 ) , methylation of mgmt promoter analyzed by msp ( hr 9.856 ; p=0.013 ) , and methylation of mgmt promoter analyzed by psq ( hr 18.007 ; p<0.001 ) were associated with os . however , sex ( p=0.572 ) , concurrent temozolomide chemoradiotherapy ( p=0.063 ) and method of salvage treatment ( p>0.05 ) were not associated with os . multi - factor adjustment by the cox - regression model was performed for the factors that were associated with os in univariate analysis and additional factors of interest to the authors ( such as method of salvage treatment ) ( table 6 ) . in multivariate analysis , we found that age ( < 50 years vs. 50 years ; hr 3.386 , p=0.048 ) , kps ( 70 vs. < 70 ; hr 9.417 , p=0.002 ) , extent of surgical resection ( gross total resection vs. subtotal resection , hr 8.045 ) , method of adjuvant treatment ( combination therapy vs. other treatment , hr 14.128 , p<0.001 ) , methylation status of mgmt promoter by msp ( methylated vs. unmethylated , hr 4.927 , p=0.034 ) , and methylation status of mgmt promoter by psq ( methylated vs. unmethylated , hr 9.208 , p=0.001 ) were independently associated with os . methods of salvage treatment and concurrent temozolomide chemoradiotherapy that were potentially associated with os did not have any association in multivariate analysis ( p>0.05 ) . in this study , we performed psq analysis to determine mgmt promoter methylation status and estimate the extent of methylation using 1- to 13-year - old archival tissue samples of gbms . we also investigated the prognostic significance of mgmt promoter methylation and other factors associated with os in patients who were treated in the routine clinic . recently , stupp et al.19 ) reported the results of 5-year analysis of the eortc - ncic trial for survival in gbm patients . their randomized phase iii study showed that the following factors had an association with outcome : concurrent chemoradiotherapy with tmz , complete resection , age < 50 , good functional status ( recursive partitioning analysis class iii > iv > v ) , and methylated mgmt promoter . we obtained similar results in our study ; age < 50 , good functional status ( kps 70 ) , gross total resection , and methylated mgmt promoter analyzed by both msp and psq were all associated with os . therefore , this study provided good verification of the published literature . in terms of the frequency of mgmt promoter methylation in gbm , hegi et al.7 ) reported that 45% of gbms showed mgmt promoter methylation in their randomized trial , and that the median success rate of msp analysis in ffpe samples was 75% . in contrast , the success rate of psq analysis in our study was 100% for 104 samples from 1- to 13-year - old archival specimens , and 41.3% of the gbms were methylated . these results , and data from several other researchers , indicate that 4050% of gbms are potentially methylated , which might be of clinical benefit regarding treatment with alkylating agents . several methods for determining promoter methylation have been described , including the use of restriction enzymes18 ) or genomic bisulfite sequencing6 ) . among these methods , some are quantitative , some are semi - quantitative , and some are qualitative . the msp assay is highly sensitive and has been used widely in this context18 ) . however , as there are no inbuilt measures of the adequacy of bisulfite treatment , the possibility of false positives due to inadequate conversion of non - methylated cytosine to uracil exists , particularly for dna of poor quality or low quantity . another potential source of false positives is mis - priming , which may be a greater problem when high numbers of pcr cycles or nested primers are used . an accurate quantitative approach is very important for dna methylation analysis , and msp has many weaknesses in this regard . one of the major concerns with msp is the use of relative quantification by means of an external control using a different pcr for the control gene . the accuracy and reproducibility of this approach for the large diversity of genes has still not been adequately addressed . development of dna methylation risk prediction panels based on diverse genes will be required to develop and optimize an accurate and reproducible method . in this study , we present results of psq analysis of 1- to 13-year - old archive specimens . as described above , psq is a simple technique based on the sequencing - by - synthesis principle and allows accurate and quantitative analysis of dna sequences . the percentage of methylation was evenly distributed over the years of this study and no increasing or decreasing pattern of methylation with specimen age was observed , regardless of the age of the samples . this might represent a huge improvement in the success rate when compared with data reported using msp by hegi et al.7 ) . using the same method as in the current study , dunn et al.2 ) analyzed 264 of 287 tumor samples from 121 cases by psq . some failures occurred , most likely as a result of the small sample volume ( < 1 mm ) with low dna yield or poor dna integrity in the ffpe samples . nonetheless , despite these failures the psq data were reproducible and showed a good correlation between duplicate pcr reactions from the same bisulfite modification and between two independent bisulfite modifications of the same dna extract2 ) . more recently , a quantitative real - time msp assay in which the copy number of methylated mgmt alleles was calculated showed improvement over the gel - based msp assay in terms of reproducibility and use with archival samples , but did not provide methylation data at individual cpg sites21 ) . therefore , pcr followed by the psq method is a stable technique that produces consistent data . these results support the need for a large nationwide retrospective trial to evaluate the mgmt promoter methylation status of old archived material from multiple institutions . in terms of the relationship between the extent of methylation estimated by psq and clinical outcome in gbm , this study showed that gbm patients with a more methylated mgmt promoter had longer overall survival than those with a less methylated mgmt promoter . similarly , dunn et al.2 ) reported an interesting clustering analysis based on the extent of methylation . progression - free survival was significantly different between low ( mean percentage of methylation 929% ) and high ( 29% ) methylation groups of gbm , and between high methylated and unmethylated cases . log - rank tests showed significant differences in os between unmethylated and methylated groups and between cases with a low versus high percentage of methylation . this result suggested that qualitative and quantitative data should be simultaneously obtained from the mgmt promoter methylation analysis to predict progression - free survival , overall survival , or chemoresponsiveness in clinical practice . although we did not obtain exactly the same results as those of dunn , we found a similar tendency for a higher percentage of methylation to be associated with longer overall survival . the discrepancy in results probably originates from different sample size and different follow - up duration . moreover , our archives included a relatively small number of samples with a low percentage of methylation . another issue for mgmt promoter methylation analysis is the relevant cpg sites for methylation status . the pattern of mgmt promoter methylation is very heterogeneous from tumor to tumor , and the specific cpg sites that need to be methylated to silence transcription and provide a favorable outcome have not been established . several studies have investigated the methylation status at individual cpgs within mgmt cgis and compared them with gene expression . watts et al.22 ) studied the methylation status of 108 cpgs across mgmt cgis using bisulfite sequencing and identified three distinct regions within the cgi where high methylation levels were found , but only in the mgmt non - expressing cell line . recently , karayan - tapon et al.10 ) assessed mgmt promoter methylation status by five different methods including psq , and the psq primer sets used included 5 cpgs in the dmr2 region that were also used in this study . they concluded that psq using ffpe samples predicted overall survival well in patients who were initially treated with radiotherapy plus tmz . the limitations of this study can be summarized as follows : first , the cut - off value for methylation used in this study should be verified in more detail and in a larger number of samples . second , we studied limited sites of cgi methylation using the pyro mark q96 cpg mgmt kit ( which evaluates only 5 cgis ) . therefore , further evaluation of other sites of cgi methylation is essential to increase the accuracy of the status and extent of mgmt promoter methylation , and to commercialize this kit . third , the relatively small number of gbm samples made it impossible for us to find any critical methylation status value for predicting the outcome of these tumors . finally , progression - free survival could not be estimated because there was no specific protocol for follow - up and management . this study showed that both the status and extent of methylation of the mgmt promoter analyzed by psq were found to be associated with os in patients with gbm . to date , mgmt promoter methylation analysis is one of the most recommended molecular assays in clinical neurooncology . a range of new methodologies are available for mgmt testing that potentially allow higher levels of sensitivity , specificity , robustness , and reproducibility . in this regard , among many methods for determining the characteristics of gene promoter methylation , psq is a quantitative approach that overcomes the problems associated with msp , and is a simple technique for accurate analysis of dna sequences . as a result , psq might be a promising technique for mgmt evaluation in daily practice . furthermore , the development of new biomarkers for malignant brain tumors using molecular methods such as psq might be helpful for improvement of therapeutic outcome in patients with malignant brain tumors .

objectivethis study investigated whether pyrosequencing can be used to determine the methylation status of the mgmt promoter as a clinical biomarker using relatively old archival tissue samples of glioblastoma . we also examined other prognostic factors for survival of glioblastoma patients.methodsthe available study set included formalin - fixed paraffin - embedded ( ffpe ) tissue from 104 patients at two institutes from 1997 to 2012 , all of which were diagnosed histopathologically as glioblastoma . clinicopathologic data were collected by review of medical records . for pyrosequencing analysis , the pyromark q96 cpg mgmt kit ( qiagen , hilden , germany ) was used to detect the level of methylation at exon 1 positions 1739 of the mgmt gene , which contains 5 cpgs.resultsmethylation of the mgmt promoter was detected in 43 ( 41.3% ) of 104 samples . the average percentage methylation was 14.016.8% overall and 39.014.7% for methylated cases . there was no significant pattern of linear increase or decrease according to the age of the ffpe block ( p=0.687 ) . in multivariate analysis , age , performance status , extent of surgery , method of adjuvant therapy , and methylation status estimated by pyrosequencing were independently associated with overall survival . additionally , patients with a high level of methylation survived longer than those with low methylation ( p=0.016).conclusionin this study , the status and extent of methylation of the mgmt promoter analyzed by pyrosequencing were associated with overall survival in glioblastoma patients . pyrosequencing is a quantitative method that overcomes the problems of msp and a simple technique for accurate analysis of dna sequences .

INTRODUCTION MATERIALS AND METHODS Sample selection Clinical and radiological data DNA extraction and bisulfite conversion PCR and PSQ Data analysis and statistics RESULTS Clinical characteristics Methylation status of Survival of GBM patients Factors associated with overall survival DISCUSSION CONCLUSION

the success rate of msp on formalin - fixed paraffin - embedded ( ffpe ) tumor samples was highly variable and the median success rate was 75.0% ( range , 0% to 100% ) . an alternative technique that overcomes the problems associated with msp is pyrosequencing ( psq ) , which is based on the sequencing - by - synthesis principle and involves a simple technique for accurate and quantitative analysis of dna sequences . the objective of this study was to determine whether psq could be used to determine quantitatively the methylation status of the mgmt promoter as a clinical biomarker in routine practice using 2- to 15-year - old archival tissue samples of gbms . we also estimated the methylation status of the mgmt promoter using msp , which is already known to be a prognostic marker for overall survival and a predictive marker for conventional treatment of gbm patients , and verified the results obtained with psq . the study set included ffpe brain tumor tissue of 129 patients from the department of pathology archives of one hospital ( collected from 19972010 ) or from another hospital ( collected from 20002012 ) , of which all were diagnosed as gbm . epidemiologic characteristics [ including sex , age at the time of surgery , and karnofsky performance scale ( kps ) ] , the type of postoperative treatment , therapeutic type after recurrence , duration of follow - up , and the time of death were retrospectively reviewed for each patient using medical records . genomic dna was extracted from three 10-m - thick slices of ffpe material using the qiaamp dna ffpe extraction kit and the qiacube automated dna extraction machine ( qiagen , hilden , germany ) and quantified by uv absorption ( nanodrop , thermo scientific , wilmington , de , usa ) , typically yielding a total of > 1 g of gdna per specimen . gdna ( 200 ng ) was used in the bisulfite conversion reactions , in which unmethylated cytosine was converted to uracil with the epitect bisulfite kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . the pyromark q96 cpg mgmt kit510 ) ( ensembl i d : otthumt00000051009 ) ( qiagen , hilden , germany ) is available as a ready - to - use research kit ; this kit was able to detect the level of methylation at positions 1739 in exon 1 of the mgmt gene , which contains 5 cpgs . pcr was performed using a converted gdna equivalent of approximately 5000 cells and the pyromark pcr kit ( qiagen , hilden , germany ) . briefly , 12.5 l master mix , 2.5 l coral red , 5 pmol of each primer , 7 l of water , and 2 l sample were mixed for each reaction and run under the following thermal cycling conditions : 95 for 15 minutes , followed by 45 cycles of 30 seconds at 94 , 30 seconds at the optimized primer - specific annealing temperature , and 30 seconds at 72 , followed by a final extension for 10 minutes at 72. amplification of the correct dna product was confirmed by electrophoresis on a 2% low melting point agarose gel ( sigma - aldrich , steinheim , germany ) in tris - borate - edta buffer . unmethylated ( 300 ng ; qiagen , hilden , germany ) and hypermethylated dna ( millipore , billerica , ma , usa ) were used as standard controls , and were bisulfite converted as described above . the percentage of the mean value of the five cpgs was used to determine whether methylation affects the survival of gbm patients . receiver operating characteristic ( roc ) curve analysis was used to determine the cut - off value of mean percentage of methylation at the five cpgs for predicting the longer survival3 ) . the area under the roc curve ( auc ) was used to determine the optimal threshold of the mean percentage of the methylation at the five cpgs . in addition , several variables that were of interest to the authors and were reported to be associated with survival of gbm patients in the literature also were included in multivariate analysis . in multivariate analysis , cox proportional hazard regression model was used to assess the independent effects of specific factors on overall survival and to define hazard ratios for significant covariates . the study set included ffpe brain tumor tissue of 129 patients from the department of pathology archives of one hospital ( collected from 19972010 ) or from another hospital ( collected from 20002012 ) , of which all were diagnosed as gbm . epidemiologic characteristics [ including sex , age at the time of surgery , and karnofsky performance scale ( kps ) ] , the type of postoperative treatment , therapeutic type after recurrence , duration of follow - up , and the time of death were retrospectively reviewed for each patient using medical records . genomic dna was extracted from three 10-m - thick slices of ffpe material using the qiaamp dna ffpe extraction kit and the qiacube automated dna extraction machine ( qiagen , hilden , germany ) and quantified by uv absorption ( nanodrop , thermo scientific , wilmington , de , usa ) , typically yielding a total of > 1 g of gdna per specimen . gdna ( 200 ng ) was used in the bisulfite conversion reactions , in which unmethylated cytosine was converted to uracil with the epitect bisulfite kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . the pyromark q96 cpg mgmt kit510 ) ( ensembl i d : otthumt00000051009 ) ( qiagen , hilden , germany ) is available as a ready - to - use research kit ; this kit was able to detect the level of methylation at positions 1739 in exon 1 of the mgmt gene , which contains 5 cpgs . pcr was performed using a converted gdna equivalent of approximately 5000 cells and the pyromark pcr kit ( qiagen , hilden , germany ) . briefly , 12.5 l master mix , 2.5 l coral red , 5 pmol of each primer , 7 l of water , and 2 l sample were mixed for each reaction and run under the following thermal cycling conditions : 95 for 15 minutes , followed by 45 cycles of 30 seconds at 94 , 30 seconds at the optimized primer - specific annealing temperature , and 30 seconds at 72 , followed by a final extension for 10 minutes at 72. amplification of the correct dna product was confirmed by electrophoresis on a 2% low melting point agarose gel ( sigma - aldrich , steinheim , germany ) in tris - borate - edta buffer . unmethylated ( 300 ng ; qiagen , hilden , germany ) and hypermethylated dna ( millipore , billerica , ma , usa ) were used as standard controls , and were bisulfite converted as described above . the percentage of the mean value of the five cpgs was used to determine whether methylation affects the survival of gbm patients . receiver operating characteristic ( roc ) curve analysis was used to determine the cut - off value of mean percentage of methylation at the five cpgs for predicting the longer survival3 ) . the area under the roc curve ( auc ) was used to determine the optimal threshold of the mean percentage of the methylation at the five cpgs . in addition , several variables that were of interest to the authors and were reported to be associated with survival of gbm patients in the literature also were included in multivariate analysis . in multivariate analysis , cox proportional hazard regression model was used to assess the independent effects of specific factors on overall survival and to define hazard ratios for significant covariates . for determining the methylation status of mgmt promoter methylation by psq , roc curve analysis of using the mean percentage of methylation at the five cpgs to predict the longer survival showed that the auc was 0.68 . using psq analysis , mgmt promoter methylation was detected in 43 ( 41.3% ) of the gbm samples . the average percentage methylation for all samples of the methylated cases over the study period of 13 years was also investigated ( table 2 ) . the average methylation percentage of all gbm samples was 14.016.8% ; the percentage for methylated cases was 39.014.7% and that for unmethylated cases was 3.21.8% ( p<0.001 ) . the percentage methylation values for each year were not significantly different ( p=0.687 ) and did not show an increasing or decreasing linear pattern according to the age of the ffpe block . six samples ( 5.8% ) had an opposite methylation status of the mgmt promoter according to the two assays ( table 3 ) ; all 6 samples were obtained before 2003 and therefore were at least 10 years old and included one individual case for every year from 1997 to 2002 . for analysis by msp , the mean os of gbm patients with an unmethylated mgmt promoter was 16.2 months ( 95% ci 11.321.1 months ) , and that of patients with a methylated mgmt promoter was 19.6 months ( 95% ci 14.824.2 months , p=0.004 ) . for analysis by msp , the mean os of these patients with an unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) , and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months , p=0.002 ) ( table 4 ) . according to analysis by psq , the mean os of total 104 gbm patients with unmethylated mgmt promoter was 14.1 months ( 95% ci 11.616.9 months ) and that of patients with a methylated mgmt promoter was 20.9 months ( 95% ci 17.324.4 months ) . more detailed categorization according to the percentage of methylation showed a significant difference in os even among patients with a methylated mgmt promoter ( p=0.012 ) ; the mean os of gbm patients with a less methylated mgmt promoter ( from 939% ) was 17.8 months ( 95% ci 13.921.7 months ) , whereas that of patients with a more methylated mgmt promoter ( above 39% ) was 24.5 months ( 95% ci 19.229.8 months ) . actually , in terms of 44 patients who were treated with concurrent temozolomide chemoradiotherapy , the methylation status analyzed by psq was completely identical to that by msp in 44 patients who were treated with temozolomide concurrent chemoradiotherapy . therefore , there was no difference of overall survival between two groups according to the analyzing technique ; the patients with unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months ) ( table 4 ) . age < 50 [ hazard ratio ( hr ) 2.912 ; p=0.042 ] , kps 70 ( hr 13.329 ; p=0.001 ) , extent of surgical resection ( p<0.05 ) , type of adjuvant therapy ( p<0.05 ) , methylation of mgmt promoter analyzed by msp ( hr 9.856 ; p=0.013 ) , and methylation of mgmt promoter analyzed by psq ( hr 18.007 ; p<0.001 ) were associated with os . in multivariate analysis , we found that age ( < 50 years vs. 50 years ; hr 3.386 , p=0.048 ) , kps ( 70 vs. < 70 ; hr 9.417 , p=0.002 ) , extent of surgical resection ( gross total resection vs. subtotal resection , hr 8.045 ) , method of adjuvant treatment ( combination therapy vs. other treatment , hr 14.128 , p<0.001 ) , methylation status of mgmt promoter by msp ( methylated vs. unmethylated , hr 4.927 , p=0.034 ) , and methylation status of mgmt promoter by psq ( methylated vs. unmethylated , hr 9.208 , p=0.001 ) were independently associated with os . for determining the methylation status of mgmt promoter methylation by psq , roc curve analysis of using the mean percentage of methylation at the five cpgs to predict the longer survival showed that the auc was 0.68 . the optimal threshold of mean percentage of methylation for distinguishing between the patients with long survival and those with short survival was 9% , and this yielded a sensitivity of 53.8% [ 95% confidence interval ( ci ) 44.662.9% ] , a specificity of 66.7% ( 95% ci 52.980.5% ) , and an accuracy of 68.0% . the average percentage methylation for all samples of the methylated cases over the study period of 13 years was also investigated ( table 2 ) . the average methylation percentage of all gbm samples was 14.016.8% ; the percentage for methylated cases was 39.014.7% and that for unmethylated cases was 3.21.8% ( p<0.001 ) . the percentage methylation values for each year were not significantly different ( p=0.687 ) and did not show an increasing or decreasing linear pattern according to the age of the ffpe block . six samples ( 5.8% ) had an opposite methylation status of the mgmt promoter according to the two assays ( table 3 ) ; all 6 samples were obtained before 2003 and therefore were at least 10 years old and included one individual case for every year from 1997 to 2002 . for analysis by msp , the mean os of gbm patients with an unmethylated mgmt promoter was 16.2 months ( 95% ci 11.321.1 months ) , and that of patients with a methylated mgmt promoter was 19.6 months ( 95% ci 14.824.2 months , p=0.004 ) . for analysis by msp , the mean os of these patients with an unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) , and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months , p=0.002 ) ( table 4 ) . according to analysis by psq , the mean os of total 104 gbm patients with unmethylated mgmt promoter was 14.1 months ( 95% ci 11.616.9 months ) and that of patients with a methylated mgmt promoter was 20.9 months ( 95% ci 17.324.4 months ) . more detailed categorization according to the percentage of methylation showed a significant difference in os even among patients with a methylated mgmt promoter ( p=0.012 ) ; the mean os of gbm patients with a less methylated mgmt promoter ( from 939% ) was 17.8 months ( 95% ci 13.921.7 months ) , whereas that of patients with a more methylated mgmt promoter ( above 39% ) was 24.5 months ( 95% ci 19.229.8 months ) . therefore , there was no difference of overall survival between two groups according to the analyzing technique ; the patients with unmethylated mgmt promoter was 17.1 months ( 95% ci 9.623.4 months ) and that of patients with a methylated mgmt promoter was 20.3 months ( 95% ci 15.326.0 months ) ( table 4 ) . age < 50 [ hazard ratio ( hr ) 2.912 ; p=0.042 ] , kps 70 ( hr 13.329 ; p=0.001 ) , extent of surgical resection ( p<0.05 ) , type of adjuvant therapy ( p<0.05 ) , methylation of mgmt promoter analyzed by msp ( hr 9.856 ; p=0.013 ) , and methylation of mgmt promoter analyzed by psq ( hr 18.007 ; p<0.001 ) were associated with os . in multivariate analysis , we found that age ( < 50 years vs. 50 years ; hr 3.386 , p=0.048 ) , kps ( 70 vs. < 70 ; hr 9.417 , p=0.002 ) , extent of surgical resection ( gross total resection vs. subtotal resection , hr 8.045 ) , method of adjuvant treatment ( combination therapy vs. other treatment , hr 14.128 , p<0.001 ) , methylation status of mgmt promoter by msp ( methylated vs. unmethylated , hr 4.927 , p=0.034 ) , and methylation status of mgmt promoter by psq ( methylated vs. unmethylated , hr 9.208 , p=0.001 ) were independently associated with os . in this study , we performed psq analysis to determine mgmt promoter methylation status and estimate the extent of methylation using 1- to 13-year - old archival tissue samples of gbms . we obtained similar results in our study ; age < 50 , good functional status ( kps 70 ) , gross total resection , and methylated mgmt promoter analyzed by both msp and psq were all associated with os . in contrast , the success rate of psq analysis in our study was 100% for 104 samples from 1- to 13-year - old archival specimens , and 41.3% of the gbms were methylated . as described above , psq is a simple technique based on the sequencing - by - synthesis principle and allows accurate and quantitative analysis of dna sequences . the percentage of methylation was evenly distributed over the years of this study and no increasing or decreasing pattern of methylation with specimen age was observed , regardless of the age of the samples . in terms of the relationship between the extent of methylation estimated by psq and clinical outcome in gbm , this study showed that gbm patients with a more methylated mgmt promoter had longer overall survival than those with a less methylated mgmt promoter . recently , karayan - tapon et al.10 ) assessed mgmt promoter methylation status by five different methods including psq , and the psq primer sets used included 5 cpgs in the dmr2 region that were also used in this study . therefore , further evaluation of other sites of cgi methylation is essential to increase the accuracy of the status and extent of mgmt promoter methylation , and to commercialize this kit . this study showed that both the status and extent of methylation of the mgmt promoter analyzed by psq were found to be associated with os in patients with gbm . in this regard , among many methods for determining the characteristics of gene promoter methylation , psq is a quantitative approach that overcomes the problems associated with msp , and is a simple technique for accurate analysis of dna sequences .

we enrolled participants with tricuspid aortic valves and moderate - to - severe as , based on clinically indicated transthoracic echocardiographic findings and european association of echocardiography / american society of echocardiography recommendations for assessment of valve stenosis,21 including any of : maximum aortic velocity 3.0 m / s ; peak gradient 35 mm hg ; mean gradient 20 mm hg ; or calculated aortic valve area 1.5 cm . in addition , subjects were required to have normal lvsf , defined as an echocardiographic biplane ejection fraction of 50% or higher , and stable clinical features . participants were identified through electronic search of the echocardiographic database in dunedin hospital , which provides secondary and tertiary cardiology and cardiac surgery services . echocardiograms and electronic health records were reviewed by one of the investigating physicians , a european association of cardiovascular imaging accredited echocardiographer , to ensure that the potential participant met the criteria . all procedures were approved by new zealand lower south and multi - region ethics committees and in accord with the helsinki declaration of 1975 , as revised in 2000 . we excluded participants with bicuspid aortic valve ( bav ) owing to the additional complexities of the different bav phenotypes and excluded patients with other congenital cardiac abnormalities or other significant structural heart diseases , such as valvular regurgitation of more than mild severity . subjects with a past history of rheumatic heart disease or current major systemic disease ( such as advanced chronic kidney disease ) were also excluded . a recruitment flow chart for as participants along with reasons for exclusion is shown in figure s1 . the first was a control cohort recruited from the community by newspaper and poster advertising . they were aged predominantly 60 years and older without a history of significant cvd and in good general health this cohort was used for both the discovery and validation phase of the study . an assumption of 1.3% to 2.8% population prevalence with significant as was made,22 and the inference drawn that such a low rate of potential false negatives would have minimal effect on the study result . because we wished to examine the effect of cad on our analysis , the second group consisted of randomly selected participants undergoing coronary angiography , who were enrolled in hospital preprocedurally . clinically significant cad was diagnosed based on either a history of previous coronary disease or a coronary revascularisation procedure or angiographic demonstration of luminal obstruction greater than 50% . a number of the participants recruited as controls ( 9 of 110 controls ) were found to have as on echocardiography these were analyzed in the as group . edta blood samples were centrifuged within 1 hour of collection at 3000 revolutions per minute for 8 minutes and frozen for batch processing . for those undergoing angiography , samples were obtained before administration of heparin . total rna , including mirna , was extracted using mirna purification kits ( norgen biotek , thorold , ontario , canada ) , according to the manufacturer s instructions . mirna was analyzed using affymetrix mirna v2.0 genechips ( affymetrix , santa clara , ca ) . the array platform was used according to manufacturer instructions and run in an accredited service laboratory ( otago genomics & bioinformatics facility , university of otago , dunedin , new zealand ) . array data are available in the arrayexpress database ( www.ebi.ac.uk/arrayexpress ) under accession number e - mtab-2785 . we examined differentially expressed mirnas using quantitative polymerase chain reaction ( qpcr ) . for qpcr analysis , extracted rna was initially reverse transcribed to cdna using the qscript microrna cdna synthesis kit ( quanta biosciences inc , gaithersburg , md ) . sybr green pcr probes ( quanta biosciences ) were used for qpcr confirmation of specific mirnas . any probe assay with a cycle threshold ( ct ) over 35 two normalization controls were used : mir-16 - 5p ( based on previous use as a normalization control ) and mir-151 - 5p ( based on the normfinder stability score of microarray data).23,24 two separate runs were performed ( mir-22 - 3p and mir-24 - 3p in the first run , and mir-382 - 3p , mir-451a , and mir-21 - 5p in the second ) , with both normalization controls measured in each run . there was excellent intrasample concordance in levels of normalization controls between runs ( figure s2 ) . demographic and clinical variables were compared between groups using the t test for continuous variables and the chi - square test or fisher s exact test for categorical variables . normalized values were loaded into qlucore omics explorer ( version 2.3 ; qlucore ab , lund , sweden ) and analyzed using principal components analysis ( pca ) and unsupervised hierarchical clustering . the raw p value was adjusted for multiple testing using the benjamini - hochberg method . age was adjusted for using linear regression with qlucore omic explorer s built - in factor elimination . statistical analysis of qpcr results was conducted using stata / se ( v12.1 ; statacorp lp , college station , tx ) , with p<0.05 considered to be significant . expression levels were normalized to the mean of the normalization controls using the delta ct method.25 because qpcr distributions were not normally distributed , we used nonparametric tests ( for continuous variables : mann whitney u test ; for categorical variables : fisher s exact test ) . spearman s correlation was used to examine associations with echocardiographic measures in participants with as and with age in controls . to show the main body of data without log transformation , figures with qpcr data had extreme outliers removed , but all qpcr data were used for statistical testing . given previously demonstrated differences in mirna levels in different age groups,26 we used spearman s rank - correlation coefficient to look for statistically significant associations between age and circulating levels of these mirnas in control participants . we enrolled participants with tricuspid aortic valves and moderate - to - severe as , based on clinically indicated transthoracic echocardiographic findings and european association of echocardiography / american society of echocardiography recommendations for assessment of valve stenosis,21 including any of : maximum aortic velocity 3.0 m / s ; peak gradient 35 mm hg ; mean gradient 20 mm hg ; or calculated aortic valve area 1.5 cm . in addition , subjects were required to have normal lvsf , defined as an echocardiographic biplane ejection fraction of 50% or higher , and stable clinical features . participants were identified through electronic search of the echocardiographic database in dunedin hospital , which provides secondary and tertiary cardiology and cardiac surgery services . echocardiograms and electronic health records were reviewed by one of the investigating physicians , a european association of cardiovascular imaging accredited echocardiographer , to ensure that the potential participant met the criteria . all procedures were approved by new zealand lower south and multi - region ethics committees and in accord with the helsinki declaration of 1975 , as revised in 2000 . we excluded participants with bicuspid aortic valve ( bav ) owing to the additional complexities of the different bav phenotypes and excluded patients with other congenital cardiac abnormalities or other significant structural heart diseases , such as valvular regurgitation of more than mild severity . subjects with a past history of rheumatic heart disease or current major systemic disease ( such as advanced chronic kidney disease ) were also excluded . a recruitment flow chart for as participants along with reasons for exclusion is shown in figure s1 . the first was a control cohort recruited from the community by newspaper and poster advertising . they were aged predominantly 60 years and older without a history of significant cvd and in good general health this cohort was used for both the discovery and validation phase of the study . an assumption of 1.3% to 2.8% population prevalence with significant as was made,22 and the inference drawn that such a low rate of potential false negatives would have minimal effect on the study result . because we wished to examine the effect of cad on our analysis , the second group consisted of randomly selected participants undergoing coronary angiography , who were enrolled in hospital preprocedurally . clinically significant cad was diagnosed based on either a history of previous coronary disease or a coronary revascularisation procedure or angiographic demonstration of luminal obstruction greater than 50% . a number of the participants recruited as controls ( 9 of 110 controls ) were found to have as on echocardiography these were analyzed in the as group . edta blood samples were centrifuged within 1 hour of collection at 3000 revolutions per minute for 8 minutes and frozen for batch processing . for those undergoing angiography , total rna , including mirna , was extracted using mirna purification kits ( norgen biotek , thorold , ontario , canada ) , according to the manufacturer s instructions . mirna was analyzed using affymetrix mirna v2.0 genechips ( affymetrix , santa clara , ca ) . the array platform was used according to manufacturer instructions and run in an accredited service laboratory ( otago genomics & bioinformatics facility , university of otago , dunedin , new zealand ) . array data are available in the arrayexpress database ( www.ebi.ac.uk/arrayexpress ) under accession number e - mtab-2785 . we examined differentially expressed mirnas using quantitative polymerase chain reaction ( qpcr ) . for qpcr analysis , extracted rna was initially reverse transcribed to cdna using the qscript microrna cdna synthesis kit ( quanta biosciences inc , gaithersburg , md ) . sybr green pcr probes ( quanta biosciences ) were used for qpcr confirmation of specific mirnas . any probe assay with a cycle threshold ( ct ) over 35 two normalization controls were used : mir-16 - 5p ( based on previous use as a normalization control ) and mir-151 - 5p ( based on the normfinder stability score of microarray data).23,24 two separate runs were performed ( mir-22 - 3p and mir-24 - 3p in the first run , and mir-382 - 3p , mir-451a , and mir-21 - 5p in the second ) , with both normalization controls measured in each run . there was excellent intrasample concordance in levels of normalization controls between runs ( figure s2 ) . demographic and clinical variables were compared between groups using the t test for continuous variables and the chi - square test or fisher s exact test for categorical variables . normalized values were loaded into qlucore omics explorer ( version 2.3 ; qlucore ab , lund , sweden ) and analyzed using principal components analysis ( pca ) and unsupervised hierarchical clustering . the raw p value was adjusted for multiple testing using the benjamini - hochberg method . age was adjusted for using linear regression with qlucore omic explorer s built - in factor elimination . statistical analysis of qpcr results was conducted using stata / se ( v12.1 ; statacorp lp , college station , tx ) , with p<0.05 considered to be significant . expression levels were normalized to the mean of the normalization controls using the delta ct method.25 because qpcr distributions were not normally distributed , we used nonparametric tests ( for continuous variables : mann whitney u test ; for categorical variables : fisher s exact test ) . spearman s correlation was used to examine associations with echocardiographic measures in participants with as and with age in controls . to show the main body of data without log transformation , figures with qpcr data had extreme outliers removed , but all qpcr data were used for statistical testing . given previously demonstrated differences in mirna levels in different age groups,26 we used spearman s rank - correlation coefficient to look for statistically significant associations between age and circulating levels of these mirnas in control participants . the plasma mirna profile of 24 participants with as was compared to 27 healthy elderly controls using microarray . despite attempts to use an elderly control group , the as participants were still significantly older ( mean age , 77.9 years in as group compared to 67.0 years in controls ; t test , p<0.001 ) . there was no significant gender difference between groups ( 37% female in as group compared to 50% female in control group ; fisher s exact p=0.40 ) . participants with as had severe disease , with mean aortic valve maximum velocity of 4.2 m / s ( sd , 0.6 m / s ) , mean pressure gradient 46 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 0.8 cm ( sd , 0.3 cm ) . in edta plasma samples , 16 mirnas were significantly differentially expressed between the as and control groups ( adjusted p<0.05 ) . pca and hierarchical clustering showed partial clustering according to disease status using these mirnas ( figure 1 ) . two upregulated mirnas , mir-451a and mir-22 - 3p , and 2 downregulated mirnas , mir-24 - 3p and mir-382 - 5p remained significantly different after adjusting for age , leading to incomplete clustering according to disease status ( table 1 and figure 2 ) . plasma microarray analysis using micrornas differentially expressed at adjusted p<0.05 showed some , but not complete , clustering on heatmap ( a ) and principal components analysis ( b ) . as indicates aortic stenosis . differentially expressed micrornas in plasma of participants with aortic stenosis compared to control participants ( adjusted p<0.05 , after adjustment for age and multiple testing ) mir indicates microrna . heatmap of differentially expressed ( adjusted p<0.05 , after age adjustment ) micrornas in plasma of participants with and without as , showing some , but not complete , clustering according to disease status . as indicates aortic stenosis ; mir , microrna . after quality control , plasma samples from a total of 195 participants were used for validation qpcr analysis , consisting of 101 controls and 94 with as . those with as were older and had a greater proportion of cv comorbidities , including higher body mass index , diabetes , and hypertension ( table 2 ) . participants with as had moderate ( n=60 ) or severe ( n=34 ) disease , with mean aortic valve maximum velocity of 3.7 m / s ( sd , 0.7 m / s ) , mean pressure gradient 35 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 1.0 cm ( sd , 0.3 cm ) . demographics and baseline characteristics of participants in plasma polymerase chain reaction analysis statistical tests were performed using the t test for continuous variables and the chi - square test or fisher s exact test for categorical variables . the 4 differentially expressed mirnas on microarray analysis and mir-21 - 5p , chosen based on previous research findings , were then examined in plasma using qpcr , stratified according to the presence or absence of significant cad ( figure 3 ) . in participants with no cad , mir-22 - 3p was reduced in those with as , compared to controls ( p=0.02 ) , but there was no difference in mir-24 - 3p , mir-382 - 3p , or mir-451a levels between groups ( p=0.83 , 0.55 , and 0.06 respectively ) . as expected from previous literature , mir-21 - 5p both mir-22 - 3p and mir-24 - 3p were increased in those with as ( p=0.04 and 0.007 , respectively ) , whereas mir-382 - 3p was reduced ( p=0.04 ) . there were no differences in levels of mir-451a and mir-21 - 5p between groups ( p=0.82 and 0.30 , respectively ) . the top and bottom of the boxes indicate the 25th to 75th percentiles , respectively , and bands within boxes indicate the median of the data . whiskers indicate the tukey upper and lower adjacent values , that is , the highest data point within 1.5 times the interquartile range from the 75th percentile and the lowest data point within 1.5 times the interquartile range from the 25th percentile , respectively . whitney u test , with numbers in each group shown in parentheses . in participants without cad , mir-22 - 3p was decreased , whereas mir-21 - 5p was increased , in the as group . in participants with cad , mir-22 - 3p and mir-24 - 3p other microrna levels were similar between groups . as indicates aortic stenosis ; cad , coronary artery disease ; mir , microrna ; qpcr , quantitative polymerase chain reaction . when compared with echocardiographic measures of disease in participants with as , mir-21 - 5p and mir-382 - 5p levels showed a statistically significant correlation with maximum transvalvular velocity ( spearman s rho=0.35 , p=0.0005 , and =0.23 , p=0.02 , respectively ; figure s3 ) and mean gradient ( =0.32 , p=0.002 , and =0.31 , p=0.003 ) , but not lv mass index ( =0.14 , p=0.19 , and =0.05 , p=0.67 ) . mir-22 - 3p , mir-24 - 3p , and mir-451a did not have any statistically significant correlation with these measures . in summary , compared to microarray findings , only mir-22 - 3p and mir-382 - 5p had the expected results on qpcr , and these were observed only in those with cad . mir-21 - 5p levels were higher in those with as without cad , but showed no difference between groups in those with cad . only mir-21 - 5p and mir-382 - 5p levels correlated weakly with measures of disease severity . no statistically significant association was observed between age and any of the circulating mirnas examined by qpcr in participants without aortic stenosis ( n=101 ; age range , 54 to 89 years ; figure 4 ; table s1 ) . no association between age and circulating microrna levels was observed in control participants ( n=101 ) . age is shown on the x - axis , and expression relative to mean of the normalization controls is shown on the y - axis . outside of mir-382 - 5p , the plasma mirna profile of 24 participants with as was compared to 27 healthy elderly controls using microarray . despite attempts to use an elderly control group , the as participants were still significantly older ( mean age , 77.9 years in as group compared to 67.0 years in controls ; t test , p<0.001 ) . there was no significant gender difference between groups ( 37% female in as group compared to 50% female in control group ; fisher s exact p=0.40 ) . participants with as had severe disease , with mean aortic valve maximum velocity of 4.2 m / s ( sd , 0.6 m / s ) , mean pressure gradient 46 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 0.8 cm ( sd , 0.3 cm ) . in edta plasma samples , 16 mirnas were significantly differentially expressed between the as and control groups ( adjusted p<0.05 ) . pca and hierarchical clustering showed partial clustering according to disease status using these mirnas ( figure 1 ) . two upregulated mirnas , mir-451a and mir-22 - 3p , and 2 downregulated mirnas , mir-24 - 3p and mir-382 - 5p remained significantly different after adjusting for age , leading to incomplete clustering according to disease status ( table 1 and figure 2 ) . plasma microarray analysis using micrornas differentially expressed at adjusted p<0.05 showed some , but not complete , clustering on heatmap ( a ) and principal components analysis ( b ) . as indicates aortic stenosis . differentially expressed micrornas in plasma of participants with aortic stenosis compared to control participants ( adjusted p<0.05 , after adjustment for age and multiple testing ) mir indicates microrna . heatmap of differentially expressed ( adjusted p<0.05 , after age adjustment ) micrornas in plasma of participants with and without as , showing some , but not complete , clustering according to disease status . as indicates aortic stenosis ; mir , microrna . after quality control , plasma samples from a total of 195 participants were used for validation qpcr analysis , consisting of 101 controls and 94 with as . those with as were older and had a greater proportion of cv comorbidities , including higher body mass index , diabetes , and hypertension ( table 2 ) . participants with as had moderate ( n=60 ) or severe ( n=34 ) disease , with mean aortic valve maximum velocity of 3.7 m / s ( sd , 0.7 m / s ) , mean pressure gradient 35 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 1.0 cm ( sd , 0.3 cm ) . demographics and baseline characteristics of participants in plasma polymerase chain reaction analysis statistical tests were performed using the t test for continuous variables and the chi - square test or fisher s exact test for categorical variables . the 4 differentially expressed mirnas on microarray analysis and mir-21 - 5p , chosen based on previous research findings , were then examined in plasma using qpcr , stratified according to the presence or absence of significant cad ( figure 3 ) . in participants with no cad , mir-22 - 3p was reduced in those with as , compared to controls ( p=0.02 ) , but there was no difference in mir-24 - 3p , mir-382 - 3p , or mir-451a levels between groups ( p=0.83 , 0.55 , and 0.06 respectively ) . as expected from previous literature , mir-21 - 5p both mir-22 - 3p and mir-24 - 3p were increased in those with as ( p=0.04 and 0.007 , respectively ) , whereas mir-382 - 3p was reduced ( p=0.04 ) . there were no differences in levels of mir-451a and mir-21 - 5p between groups ( p=0.82 and 0.30 , respectively ) . as measured by qpcr in participants ( a ) without and ( b ) with coronary artery disease . the top and bottom of the boxes indicate the 25th to 75th percentiles , respectively , and bands within boxes indicate the median of the data . whiskers indicate the tukey upper and lower adjacent values , that is , the highest data point within 1.5 times the interquartile range from the 75th percentile and the lowest data point within 1.5 times the interquartile range from the 25th percentile , respectively . whitney u test , with numbers in each group shown in parentheses . in participants without cad , mir-22 - 3p was decreased , whereas mir-21 - 5p was increased , in the as group . in participants with cad , mir-22 - 3p and mir-24 - 3p other microrna levels were similar between groups . as indicates aortic stenosis ; cad , coronary artery disease ; mir , microrna ; qpcr , quantitative polymerase chain reaction . when compared with echocardiographic measures of disease in participants with as , mir-21 - 5p and mir-382 - 5p levels showed a statistically significant correlation with maximum transvalvular velocity ( spearman s rho=0.35 , p=0.0005 , and =0.23 , p=0.02 , respectively ; figure s3 ) and mean gradient ( =0.32 , p=0.002 , and =0.31 , p=0.003 ) , but not lv mass index ( =0.14 , p=0.19 , and =0.05 , p=0.67 ) . mir-22 - 3p , mir-24 - 3p , and mir-451a did not have any statistically significant correlation with these measures . in summary , compared to microarray findings , only mir-22 - 3p and mir-382 - 5p had the expected results on qpcr , and these were observed only in those with cad . mir-21 - 5p levels were higher in those with as without cad , but showed no difference between groups in those with cad . only mir-21 - 5p and mir-382 - 5p levels correlated weakly with measures of disease severity . no statistically significant association was observed between age and any of the circulating mirnas examined by qpcr in participants without aortic stenosis ( n=101 ; age range , 54 to 89 years ; figure 4 ; table s1 ) . no association between age and circulating microrna levels was observed in control participants ( n=101 ) . age is shown on the x - axis , and expression relative to mean of the normalization controls is shown on the y - axis . outside of mir-382 - 5p , the y - axis scale is log - transformed . in this study , we have shown that circulating whole mirnome profiles discriminate , albeit incompletely , between participants with as and those without . age - adjusted analysis identified 4 mirnas that were differentially expressed between the 2 groups . even these 2 mirnas had different results according to cad status , with mir-22 - 3p being increased in as participants with cad , but decreased in those without . previous studies have looked at specific circulating mirnas in as , but only 1 examined those we identified as suitable for qpcr validation.19 no difference was found in mir-22 - 3p between groups in that study.19 this is in contrast to our study , but it should be noted that 67% of the rsj et al . as group had cad , and their control group was relatively small . in addition to those mirnas identified by microarray , we also examined circulating levels of mir-21 - 5p , based on previous research showing that circulating mir-21 - 5p is increased in patients with as , and correlates with both myocardial mir-21 - 5p and degree of myocardial fibrosis.15 furthermore , there is strong preclinical evidence that pressure overload leads to increased myocardial mir-21 - 5p in both animal and human models.27,28 whereas we found that mir-21 - 5p was increased in as participants without cad , there was no difference between groups in those with cad . two other studies , using different analysis methods ( qpcr using an exogenous control and a pcr array ) , have also found no difference in this mirna between as and control groups.16,20 therefore , despite the modest correlation with disease severity , it is unlikely that mir-21 - 5p will provide sufficient discrimination between heterogeneous groups of patients . our discrepant findings lead us to suggest that mirnas are unlikely to be useful as biomarkers in the near future . the as group had a number of different clinical features compared to the non - as group , such as having more males and more participants taking statin medications ( table 2 ) . another possibility is that the mirna profile changes little until ventricular decompensation occurs all of our participants with as had preserved ejection fraction . however , the recent study by derda et al . showed no difference in mir-21 - 5p levels , despite 41% of as participants having reduced ejection fraction.16 it is possible that confounding factors such as these make the already noisy signal from circulating mirna profiles harder to detect . even within the non - as group , there were large variations in mirna levels assessed by qpcr ( figure 4 ) . alternative techniques for examining circulating mirnas may improve the signal - to - noise ratio . these include the use of platelet - poor plasma,29 examining argonaute or high - density lipoprotein - bound mirnas,30,31 or , perhaps most promisingly , focusing on mirnas contained within circulating microparticles.32 this study highlights the difficulties translating mirnas into clinically useful biomarkers . the wide variation in levels , even among participants without significant disease , means that finding differences between groups is difficult . these findings are not exclusive to as , with , for example , a previous review noting that mirnas were not yet suitable for clinical use in cad owing to , among other issues , the inherent variability in qpcr.14 a well - recognized limitation when performing mirna qpcr is the lack of agreed normalization controls , with exogenous mirnas , endogenous mirnas , and endogenous rnas of similar size all being used previously.33 exogenous mirnas provide information primarily on mirna extraction efficiency ( rather than amplification ) and generate relative , rather than absolute , results . the use of non - mirna rna ( such as rnu6 ) as endogenous normalizers has also been proposed , but this marker has been shown to differ in different disease conditions , including inflammatory disease.34 we therefore used 2 endogenous mirnas , identified from both previous literature and our own microarray experiment , as neutrally expressed normalization markers . a different approach , using an endogenous control that most closely follows the mean intrasample expression ( rather than the intersample expression assessed by normfinder ) may be a superior method.35 we had considered using this method , but the most suitable mirna identified , mir-103a-3p , has previously been investigated for heart failure diagnosis,36 making its use as a normalization control contentious in this case . another limitation is that we were not able to enroll a group of control participants with a mean age of almost 79 years and without significant comorbidity , in part influenced by the high population prevalence of aortic sclerosis and as in such age groups.2,22 circulating mirna levels are known to alter with age , and indeed our microarray results showed a number of differentially expressed mirnas losing statistical significance once adjusted for age . however , in those without aortic stenosis , despite a wide age range , none of those mirnas examined by qpcr showed an association with age . finally , as with any observational study , there is a potential for recall bias , in particular in misclassification of the major confounder , cad . in conclusion , this study identified different circulating mirna profiles in participants with as , compared to those without . these results indicate that translating circulating mirnas into useful prognostic biomarkers for as will be a challenging process requiring considerable further refinement . this work was supported by a university of otago research grant , healthcare otago charitable trust grant , and the health research council of new zealand . dr coffey was supported by a tony hocken research award from the department of medicine , university of otago , and by the national institute of health research oxford biomedical research center program . scatter plots of levels of individual mirnas measured by quantitative polymerase chain reaction and echocardiographic parameters in participants with aortic stenosis .

backgroundaortic stenosis ( as ) is a progressive condition leading to heart failure and death without treatment . no medical therapy currently exists for as , and a major management challenge is deciding on the correct timing of aortic valve replacement . micrornas ( mirnas ) are short noncoding rnas that are stable in the circulation . we wished to use mirnas as biomarkers of disease in as.methods and resultswe performed microarray - based whole mirnome profiling of 24 participants with as and 27 control participants . after adjustment for age and multiple testing , we identified 4 mirnas significantly different between groups . these findings were then examined using quantitative polymerase chain reaction in a larger validation cohort of 101 controls and 94 participants with as , stratified in a prespecified analysis by presence of coexisting coronary artery disease ( cad ) . we obtained mixed results for mir-22 - 3p , mir-24 - 3p , mir-382 - 5p , and mir-451a in the validation cohort , with differing associations according to cad status . mir-21 - 5p was increased in as patients without cad , but there was no difference between groups with cad.conclusiondespite holding great promise , circulating mirna profiling requires further refinement before translation into clinical use as a biomarker in aortic stenosis .

Methods Participant Selection miRNA Microarray Analysis Quantitative Polymerase Chain Reaction (qPCR) Analysis Statistical Analysis Results Demographics of Participants in Microarray Analysis Microarray Results Demographics of Participants in qPCR Validation qPCR Validation Association Between Age and Circulating miRNA Levels Discussion Conclusion Sources of Funding Disclosures Supporting Information

we enrolled participants with tricuspid aortic valves and moderate - to - severe as , based on clinically indicated transthoracic echocardiographic findings and european association of echocardiography / american society of echocardiography recommendations for assessment of valve stenosis,21 including any of : maximum aortic velocity 3.0 m / s ; peak gradient 35 mm hg ; mean gradient 20 mm hg ; or calculated aortic valve area 1.5 cm . we excluded participants with bicuspid aortic valve ( bav ) owing to the additional complexities of the different bav phenotypes and excluded patients with other congenital cardiac abnormalities or other significant structural heart diseases , such as valvular regurgitation of more than mild severity . because we wished to examine the effect of cad on our analysis , the second group consisted of randomly selected participants undergoing coronary angiography , who were enrolled in hospital preprocedurally . we examined differentially expressed mirnas using quantitative polymerase chain reaction ( qpcr ) . any probe assay with a cycle threshold ( ct ) over 35 two normalization controls were used : mir-16 - 5p ( based on previous use as a normalization control ) and mir-151 - 5p ( based on the normfinder stability score of microarray data).23,24 two separate runs were performed ( mir-22 - 3p and mir-24 - 3p in the first run , and mir-382 - 3p , mir-451a , and mir-21 - 5p in the second ) , with both normalization controls measured in each run . spearman s correlation was used to examine associations with echocardiographic measures in participants with as and with age in controls . given previously demonstrated differences in mirna levels in different age groups,26 we used spearman s rank - correlation coefficient to look for statistically significant associations between age and circulating levels of these mirnas in control participants . we enrolled participants with tricuspid aortic valves and moderate - to - severe as , based on clinically indicated transthoracic echocardiographic findings and european association of echocardiography / american society of echocardiography recommendations for assessment of valve stenosis,21 including any of : maximum aortic velocity 3.0 m / s ; peak gradient 35 mm hg ; mean gradient 20 mm hg ; or calculated aortic valve area 1.5 cm . we excluded participants with bicuspid aortic valve ( bav ) owing to the additional complexities of the different bav phenotypes and excluded patients with other congenital cardiac abnormalities or other significant structural heart diseases , such as valvular regurgitation of more than mild severity . because we wished to examine the effect of cad on our analysis , the second group consisted of randomly selected participants undergoing coronary angiography , who were enrolled in hospital preprocedurally . we examined differentially expressed mirnas using quantitative polymerase chain reaction ( qpcr ) . any probe assay with a cycle threshold ( ct ) over 35 two normalization controls were used : mir-16 - 5p ( based on previous use as a normalization control ) and mir-151 - 5p ( based on the normfinder stability score of microarray data).23,24 two separate runs were performed ( mir-22 - 3p and mir-24 - 3p in the first run , and mir-382 - 3p , mir-451a , and mir-21 - 5p in the second ) , with both normalization controls measured in each run . spearman s correlation was used to examine associations with echocardiographic measures in participants with as and with age in controls . given previously demonstrated differences in mirna levels in different age groups,26 we used spearman s rank - correlation coefficient to look for statistically significant associations between age and circulating levels of these mirnas in control participants . the plasma mirna profile of 24 participants with as was compared to 27 healthy elderly controls using microarray . despite attempts to use an elderly control group , the as participants were still significantly older ( mean age , 77.9 years in as group compared to 67.0 years in controls ; t test , p<0.001 ) . there was no significant gender difference between groups ( 37% female in as group compared to 50% female in control group ; fisher s exact p=0.40 ) . participants with as had severe disease , with mean aortic valve maximum velocity of 4.2 m / s ( sd , 0.6 m / s ) , mean pressure gradient 46 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 0.8 cm ( sd , 0.3 cm ) . two upregulated mirnas , mir-451a and mir-22 - 3p , and 2 downregulated mirnas , mir-24 - 3p and mir-382 - 5p remained significantly different after adjusting for age , leading to incomplete clustering according to disease status ( table 1 and figure 2 ) . differentially expressed micrornas in plasma of participants with aortic stenosis compared to control participants ( adjusted p<0.05 , after adjustment for age and multiple testing ) mir indicates microrna . heatmap of differentially expressed ( adjusted p<0.05 , after age adjustment ) micrornas in plasma of participants with and without as , showing some , but not complete , clustering according to disease status . after quality control , plasma samples from a total of 195 participants were used for validation qpcr analysis , consisting of 101 controls and 94 with as . those with as were older and had a greater proportion of cv comorbidities , including higher body mass index , diabetes , and hypertension ( table 2 ) . participants with as had moderate ( n=60 ) or severe ( n=34 ) disease , with mean aortic valve maximum velocity of 3.7 m / s ( sd , 0.7 m / s ) , mean pressure gradient 35 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 1.0 cm ( sd , 0.3 cm ) . demographics and baseline characteristics of participants in plasma polymerase chain reaction analysis statistical tests were performed using the t test for continuous variables and the chi - square test or fisher s exact test for categorical variables . the 4 differentially expressed mirnas on microarray analysis and mir-21 - 5p , chosen based on previous research findings , were then examined in plasma using qpcr , stratified according to the presence or absence of significant cad ( figure 3 ) . in participants with no cad , mir-22 - 3p was reduced in those with as , compared to controls ( p=0.02 ) , but there was no difference in mir-24 - 3p , mir-382 - 3p , or mir-451a levels between groups ( p=0.83 , 0.55 , and 0.06 respectively ) . as expected from previous literature , mir-21 - 5p both mir-22 - 3p and mir-24 - 3p were increased in those with as ( p=0.04 and 0.007 , respectively ) , whereas mir-382 - 3p was reduced ( p=0.04 ) . there were no differences in levels of mir-451a and mir-21 - 5p between groups ( p=0.82 and 0.30 , respectively ) . in participants without cad , mir-22 - 3p was decreased , whereas mir-21 - 5p was increased , in the as group . in participants with cad , mir-22 - 3p and mir-24 - 3p other microrna levels were similar between groups . as indicates aortic stenosis ; cad , coronary artery disease ; mir , microrna ; qpcr , quantitative polymerase chain reaction . when compared with echocardiographic measures of disease in participants with as , mir-21 - 5p and mir-382 - 5p levels showed a statistically significant correlation with maximum transvalvular velocity ( spearman s rho=0.35 , p=0.0005 , and =0.23 , p=0.02 , respectively ; figure s3 ) and mean gradient ( =0.32 , p=0.002 , and =0.31 , p=0.003 ) , but not lv mass index ( =0.14 , p=0.19 , and =0.05 , p=0.67 ) . mir-22 - 3p , mir-24 - 3p , and mir-451a did not have any statistically significant correlation with these measures . in summary , compared to microarray findings , only mir-22 - 3p and mir-382 - 5p had the expected results on qpcr , and these were observed only in those with cad . mir-21 - 5p levels were higher in those with as without cad , but showed no difference between groups in those with cad . only mir-21 - 5p and mir-382 - 5p levels correlated weakly with measures of disease severity . no statistically significant association was observed between age and any of the circulating mirnas examined by qpcr in participants without aortic stenosis ( n=101 ; age range , 54 to 89 years ; figure 4 ; table s1 ) . no association between age and circulating microrna levels was observed in control participants ( n=101 ) . age is shown on the x - axis , and expression relative to mean of the normalization controls is shown on the y - axis . outside of mir-382 - 5p , the plasma mirna profile of 24 participants with as was compared to 27 healthy elderly controls using microarray . despite attempts to use an elderly control group , the as participants were still significantly older ( mean age , 77.9 years in as group compared to 67.0 years in controls ; t test , p<0.001 ) . there was no significant gender difference between groups ( 37% female in as group compared to 50% female in control group ; fisher s exact p=0.40 ) . participants with as had severe disease , with mean aortic valve maximum velocity of 4.2 m / s ( sd , 0.6 m / s ) , mean pressure gradient 46 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 0.8 cm ( sd , 0.3 cm ) . two upregulated mirnas , mir-451a and mir-22 - 3p , and 2 downregulated mirnas , mir-24 - 3p and mir-382 - 5p remained significantly different after adjusting for age , leading to incomplete clustering according to disease status ( table 1 and figure 2 ) . differentially expressed micrornas in plasma of participants with aortic stenosis compared to control participants ( adjusted p<0.05 , after adjustment for age and multiple testing ) mir indicates microrna . heatmap of differentially expressed ( adjusted p<0.05 , after age adjustment ) micrornas in plasma of participants with and without as , showing some , but not complete , clustering according to disease status . after quality control , plasma samples from a total of 195 participants were used for validation qpcr analysis , consisting of 101 controls and 94 with as . those with as were older and had a greater proportion of cv comorbidities , including higher body mass index , diabetes , and hypertension ( table 2 ) . participants with as had moderate ( n=60 ) or severe ( n=34 ) disease , with mean aortic valve maximum velocity of 3.7 m / s ( sd , 0.7 m / s ) , mean pressure gradient 35 mm hg ( sd , 13 mm hg ) , and calculated aortic valve area 1.0 cm ( sd , 0.3 cm ) . demographics and baseline characteristics of participants in plasma polymerase chain reaction analysis statistical tests were performed using the t test for continuous variables and the chi - square test or fisher s exact test for categorical variables . the 4 differentially expressed mirnas on microarray analysis and mir-21 - 5p , chosen based on previous research findings , were then examined in plasma using qpcr , stratified according to the presence or absence of significant cad ( figure 3 ) . in participants with no cad , mir-22 - 3p was reduced in those with as , compared to controls ( p=0.02 ) , but there was no difference in mir-24 - 3p , mir-382 - 3p , or mir-451a levels between groups ( p=0.83 , 0.55 , and 0.06 respectively ) . as expected from previous literature , mir-21 - 5p both mir-22 - 3p and mir-24 - 3p were increased in those with as ( p=0.04 and 0.007 , respectively ) , whereas mir-382 - 3p was reduced ( p=0.04 ) . there were no differences in levels of mir-451a and mir-21 - 5p between groups ( p=0.82 and 0.30 , respectively ) . as measured by qpcr in participants ( a ) without and ( b ) with coronary artery disease . in participants without cad , mir-22 - 3p was decreased , whereas mir-21 - 5p was increased , in the as group . in participants with cad , mir-22 - 3p and mir-24 - 3p other microrna levels were similar between groups . as indicates aortic stenosis ; cad , coronary artery disease ; mir , microrna ; qpcr , quantitative polymerase chain reaction . when compared with echocardiographic measures of disease in participants with as , mir-21 - 5p and mir-382 - 5p levels showed a statistically significant correlation with maximum transvalvular velocity ( spearman s rho=0.35 , p=0.0005 , and =0.23 , p=0.02 , respectively ; figure s3 ) and mean gradient ( =0.32 , p=0.002 , and =0.31 , p=0.003 ) , but not lv mass index ( =0.14 , p=0.19 , and =0.05 , p=0.67 ) . mir-22 - 3p , mir-24 - 3p , and mir-451a did not have any statistically significant correlation with these measures . in summary , compared to microarray findings , only mir-22 - 3p and mir-382 - 5p had the expected results on qpcr , and these were observed only in those with cad . mir-21 - 5p levels were higher in those with as without cad , but showed no difference between groups in those with cad . only mir-21 - 5p and mir-382 - 5p levels correlated weakly with measures of disease severity . no statistically significant association was observed between age and any of the circulating mirnas examined by qpcr in participants without aortic stenosis ( n=101 ; age range , 54 to 89 years ; figure 4 ; table s1 ) . no association between age and circulating microrna levels was observed in control participants ( n=101 ) . age is shown on the x - axis , and expression relative to mean of the normalization controls is shown on the y - axis . outside of mir-382 - 5p , the y - axis scale is log - transformed . in this study , we have shown that circulating whole mirnome profiles discriminate , albeit incompletely , between participants with as and those without . age - adjusted analysis identified 4 mirnas that were differentially expressed between the 2 groups . even these 2 mirnas had different results according to cad status , with mir-22 - 3p being increased in as participants with cad , but decreased in those without . previous studies have looked at specific circulating mirnas in as , but only 1 examined those we identified as suitable for qpcr validation.19 no difference was found in mir-22 - 3p between groups in that study.19 this is in contrast to our study , but it should be noted that 67% of the rsj et al . as group had cad , and their control group was relatively small . in addition to those mirnas identified by microarray , we also examined circulating levels of mir-21 - 5p , based on previous research showing that circulating mir-21 - 5p is increased in patients with as , and correlates with both myocardial mir-21 - 5p and degree of myocardial fibrosis.15 furthermore , there is strong preclinical evidence that pressure overload leads to increased myocardial mir-21 - 5p in both animal and human models.27,28 whereas we found that mir-21 - 5p was increased in as participants without cad , there was no difference between groups in those with cad . two other studies , using different analysis methods ( qpcr using an exogenous control and a pcr array ) , have also found no difference in this mirna between as and control groups.16,20 therefore , despite the modest correlation with disease severity , it is unlikely that mir-21 - 5p will provide sufficient discrimination between heterogeneous groups of patients . our discrepant findings lead us to suggest that mirnas are unlikely to be useful as biomarkers in the near future . another possibility is that the mirna profile changes little until ventricular decompensation occurs all of our participants with as had preserved ejection fraction . showed no difference in mir-21 - 5p levels , despite 41% of as participants having reduced ejection fraction.16 it is possible that confounding factors such as these make the already noisy signal from circulating mirna profiles harder to detect . these findings are not exclusive to as , with , for example , a previous review noting that mirnas were not yet suitable for clinical use in cad owing to , among other issues , the inherent variability in qpcr.14 a well - recognized limitation when performing mirna qpcr is the lack of agreed normalization controls , with exogenous mirnas , endogenous mirnas , and endogenous rnas of similar size all being used previously.33 exogenous mirnas provide information primarily on mirna extraction efficiency ( rather than amplification ) and generate relative , rather than absolute , results . a different approach , using an endogenous control that most closely follows the mean intrasample expression ( rather than the intersample expression assessed by normfinder ) may be a superior method.35 we had considered using this method , but the most suitable mirna identified , mir-103a-3p , has previously been investigated for heart failure diagnosis,36 making its use as a normalization control contentious in this case . another limitation is that we were not able to enroll a group of control participants with a mean age of almost 79 years and without significant comorbidity , in part influenced by the high population prevalence of aortic sclerosis and as in such age groups.2,22 circulating mirna levels are known to alter with age , and indeed our microarray results showed a number of differentially expressed mirnas losing statistical significance once adjusted for age . in conclusion , this study identified different circulating mirna profiles in participants with as , compared to those without . these results indicate that translating circulating mirnas into useful prognostic biomarkers for as will be a challenging process requiring considerable further refinement . scatter plots of levels of individual mirnas measured by quantitative polymerase chain reaction and echocardiographic parameters in participants with aortic stenosis .

multiple sclerosis ( ms ) is a progressive disease of the central nervous system in which communication between the brain and other parts of the body is disrupted . its effects can range from relatively benign in most cases to somewhat disabling and then to devastating for some people . during an ms attack , inflammation occurs in areas of the white matter of the central nervous system in random patches ; these are called plaques . myelin allows for smooth , high - speed transmission of electrochemical messages between the brain , the spinal cord , and the rest of the body . when myelin is damaged , neurological transmission of messages may be slowed or blocked completely , resulting in some body functions being diminished or lost . approximately 520 000 people in the americas , 630 000 in europe , 66 000 in the eastern mediterranean , 56 000 in the western pacific , 31 500 in southeast asia , 11 000 in africa , and 1.3 million people worldwide have been diagnosed with ms . it primarily affects adults , has an age of onset typically between 20 and 40 years , and is twice as common in women than in men . symptoms and signs of ms vary widely depending on the location of affected myelin sheaths . the most common symptoms of ms are weakness , spasticity in one or more limbs seen in 50% of ms patients followed by numbness , tingling , and fatigue ( 40% ) , partial or complete loss of vision , double or blurred vision ( 31% ) , incontinence of urine and dysfunction of the bowel ( 17.5% ) , pain ( 15% ) , cognitive or behavioral problems , and sexual dysfunction ( 10% ) . in the worst cases it is a mild disease , but in others , it can lead to permanent disability . although symptoms may resolve completely between the episodes , permanent neurological problems usually persist , especially as the disease progresses . many risk factors for ms have been identified , but no definitive cause has been found . however , a number of alternative or complementary therapies have been used worldwide to treat the disease symptomatically and convert ms into remission . the five most prevalent alternative or complementary approaches are diet and nutrition ( 88.3% ) , acupuncture ( 86.7% ) , herbal medicine ( 81.7% ) , massage ( 78.3% ) , and homeopathy ( 73.3% ) . chinese scalp acupuncture is a contemporary acupuncture technique integrating traditional chinese needling methods with western medical knowledge of the cerebral cortex ( figures 1 and 2 ) . scalp acupuncture has been proven to be a very effective technique for treating central nervous system disorders ranging from ms , strokes , parkinson 's disease , traumatic brain injury , and posttraumatic stress disorder to phantom pain and complex regional pain . the scalp somato - topic system appears to manifest the convergence of the central nervous system and the endocrine system . the scalp somatotopic system seems to operate as a miniature transmitter - receiver in direct contact with the central nervous system and endocrine system . by stimulating those reflex areas , acupuncture can have a direct effect on the cerebral cortex , cerebellum , thalamocortical circuits , thalamus , hypothalamus , and pineal body . the scalp 's unique neurologic and endocrinal composition makes it an ideal external stimulating field for internal activities of the brain . using a small number of needles , scalp acupuncture can often produce remarkable results almost immediately , sometimes taking only several minutes to complete . charles , a 58-year - old lawyer , came to the clinic in albuquerque , new mexico , in 2010 . his initial symptoms were an onset of numbness in the right arm that was followed by subsequent numbness descending down both legs . over the past 20 years , charles had multiple relapses and remissions with episodes of lower extremity weakness , stiffness and muscle spasm , incontinence of urine , loss of balance , double vision , and fatigue . twelve years ago , he had a dramatic neurological decline during which he was unable to walk steadily and lost strength and sensation in his lower extremities . for the last 3 years , up to when he came to the clinic , it was more difficult for him to walk due to weakness in his legs and loss of balance . charles also complained of numbness , tingling , and spasms in his legs accompanied by incontinence of urine , double vision , poor memory and concentration , dizziness and vertigo , heat intolerance , and severe fatigue . his diagnosis had been confirmed by several mris that showed lesions in the brain and spine . family history revealed that a brother with ms had died in 1999 , and two sisters have been diagnosed with ms . for the first scalp acupuncture session , general and neurological examinations were carried out . motor strength was 4/5 in both legs and 5/5 in both arms and in hand grip the patient 's finger - to - nose tests on both right and left sides were abnormal . charles had difficulty getting out of a chair and presented with spastic and ataxic gait . he ambulated with stiffness in both legs , was unsteady , and had a wide - based stance . the patient failed the heel - toe walking test and could not stand on one leg . chinese medical examination showed that his tongue was red and slightly purple with a thin yellow coating ; his pulse on the left side was wiry and rolling ; on the right side , it was wiry and thready , and weak pulses were also noticed in the kidney positions . when palpating points , lr-3 ( taichong ) , gb-34 ( yanglingquan ) , ub-18 ( ganshu ) , and sp-9 ( yinlingquan ) were very tender and resulted in sharp pain , and ub-23 ( shenshu ) , and ub-15 ( xinshu ) were tender and showed dull soreness . primary scalp areas were motor area , sensory area , and foot motor and sensory area . secondary scalp areas were balance area , hearing and dizziness area , and tremor area ( figures 3 - 5 ) . the motor area , sensory area , and foot motor and sensory area received needles that were stimulated bilaterally . the needles were rotated at least 200 times per minute with thumb and index finger for 1 to 3 minutes . he was amazed to feel the dizziness , balance , stiffness , and weakness in his legs improve just minutes after a few needles were inserted in his scalp . he was initially very suspicious and nervous when the doctor asked him to stand up with his eyes closed . he was not only able to stand up with improved stability , but he also could walk much better . at the second treatment , charles reported that he had no more incontinence of urine and that the numbness , spasms , and weakness of both legs showed some improvement as well . , charles 's vision had significantly improved , and he no longer had double vision and heat intolerance . he also had more energy and was able to do more work at his law office . by the fifth treatment , charles reported that he was able to walk around his home and office without problems and that he could walk much longer distances . he had more energy and had not experienced incontinence of urine since the first treatment . what is a longtime local lawyer doing , writing about serious medical problems like multiple sclerosis ( ms ) and parkinson 's disease ? it is because i have had the rare opportunity of taking part in what i unhesitatingly tell everyone is nothing less than a miracle . what was uppermost was that 20 years earlier i had been diagnosed with multiple sclerosis ms took my brother in 1999 and has crippled one of my sisters and compromised the life of my youngest sister . i had managed to keep the disease under control and under wraps by wisely following my wife 's advice on nutrition , exercise , and a common - sense approach to ms . together we made trips to leading medical centers where i became a voluntary guinea pig for new medications . on top of that , i was tested , scanned , poked , and prodded over and [ over ] again by some of the best neurologists and medical professionals studying the disease . finally , despite all that , the symptoms were taking their toll , especially in the heat of the mesilla valley 's long summer . i realized i could no longer continue as head of the law firm i had helped to build over 3 decades . when my firm dissolved , i joined another law firm on a part - time basis to comply with limited energy and finish up so on a hot july day i awaited my first client at my new office that client was dr vittal pai , whom i 'd known for more than 20 years . i had not seen dr pai in a couple of years and always enjoyed visiting with him . dr pai , an ear , nose , and throat specialist , is a brilliant doctor known as much for his philosophical approach to life and his quick thinking and wit as he is for his substantial skills as a physician . dr pai observed my condition and placed a card on my desk saying , before we talk business , you must go see this doctor . i looked at the card and saw the name dr jason hao , doctor of oriental medicine and acupuncture . after exchanging greetings , i told dr pai that i had been a subject in medical studies at universities with the best neurologists in the world . other than steroids , i was disqualified from all the medications that had been approved to combat ms . he told me about a patient with parkinson 's disease who had incurable tremors that could not be controlled by medication . he told me about another patient with mobility damage from a stroke who seemed to have been miraculously cured with dr hao 's technique . i have had 12 treatments with dr hao at his clinic in albuquerque , new mexico . not only does he put needles in my head but he spins these needles with his fingers between 200 and 400 times a minute . dr hao told me that all of the nerves in the body can be accessed in the scalp and that the spinning of the needles clears the nerve pathways that have become broken and clogged . after these treatments , all of my major symptoms of ms are gone . my left foot , which has been numb for 18 years , is no longer numb . my vision has improved , my balance has returned , and i have not had vertigo in some time . every aspect of my life is better . i am working full - time again , although dr hao cautions me that ms is still my greatest enemy and that he has only relieved me of the symptoms brought on by the attacks . he cautions me to never use more than 70% of my energy in any 24-hour period and to take the prescribed herbs and follow basic nutritional guides . my wife and i look at each other on a daily basis and expect for this dream to end : this dream about a chinese doctor in albuquerque who spins acupuncture needles in my head to make the symptoms of ms go away . but every day i feel better , and my treatments with dr hao are now on a monthly basis rather than a weekly basis . twenty years of debilitating symptoms that compromised every aspect of my life reduced to a memory in 6 months ! those who knew me before last july and see me now at first just ca n't believe their own eyes . in view of my changes and my many inquiries to dr hao , i can not help but wonder : why is this particular form of acupuncture still not embraced coast to coast ? the treatments last about an hour and are relatively inexpensive and completely painless . for the tenth scalp acupuncture session , charles was awake , alert , cooperative , very attentive , and gave quicker responses . motor strength increased to 5/5 in both legs , both arms , and hand grip . the patient 's finger - to - nose tests on both right and left sides returned to normal . charles got out of a chair without any challenge and ambulated with a normal gait . the patient peformed the heel - toe walking test with no problems and could stand on either his left or right leg steadily . chinese medical examination at the tenth session showed that his tongue had changed to slightly red with a thin white coating ; his pulses changed to soft on both left and right sides , and pulses in the kidney positions changed to thready . when palpating points , all sensitive points including lr-3 ( taichong ) , gb-34 ( yanglingquan ) , ub-18 ( ganshu ) , sp-9 ( yinlingquan ) , ub-23(shenshu ) , and ub-15 ( xinshu ) showed neither tenderness nor pain and soreness . after the tenth treatment , charles was able to work full - time and started to take vacations again . he reported that he enjoyed standing on one leg during his work breaks and that doing so helped him feel like a normal person . although charles returned to a normal life , he prefers to come to the clinic every 4 to 6 weeks for maintenance treatments . primary scalp areas were motor area , sensory area , and foot motor and sensory area . secondary scalp areas were balance area , hearing and dizziness area , and tremor area ( figures 3 - 5 ) . the motor area , sensory area , and foot motor and sensory area received needles that were stimulated bilaterally . the needles were rotated at least 200 times per minute with thumb and index finger for 1 to 3 minutes . he was amazed to feel the dizziness , balance , stiffness , and weakness in his legs improve just minutes after a few needles were inserted in his scalp . he was initially very suspicious and nervous when the doctor asked him to stand up with his eyes closed . he was not only able to stand up with improved stability , but he also could walk much better . at the second treatment , charles reported that he had no more incontinence of urine and that the numbness , spasms , and weakness of both legs showed some improvement as well . charles 's vision had significantly improved , and he no longer had double vision and heat intolerance . he also had more energy and was able to do more work at his law office . by the fifth treatment , charles reported that he was able to walk around his home and office without problems and that he could walk much longer distances . he had more energy and had not experienced incontinence of urine since the first treatment . you are likely to wonder , what is a longtime local lawyer doing , writing about serious medical problems like multiple sclerosis ( ms ) and parkinson 's disease ? it is because i have had the rare opportunity of taking part in what i unhesitatingly tell everyone is nothing less than a miracle . what was uppermost was that 20 years earlier i had been diagnosed with multiple sclerosis ms took my brother in 1999 and has crippled one of my sisters and compromised the life of my youngest sister . i had managed to keep the disease under control and under wraps by wisely following my wife 's advice on nutrition , exercise , and a common - sense approach to ms . together we made trips to leading medical centers where i became a voluntary guinea pig for new medications . on top of that , i was tested , scanned , poked , and prodded over and [ over ] again by some of the best neurologists and medical professionals studying the disease . finally , despite all that , the symptoms were taking their toll , especially in the heat of the mesilla valley 's long summer . i realized i could no longer continue as head of the law firm i had helped to build over 3 decades . when my firm dissolved , i joined another law firm on a part - time basis to comply with limited energy and finish up so on a hot july day i awaited my first client at my new office that client was dr vittal pai , whom i 'd known for more than 20 years . i had not seen dr pai in a couple of years and always enjoyed visiting with him . dr pai , an ear , nose , and throat specialist , is a brilliant doctor known as much for his philosophical approach to life and his quick thinking and wit as he is for his substantial skills as a physician . dr pai observed my condition and placed a card on my desk saying , before we talk business , you must go see this doctor . i looked at the card and saw the name dr jason hao , doctor of oriental medicine and acupuncture . after exchanging greetings , i told dr pai that i had been a subject in medical studies at universities with the best neurologists in the world . other than steroids , i was disqualified from all the medications that had been approved to combat ms . he told me about a patient with parkinson 's disease who had incurable tremors that could not be controlled by medication . he told me about another patient with mobility damage from a stroke who seemed to have been miraculously cured with dr hao 's technique . i have had 12 treatments with dr hao at his clinic in albuquerque , new mexico . not only does he put needles in my head but he spins these needles with his fingers between 200 and 400 times a minute . dr hao told me that all of the nerves in the body can be accessed in the scalp and that the spinning of the needles clears the nerve pathways that have become broken and clogged . my left foot , which has been numb for 18 years , is no longer numb . my vision has improved , my balance has returned , and i have not had vertigo in some time . every aspect of my life is better . i am working full - time again , although dr hao cautions me that ms is still my greatest enemy and that he has only relieved me of the symptoms brought on by the attacks . he cautions me to never use more than 70% of my energy in any 24-hour period and to take the prescribed herbs and follow basic nutritional guides . my wife and i look at each other on a daily basis and expect for this dream to end : this dream about a chinese doctor in albuquerque who spins acupuncture needles in my head to make the symptoms of ms go away . but every day i feel better , and my treatments with dr hao are now on a monthly basis rather than a weekly basis . twenty years of debilitating symptoms that compromised every aspect of my life reduced to a memory in 6 months ! those who knew me before last july and see me now at first just ca n't believe their own eyes . in view of my changes and my many inquiries to dr hao , i can not help but wonder : why is this particular form of acupuncture still not embraced coast to coast ? the treatments last about an hour and are relatively inexpensive and completely painless . for the tenth scalp acupuncture session charles was awake , alert , cooperative , very attentive , and gave quicker responses . motor strength increased to 5/5 in both legs , both arms , and hand grip . the patient 's finger - to - nose tests on both right and left sides returned to normal . charles got out of a chair without any challenge and ambulated with a normal gait . the patient peformed the heel - toe walking test with no problems and could stand on either his left or right leg steadily . chinese medical examination at the tenth session showed that his tongue had changed to slightly red with a thin white coating ; his pulses changed to soft on both left and right sides , and pulses in the kidney positions changed to thready . when palpating points , all sensitive points including lr-3 ( taichong ) , gb-34 ( yanglingquan ) , ub-18 ( ganshu ) , sp-9 ( yinlingquan ) , ub-23(shenshu ) , and ub-15 ( xinshu ) showed neither tenderness nor pain and soreness . after the tenth treatment , charles was able to work full - time and started to take vacations again . he reported that he enjoyed standing on one leg during his work breaks and that doing so helped him feel like a normal person . although charles returned to a normal life , he prefers to come to the clinic every 4 to 6 weeks for maintenance treatments . you are likely to wonder , what is a longtime local lawyer doing , writing about serious medical problems like multiple sclerosis ( ms ) and parkinson 's disease ? it is because i have had the rare opportunity of taking part in what i unhesitatingly tell everyone is nothing less than a miracle . what was uppermost was that 20 years earlier i had been diagnosed with multiple sclerosis ms took my brother in 1999 and has crippled one of my sisters and compromised the life of my youngest sister . i had managed to keep the disease under control and under wraps by wisely following my wife 's advice on nutrition , exercise , and a common - sense approach to ms . together we made trips to leading medical centers where i became a voluntary guinea pig for new medications . on top of that , i was tested , scanned , poked , and prodded over and [ over ] again by some of the best neurologists and medical professionals studying the disease . finally , despite all that , the symptoms were taking their toll , especially in the heat of the mesilla valley 's long summer . i realized i could no longer continue as head of the law firm i had helped to build over 3 decades . when my firm dissolved , i joined another law firm on a part - time basis to comply with limited energy and finish up so on a hot july day i awaited my first client at my new office that client was dr vittal pai , whom i 'd known for more than 20 years . i had not seen dr pai in a couple of years and always enjoyed visiting with him . dr pai , an ear , nose , and throat specialist , is a brilliant doctor known as much for his philosophical approach to life and his quick thinking and wit as he is for his substantial skills as a physician . dr pai observed my condition and placed a card on my desk saying , before we talk business , you must go see this doctor . i looked at the card and saw the name dr jason hao , doctor of oriental medicine and acupuncture . after exchanging greetings , i told dr pai that i had been a subject in medical studies at universities with the best neurologists in the world . other than steroids , i was disqualified from all the medications that had been approved to combat ms . those of you who know dr pai also know his serene smile . he told me about a patient with parkinson 's disease who had incurable tremors that could not be controlled by medication . he told me about another patient with mobility damage from a stroke who seemed to have been miraculously cured with dr hao 's technique . i have had 12 treatments with dr hao at his clinic in albuquerque , new mexico . not only does he put needles in my head but he spins these needles with his fingers between 200 and 400 times a minute . dr hao told me that all of the nerves in the body can be accessed in the scalp and that the spinning of the needles clears the nerve pathways that have become broken and clogged . after these treatments , all of my major symptoms of ms are gone . my left foot , which has been numb for 18 years , is no longer numb . my vision has improved , my balance has returned , and i have not had vertigo in some time . every aspect of my life is better . i am working full - time again , although dr hao cautions me that ms is still my greatest enemy and that he has only relieved me of the symptoms brought on by the attacks . he cautions me to never use more than 70% of my energy in any 24-hour period and to take the prescribed herbs and follow basic nutritional guides . my wife and i look at each other on a daily basis and expect for this dream to end : this dream about a chinese doctor in albuquerque who spins acupuncture needles in my head to make the symptoms of ms go away . but every day i feel better , and my treatments with dr hao are now on a monthly basis rather than a weekly basis . twenty years of debilitating symptoms that compromised every aspect of my life reduced to a memory in 6 months ! those who knew me before last july and see me now at first just ca n't believe their own eyes . in view of my changes and my many inquiries to dr hao , i can not help but wonder : why is this particular form of acupuncture still not embraced coast to coast ? scalp acupuncture has proven to have superior success in treating ms and other central nervous system damage as compared to other acupuncture modalities including acupuncture on the ear , body , and hand . it not only can improve the symptoms and the patient 's quality of life and slow and reverse the progression of physical disability but also reduce the number of relapses . patients should get acupuncture treatment as soon as possible ; the earlier the treatment , the better the prognosis . scalp acupuncture treatment for ms has had much success in reducing numbness and pain , decreasing spasms , improving weakness and paralysis of limbs , and improving balance . many patients also have reported that their bladder and bowel control , fatigue , and overall sense of well - being significantly improved after treatment . as stated above , recent studies have shown that scalp acupuncture can be a very effective modality in controlling ms . it usually relieves symptoms immediately , and sometimes in just several minutes noticeable results are achieved . the primary acupuncture areas for patients with motor problems such as paralysis , weakness of limbs , or abnormal sensations in limbs , including tingling , numbness , or pain , are the motor area , sensory area , and foot motor and sensory area . those areas should be inserted with needles and stimulated unilaterally or bilaterally , according to the patient 's manifestations . select the balance area or dizziness area of the scalp , depending on which symptom(s ) the patient exhibits . many patients have a very quick positive response in controlling urine and bowel functions when the foot motor and sensory area is stimulated . although scalp acupuncture has the fastest track record for improving symptoms , sometimes other techniques also are necessary for further improvement . regular body acupuncture , electrical stimulation , moxibustion , and chinese herbs , as well as physical therapy and massage can be combined with scalp acupuncture to speed recovery . regular acupuncture treatment has been found to have a positive therapeutic effect on the recovery of movement and reducing abnormal sensations of the hands , fingers , feet , and toes . commonly used points are gb-34 , li-3 , ki-3 , ba feng ( extra point ) for lower limbs at li-11 , li-4 , te-5 , and ba xie ( extra point ) for upper - limb work . electrical stimulation is very helpful if the practitioner has difficulty performing the needle rotation more than 200 times per minute . it is suggested that no more than two of the scalp needles be stimulated at any session so the brain does not become too confused to respond . moxibustion can enhance the therapeutic results of scalp acupuncture , especially for older or weak patients . when treating chronic progressive diseases like ms , parkinson 's , and amyotrophic lateral sclerosis , the effects are sometimes temporary . they may last for hours , days , weeks , or months , but follow - up treatments will be necessary on an ongoing basis . when treating paralysis , whether from stroke or trauma , the improvements in movement often are permanent . the practitioner should consider scalp acupuncture as the primary approach rather than as a complementary approach for patients with ms . ms is a progressive disease of the central nervous system affecting half a million people in the americas and 1.3 million people worldwide . acupuncture , however , is one of the major complementary and alternative therapies for ms . this case report shows that scalp acupuncture seems to be a more effective modality in bringing about quicker and often effective improvements to patients with ms compared to other acupuncture modalities such as acupuncture on the ear , body , and hand . chinese scalp acupuncture is also more easily accessible , less expensive , entails less risk , can yield quicker responses , and usually causes fewer side effects than some other treatments . in the west , however , scalp acupuncture , as a useful tool for the treatment of ms , is a relatively new concept . even now , it is not surprising for a western physician to claim that it is a natural remission or coincidence if a patient recovers from ms after acupuncture . it holds the potential to expand treatment options for ms in both conventional and complementary or integrative therapies . it not only can improve the patient 's symptoms and quality of life and slow and reverse the progression of physical disabilities but it can reduce the number of relapses and help patients stay in remission . although there have been many hypotheses and research reports on scalp acupuncture for central nervous system disorders and pain management in the western medical literature over the past 40 years , there is still a long way to go in uncovering the mystery of the mechanisms of scalp acupuncture . future study is needed to investigate the mechanisms underlying acupuncture 's effect on the central nervous system dysfunctions in patients with ms . if it becomes more widely used , scalp acupuncture could have a significant impact on recovery from central nervous system disorders for thousands of patients . there is , therefore , a pressing need for chinese scalp acupuncture to be studied and perfected using modern research methods so that its potential can be fully explored and applied .

chinese scalp acupuncture is a contemporary acupuncture technique with just 40 years of history . it integrates traditional chinese needling methods with western medical knowledge of the cerebral cortex and has been proven to be a very effective technique for treating multiple sclerosis ( ms ) and other central nervous system disorders . a 65-year - old male patient who had had ms for 20 years was treated with chinese scalp acupuncture . the motor area , sensory area , foot motor and sensory area , balance area , hearing and dizziness area , and tremor area were stimulated once a week for 10 weeks , then once a month for six sessions . after the 16 treatments , the patient showed remarkable improvements . he was able to stand and walk without any problems . the numbness and tingling in his limbs did not bother him anymore . he had more energy and had not experienced incontinence of urine or dizziness after the first treatment . he was able to return to work full time . at this writing , the patient has been in remission for 26 months . this case demonstrates that chinese scalp acupuncture can be a very effective treatment for patients with ms . chinese scalp acupuncture holds the potential to expand treatment options for ms in both conventional and complementary or integrative therapies . it can not only relieve symptoms , increase the patient 's quality of life , and slow and reverse the progression of physical disability but also reduce the number of relapses and help patients with multiple sclerosis to remain in remission .

INTRODUCTION CASE PRESENTATION Treatment Outcome and Follow-up Patient's Perspective DISCUSSION CONCLUSIONS

multiple sclerosis ( ms ) is a progressive disease of the central nervous system in which communication between the brain and other parts of the body is disrupted . during an ms attack , inflammation occurs in areas of the white matter of the central nervous system in random patches ; these are called plaques . myelin allows for smooth , high - speed transmission of electrochemical messages between the brain , the spinal cord , and the rest of the body . approximately 520 000 people in the americas , 630 000 in europe , 66 000 in the eastern mediterranean , 56 000 in the western pacific , 31 500 in southeast asia , 11 000 in africa , and 1.3 million people worldwide have been diagnosed with ms . it primarily affects adults , has an age of onset typically between 20 and 40 years , and is twice as common in women than in men . the most common symptoms of ms are weakness , spasticity in one or more limbs seen in 50% of ms patients followed by numbness , tingling , and fatigue ( 40% ) , partial or complete loss of vision , double or blurred vision ( 31% ) , incontinence of urine and dysfunction of the bowel ( 17.5% ) , pain ( 15% ) , cognitive or behavioral problems , and sexual dysfunction ( 10% ) . in the worst cases it is a mild disease , but in others , it can lead to permanent disability . many risk factors for ms have been identified , but no definitive cause has been found . chinese scalp acupuncture is a contemporary acupuncture technique integrating traditional chinese needling methods with western medical knowledge of the cerebral cortex ( figures 1 and 2 ) . scalp acupuncture has been proven to be a very effective technique for treating central nervous system disorders ranging from ms , strokes , parkinson 's disease , traumatic brain injury , and posttraumatic stress disorder to phantom pain and complex regional pain . the scalp somato - topic system appears to manifest the convergence of the central nervous system and the endocrine system . the scalp somatotopic system seems to operate as a miniature transmitter - receiver in direct contact with the central nervous system and endocrine system . by stimulating those reflex areas , acupuncture can have a direct effect on the cerebral cortex , cerebellum , thalamocortical circuits , thalamus , hypothalamus , and pineal body . using a small number of needles , scalp acupuncture can often produce remarkable results almost immediately , sometimes taking only several minutes to complete . over the past 20 years , charles had multiple relapses and remissions with episodes of lower extremity weakness , stiffness and muscle spasm , incontinence of urine , loss of balance , double vision , and fatigue . twelve years ago , he had a dramatic neurological decline during which he was unable to walk steadily and lost strength and sensation in his lower extremities . for the last 3 years , up to when he came to the clinic , it was more difficult for him to walk due to weakness in his legs and loss of balance . charles also complained of numbness , tingling , and spasms in his legs accompanied by incontinence of urine , double vision , poor memory and concentration , dizziness and vertigo , heat intolerance , and severe fatigue . family history revealed that a brother with ms had died in 1999 , and two sisters have been diagnosed with ms . for the first scalp acupuncture session , general and neurological examinations were carried out . motor strength was 4/5 in both legs and 5/5 in both arms and in hand grip the patient 's finger - to - nose tests on both right and left sides were abnormal . he ambulated with stiffness in both legs , was unsteady , and had a wide - based stance . the patient failed the heel - toe walking test and could not stand on one leg . chinese medical examination showed that his tongue was red and slightly purple with a thin yellow coating ; his pulse on the left side was wiry and rolling ; on the right side , it was wiry and thready , and weak pulses were also noticed in the kidney positions . primary scalp areas were motor area , sensory area , and foot motor and sensory area . secondary scalp areas were balance area , hearing and dizziness area , and tremor area ( figures 3 - 5 ) . the motor area , sensory area , and foot motor and sensory area received needles that were stimulated bilaterally . he was amazed to feel the dizziness , balance , stiffness , and weakness in his legs improve just minutes after a few needles were inserted in his scalp . he was initially very suspicious and nervous when the doctor asked him to stand up with his eyes closed . he was not only able to stand up with improved stability , but he also could walk much better . at the second treatment , charles reported that he had no more incontinence of urine and that the numbness , spasms , and weakness of both legs showed some improvement as well . , charles 's vision had significantly improved , and he no longer had double vision and heat intolerance . he also had more energy and was able to do more work at his law office . by the fifth treatment , charles reported that he was able to walk around his home and office without problems and that he could walk much longer distances . he had more energy and had not experienced incontinence of urine since the first treatment . what is a longtime local lawyer doing , writing about serious medical problems like multiple sclerosis ( ms ) and parkinson 's disease ? what was uppermost was that 20 years earlier i had been diagnosed with multiple sclerosis ms took my brother in 1999 and has crippled one of my sisters and compromised the life of my youngest sister . on top of that , i was tested , scanned , poked , and prodded over and [ over ] again by some of the best neurologists and medical professionals studying the disease . finally , despite all that , the symptoms were taking their toll , especially in the heat of the mesilla valley 's long summer . i realized i could no longer continue as head of the law firm i had helped to build over 3 decades . when my firm dissolved , i joined another law firm on a part - time basis to comply with limited energy and finish up so on a hot july day i awaited my first client at my new office that client was dr vittal pai , whom i 'd known for more than 20 years . i had not seen dr pai in a couple of years and always enjoyed visiting with him . dr pai , an ear , nose , and throat specialist , is a brilliant doctor known as much for his philosophical approach to life and his quick thinking and wit as he is for his substantial skills as a physician . he told me about a patient with parkinson 's disease who had incurable tremors that could not be controlled by medication . not only does he put needles in my head but he spins these needles with his fingers between 200 and 400 times a minute . dr hao told me that all of the nerves in the body can be accessed in the scalp and that the spinning of the needles clears the nerve pathways that have become broken and clogged . after these treatments , all of my major symptoms of ms are gone . my left foot , which has been numb for 18 years , is no longer numb . i am working full - time again , although dr hao cautions me that ms is still my greatest enemy and that he has only relieved me of the symptoms brought on by the attacks . twenty years of debilitating symptoms that compromised every aspect of my life reduced to a memory in 6 months ! in view of my changes and my many inquiries to dr hao , i can not help but wonder : why is this particular form of acupuncture still not embraced coast to coast ? for the tenth scalp acupuncture session , charles was awake , alert , cooperative , very attentive , and gave quicker responses . motor strength increased to 5/5 in both legs , both arms , and hand grip . the patient 's finger - to - nose tests on both right and left sides returned to normal . when palpating points , all sensitive points including lr-3 ( taichong ) , gb-34 ( yanglingquan ) , ub-18 ( ganshu ) , sp-9 ( yinlingquan ) , ub-23(shenshu ) , and ub-15 ( xinshu ) showed neither tenderness nor pain and soreness . after the tenth treatment , charles was able to work full - time and started to take vacations again . although charles returned to a normal life , he prefers to come to the clinic every 4 to 6 weeks for maintenance treatments . primary scalp areas were motor area , sensory area , and foot motor and sensory area . secondary scalp areas were balance area , hearing and dizziness area , and tremor area ( figures 3 - 5 ) . the motor area , sensory area , and foot motor and sensory area received needles that were stimulated bilaterally . he was amazed to feel the dizziness , balance , stiffness , and weakness in his legs improve just minutes after a few needles were inserted in his scalp . he was initially very suspicious and nervous when the doctor asked him to stand up with his eyes closed . he was not only able to stand up with improved stability , but he also could walk much better . at the second treatment , charles reported that he had no more incontinence of urine and that the numbness , spasms , and weakness of both legs showed some improvement as well . he also had more energy and was able to do more work at his law office . by the fifth treatment , charles reported that he was able to walk around his home and office without problems and that he could walk much longer distances . he had more energy and had not experienced incontinence of urine since the first treatment . you are likely to wonder , what is a longtime local lawyer doing , writing about serious medical problems like multiple sclerosis ( ms ) and parkinson 's disease ? what was uppermost was that 20 years earlier i had been diagnosed with multiple sclerosis ms took my brother in 1999 and has crippled one of my sisters and compromised the life of my youngest sister . on top of that , i was tested , scanned , poked , and prodded over and [ over ] again by some of the best neurologists and medical professionals studying the disease . finally , despite all that , the symptoms were taking their toll , especially in the heat of the mesilla valley 's long summer . when my firm dissolved , i joined another law firm on a part - time basis to comply with limited energy and finish up so on a hot july day i awaited my first client at my new office that client was dr vittal pai , whom i 'd known for more than 20 years . dr pai , an ear , nose , and throat specialist , is a brilliant doctor known as much for his philosophical approach to life and his quick thinking and wit as he is for his substantial skills as a physician . he told me about a patient with parkinson 's disease who had incurable tremors that could not be controlled by medication . dr hao told me that all of the nerves in the body can be accessed in the scalp and that the spinning of the needles clears the nerve pathways that have become broken and clogged . my left foot , which has been numb for 18 years , is no longer numb . my vision has improved , my balance has returned , and i have not had vertigo in some time . i am working full - time again , although dr hao cautions me that ms is still my greatest enemy and that he has only relieved me of the symptoms brought on by the attacks . in view of my changes and my many inquiries to dr hao , i can not help but wonder : why is this particular form of acupuncture still not embraced coast to coast ? for the tenth scalp acupuncture session charles was awake , alert , cooperative , very attentive , and gave quicker responses . motor strength increased to 5/5 in both legs , both arms , and hand grip . the patient 's finger - to - nose tests on both right and left sides returned to normal . charles got out of a chair without any challenge and ambulated with a normal gait . the patient peformed the heel - toe walking test with no problems and could stand on either his left or right leg steadily . chinese medical examination at the tenth session showed that his tongue had changed to slightly red with a thin white coating ; his pulses changed to soft on both left and right sides , and pulses in the kidney positions changed to thready . when palpating points , all sensitive points including lr-3 ( taichong ) , gb-34 ( yanglingquan ) , ub-18 ( ganshu ) , sp-9 ( yinlingquan ) , ub-23(shenshu ) , and ub-15 ( xinshu ) showed neither tenderness nor pain and soreness . after the tenth treatment , charles was able to work full - time and started to take vacations again . although charles returned to a normal life , he prefers to come to the clinic every 4 to 6 weeks for maintenance treatments . you are likely to wonder , what is a longtime local lawyer doing , writing about serious medical problems like multiple sclerosis ( ms ) and parkinson 's disease ? what was uppermost was that 20 years earlier i had been diagnosed with multiple sclerosis ms took my brother in 1999 and has crippled one of my sisters and compromised the life of my youngest sister . on top of that , i was tested , scanned , poked , and prodded over and [ over ] again by some of the best neurologists and medical professionals studying the disease . finally , despite all that , the symptoms were taking their toll , especially in the heat of the mesilla valley 's long summer . i realized i could no longer continue as head of the law firm i had helped to build over 3 decades . when my firm dissolved , i joined another law firm on a part - time basis to comply with limited energy and finish up so on a hot july day i awaited my first client at my new office that client was dr vittal pai , whom i 'd known for more than 20 years . i had not seen dr pai in a couple of years and always enjoyed visiting with him . dr pai , an ear , nose , and throat specialist , is a brilliant doctor known as much for his philosophical approach to life and his quick thinking and wit as he is for his substantial skills as a physician . not only does he put needles in my head but he spins these needles with his fingers between 200 and 400 times a minute . dr hao told me that all of the nerves in the body can be accessed in the scalp and that the spinning of the needles clears the nerve pathways that have become broken and clogged . after these treatments , all of my major symptoms of ms are gone . my left foot , which has been numb for 18 years , is no longer numb . i am working full - time again , although dr hao cautions me that ms is still my greatest enemy and that he has only relieved me of the symptoms brought on by the attacks . but every day i feel better , and my treatments with dr hao are now on a monthly basis rather than a weekly basis . in view of my changes and my many inquiries to dr hao , i can not help but wonder : why is this particular form of acupuncture still not embraced coast to coast ? scalp acupuncture has proven to have superior success in treating ms and other central nervous system damage as compared to other acupuncture modalities including acupuncture on the ear , body , and hand . it not only can improve the symptoms and the patient 's quality of life and slow and reverse the progression of physical disability but also reduce the number of relapses . scalp acupuncture treatment for ms has had much success in reducing numbness and pain , decreasing spasms , improving weakness and paralysis of limbs , and improving balance . many patients also have reported that their bladder and bowel control , fatigue , and overall sense of well - being significantly improved after treatment . as stated above , recent studies have shown that scalp acupuncture can be a very effective modality in controlling ms . it usually relieves symptoms immediately , and sometimes in just several minutes noticeable results are achieved . the primary acupuncture areas for patients with motor problems such as paralysis , weakness of limbs , or abnormal sensations in limbs , including tingling , numbness , or pain , are the motor area , sensory area , and foot motor and sensory area . those areas should be inserted with needles and stimulated unilaterally or bilaterally , according to the patient 's manifestations . select the balance area or dizziness area of the scalp , depending on which symptom(s ) the patient exhibits . many patients have a very quick positive response in controlling urine and bowel functions when the foot motor and sensory area is stimulated . although scalp acupuncture has the fastest track record for improving symptoms , sometimes other techniques also are necessary for further improvement . regular body acupuncture , electrical stimulation , moxibustion , and chinese herbs , as well as physical therapy and massage can be combined with scalp acupuncture to speed recovery . regular acupuncture treatment has been found to have a positive therapeutic effect on the recovery of movement and reducing abnormal sensations of the hands , fingers , feet , and toes . commonly used points are gb-34 , li-3 , ki-3 , ba feng ( extra point ) for lower limbs at li-11 , li-4 , te-5 , and ba xie ( extra point ) for upper - limb work . it is suggested that no more than two of the scalp needles be stimulated at any session so the brain does not become too confused to respond . moxibustion can enhance the therapeutic results of scalp acupuncture , especially for older or weak patients . when treating chronic progressive diseases like ms , parkinson 's , and amyotrophic lateral sclerosis , the effects are sometimes temporary . they may last for hours , days , weeks , or months , but follow - up treatments will be necessary on an ongoing basis . when treating paralysis , whether from stroke or trauma , the improvements in movement often are permanent . the practitioner should consider scalp acupuncture as the primary approach rather than as a complementary approach for patients with ms . ms is a progressive disease of the central nervous system affecting half a million people in the americas and 1.3 million people worldwide . acupuncture , however , is one of the major complementary and alternative therapies for ms . this case report shows that scalp acupuncture seems to be a more effective modality in bringing about quicker and often effective improvements to patients with ms compared to other acupuncture modalities such as acupuncture on the ear , body , and hand . chinese scalp acupuncture is also more easily accessible , less expensive , entails less risk , can yield quicker responses , and usually causes fewer side effects than some other treatments . in the west , however , scalp acupuncture , as a useful tool for the treatment of ms , is a relatively new concept . it holds the potential to expand treatment options for ms in both conventional and complementary or integrative therapies . it not only can improve the patient 's symptoms and quality of life and slow and reverse the progression of physical disabilities but it can reduce the number of relapses and help patients stay in remission . although there have been many hypotheses and research reports on scalp acupuncture for central nervous system disorders and pain management in the western medical literature over the past 40 years , there is still a long way to go in uncovering the mystery of the mechanisms of scalp acupuncture . future study is needed to investigate the mechanisms underlying acupuncture 's effect on the central nervous system dysfunctions in patients with ms . if it becomes more widely used , scalp acupuncture could have a significant impact on recovery from central nervous system disorders for thousands of patients . there is , therefore , a pressing need for chinese scalp acupuncture to be studied and perfected using modern research methods so that its potential can be fully explored and applied .

insulin receptor is a tetramer protein composed by two extracellular subunits and two transmembrane subunits . the subunits have a binding site to insulin while the subunits contain an intrinsic tyrosine kinase activity towards intracellular side . insulin binding to the subunit leads to a conformational change and activation of the subunit , resulting in tyrosyl autophosphorylation of the insulin receptor . after being activated and phosphorylated , several intracellular docking proteins bind to the insulin receptor and are also tyrosyl phosphorylated , including insulin receptor substrates 1 and 2 ( irs-1 and irs-2 ) [ 1 , 2 ] , src homology collagen ( shc ) , and associated protein substrate ( aps ) . these proteins activate several signaling pathways involved in the regulation of important cellular events such as glucose uptake and metabolism , protein synthesis , gene expression , cell survival , growth , development , and differentiation [ 46 ] . irs proteins are phosphorylated on various tyrosine residues of the c - terminal region , generating specific sites for binding of proteins containing src homoly-2 ( sh2 ) domains , including phosphatidylinositol-3 kinase ( pi-3k ) , nck , and grb-2 . pi-3k is composed by a catalytic subunit ( p110 ) and a regulatory subunit ( p85 ) . this kinase is an important signaling molecule , mediating metabolic effects of the insulin . binding of p85 subunit to phosphorylated tyrosine residues of irs proteins leads to activation of the catalytic activity of p110 subunit and subsequent increase in the generation of phosphatidylinositol 3,4-bisphosphate ( pip2 ) and phosphatidylinositol 3,4,5-trisphosphate ( pip3 ) content . downstream proteins from pi3 k pathway comprehend several serine / threonine kinases , for example , phosphoinositide - dependent protein kinase-1 ( pdk-1 ) , protein kinase b ( pkb , also known as akt ) , protein kinase c ( pkc ) , p70 s6 kinase ( p70s6 k ) , and glycogen synthase kinase-3 ( gsk-3 ) . these kinases are involved in the most important biological effects induced by insulin , such as translocation of glucose transporter-4 ( glut-4 ) from intracellular vesicles to plasma membrane , glycogen and protein synthesis , antiapoptotic effects , and gene expression ( figure 1 ) [ 711 ] . other signaling pathways involved in the glucose uptake induced by insulin start with the recruitment of aps to the activated insulin receptor and subsequent association and tyrosine phosphorylation of cbl , which interacts with cbl associated protein ( cap ) through an sh3 domain and with flotillin , a constituent of lipid raft , through a sorbin domain . the complex crkii / c3 g then binds to the phosphorylated tyrosine residues of cbl , activating the c3 g activity that exchanges gdp for gtp of tc10 , a small g - protein that belongs to the rho family . after being activated , mitogen - activated protein kinase ( mapk ) cascade starts with the association of shc to insulin receptor , binding of grb-2 to shc or to irs-1 , and formation of the grb-2/sos ( son of sevenless ) in the plasma membrane [ 1719 ] . this complex leads to the activation of c - ras and raf , starting the mapk cascade . mapk pathway is involved in the differentiation , cell growth , and development induced by insulin , as well as some metabolic effects , as glycogen synthesis and glut-4 translocation to plasma membrane ( figure 1 ) [ 2224 ] . however , this cascade is not enough or even required to this later effect . disturbances in several proteins involved in the insulin signaling pathways have been found in different conditions of insulin resistance , including obesity , type 2 diabetes mellitus , metabolic syndrome , cardiovascular diseases , inflammatory disorders , and cancer [ 2628 ] . here , we will discuss possible mechanisms involved in the development of insulin resistance related to inflammatory processes . insulin resistance occurs when the insulin - sensitive tissues , mainly skeletal muscle , adipose tissue , and liver , lose the ability to respond properly to the hormone [ 29 , 30 ] . it is associated with several chronic diseases , especially those linked to obesity , such as type 2 diabetes mellitus , metabolic syndrome , dyslipidemias , cardiovascular diseases , cancer , and neurodegenerative diseases [ 3133 ] . however , the precise mechanisms involved in insulin resistance are not fully understood yet [ 3437 ] . several factors have been proposed to participate in the development of insulin resistance , including increased plasma - free fatty acid level , subclinical chronic inflammation , oxidative and nitrative stress , altered gene expression , and mitochondrial dysfunction [ 29 , 37 ] . since free fatty acids are elevated in obesity and related diseases , these metabolites have been proposed to be responsible for the impairment in the insulin action , but the mechanisms are not completely known yet [ 38 , 39 ] . high availability of fatty acids , specially long - chain saturated fatty acids , results in the establishment of insulin resistance in liver , skeletal muscle , and adipose tissue [ 40 , 41 ] . various hypothesis have been proposed to explain the insulin resistance induced by saturated fatty acids , including randle cycle , oxidative stress , modulation of gene transcription , inflammation , accumulation of intracellular lipid derivatives ( diacylglycerol and ceramides ) , and mitochondrial dysfunction [ 4247 ] ( for review , see martins et al . ) . a chronic state of inflammation in the insulin responsive tissues is a major contributor to insulin resistance in obesity and related diseases . thus , a crosstalking between inflammation and insulin resistance has been suggested by several authors . however , the precise mechanism as well as the mediators involved in this interaction is not completely defined yet . in this paper , we discuss how inflammatory signaling pathways impair insulin signaling ( see below ) . intracellular redox balance is a finely regulated process that involves several generating pathways and degrading systems . physiologically , ros participate in important biological responses , but accumulation of these molecules leads to oxidative stress condition . ros are highly oxidant molecules that can oxidize various intracellular components , including membrane phospholipids , proteins , and dna [ 49 , 50 ] . usually , these reactions cause cellular damage , reducing the function of oxidized biomolecules . in insulin resistance , increased ros production and/or decreased ros degradation is observed , leading to an oxidative stress condition and activation of signaling pathways related to stress . there is evidence that oxidative stress is also involved in muscle disorders , contributing to the insulin resistance process . transgenic mice expressing human ubiquitin protein e3 ligase , a protein that binds and promotes degradation of superoxide dismutase-1 , resulting in reduced superoxide degradation and consequently oxidative stress , present muscle dysfunction ( atrophy and sclerosis ) . activation of signaling pathways to stress has been suggested to participate in the development of insulin resistance by impairing the signaling by this hormone . several serine / threonine kinases activated by oxidative stress pathways , including jnk , pkc , gsk-3 , nf - kb , and p38 mapk , have been suggested to impair insulin signaling pathways [ 53 , 54 ] , as described below . for example , expression of genes involved in lipid and glucose metabolism , insulin signaling , inflammation , redox balance , and mitochondrial function is modified , suggesting that these processes participate in the pathophysiology of insulin resistance [ 5557 ] . disturbed mitochondrial function has been suggested to have a central role in these alterations , since this organelle participates in all these processes ( for review , see martins et al . ) . inflammation is a coordinated process evoked by the tissues in response to noxious stimuli or conditions including the presence of infection , tissue injury , or malfunction . the inflammatory response is activated by molecules released by microorganisms including microbial associated molecular patterns ( mamps ) such as lipopolysaccharide , flagellin , and peptidoglycans or produced / released by host cells including intracellular components , the so - called damage - associated molecular patterns ( damps ) , of which , hmgb1 , dna , and nucleotides are part . these inducers of inflammation bind to their respective receptors and activate biological responses by the resident cells , mainly , macrophages and mast cells . these cells act directly or indirectly on the vasculature and on leukocytes to induce , among other effects , the migration of leukocytes and extravasations of plasma proteins to the tissues . examples of proteins with this function include the receptors of the toll - like ( tlrs ) family , the c - type lectin receptors , the purinergic and advanced glycation endproductsreceptors ( rage ) , and the intracellular nucleotide oligomerization domain ( nod ) , and retinoic acid - inducible gene ( rig)-i - like receptors ( rlrs ) . after recognizing their ligands , several downstream pathways including c - jun nh(2)-terminal kinase ( jnk ) and ib kinase complex ( ikk ) are activated , resulting in changes on transcription factors activity and expression of proteins such as cytokines , enzymes , chemokines , adhesion molecules , and amplification of the inflammatory response . the activation of the inflammatory pathways described above is a hallmark of obesity , and it has been associated with the development of insulin resistance , atherosclerosis , and other tissue dysfunctions that are secondary to fat accumulation . an increased production of inflammatory mediators and activation of inflammatory pathways in several tissues including adipose tissue ( at ) , liver , pancreas , skeletal muscle , and hypothalamus are present in obese individuals and define a subclinical inflammatory process also known as meta - inflammation , little information is available regarding the inducers and sensors involved in obesity - associated inflammation . in this respect , some hypotheses to explain the initial activation / recruitment of leukocyte to the tissues ( mainly , at ) have been proposed . the release of damps by necrotic adipose cells , the increase in the flux of nonesterified fatty acids ( increased rates of lipolysis ) , the reduction in the oxygen tension ( hypoxia ) leading to activation of hypoxia - induced factor ( hif)-1 , which controls the expression of proinflammatory proteins , and the production of chemokines by adipose cells have been suggested to play a role in the initiation of inflammatory process . despite several advances in the field , the initial events involved in the beginning of inflammation in the at and the complex interactions between them are not clearly understood , and new components are continuously described and added to the puzzle such as leukotrienes and the apoptosis inhibitor of macrophage ( aim ) [ 62 , 63 ] . the release of fatty acids , through interaction with tlr4 , induces the expression of chemokines that lead to macrophage recruitment . the obesity - induced inflammation is associated with activation of resident cells such as kupffer cells in the liver and leukocyte infiltration and shift in the polarized state of tissue / recruited leukocytes . these changes are better characterized in the adipose tissue , in which an increased infiltration by proinflammatory m1-type macrophages ( classical macrophages ) , th1 , th17 and cd8 t cells and a reduction in the content of less inflammatory cells such as m2-macrophage , t reg , and th2 cells were demonstrated [ 6568 ] . the overproduction of inflammatory mediators by the infiltrating cells together with changes in adipokines production and nefa release by at contribute to the tissue dysfunctions observed in obesity , as discussed below . several chronic diseases are characterized by increased inflammatory process and insulin resistance , such as obesity , type 2 diabetes mellitus , metabolic syndrome , cardiovascular diseases , and cancer . for example , various studies suggest the involvement of some inflammatory factors in the development of insulin resistance , including cytokines ( tnf- , il-1 , il-6 ) , ros , and rns ( figure 2 ) . these factors lead to the activation of signaling pathways that ultimately impair insulin signaling ( see below ) . in addition , other factors involved in inflammatory processes also can impair insulin sensitivity , particularly lipopolysaccharides ( lpss ) and environmental stress ( hypoxia , nutrients , and ph ) ( figure 2 ) . it has been demonstrated that fatty acids , specially saturated fatty acids , are able to activate tlr-4 in skeletal muscle cells , resulting in increased activity of ikk and jnk . the first kinase degrades the inhibitor of b ( ib ) , resulting in the nuclear factor-b ( nfb ) release and migration to the cell nucleus , where it induces the transcription of proinflammatory genes . the second kinase activates members of the signal transducers and activators of transcription ( stat ) family , which are involved in several biological effects , such as expression of genes related to inflammation , apoptosis , differentiation , growth , morphogenesis , migration , and proliferation . both jnk and ikk have been proposed to be the mediators of insulin resistance induced by saturated fatty acids ( figure 2 ) . it has been shown that these kinases phosphorylate serine residues on irs proteins , blocking irs phosphorylation on tyrosine residues by the activated insulin receptor and consequently inhibiting insulin effects . moreover , phosphorylation on serine / threonine residues also increases irs protein degradation , contributing to the establishment of insulin resistance [ 7173 ] . nonfunctional tlr-4 expression protects mice from insulin resistance and inflammation induced by high - fat diet , and tlr-4 gene silencing by small interference of rna reduces inflammation in acute lung injury induced by lipopolysaccharide . obese and type 2 diabetic animals are prevented from insulin resistance and inflammation by specific inhibitors or gene mutation ( knockout or nonfunctional gene ) of ikk or jnk [ 7678 ] . these metabolites have been linked to the establishment of inflammatory process , by modulating several signaling pathways related to inflammation . for example , fatty acids can directly activate toll - like receptors ( tlrs ) , g - protein coupled receptors ( gpcrs ) , and tumor necrosis factor- ( tnf- ) receptor as well as modulate inflammatory signaling pathways involved in the increase in cytokine secretion ( tnf- , il-1 , and il-6 ) [ 60 , 79 , 80 ] , oxidative and nitrative stress , mitochondrial dysfunction , endoplasmic reticulum ( er ) stress , and proinflammatory gene expression . alterations in the expression of genes and proteins involved in the inflammatory process are clearly associated with insulin resistance and several metabolic abnormalities , including mitochondrial dysfunction , decreased fat oxidation , increased ectopic lipid storage , and impaired insulin signaling pathways ( figure 2 ) . proinflammatory cytokines were also involved in the reduction of mitochondrial function [ 79 , 82 ] . palmitate - stimulated il-1 production in macrophages occurs via nlrp3-asc inflammasome and participates in the mitochondrial dysfunction induced by this fatty acid . this dysfunction is also observed when the cells are exposed to other cytokines , such as tnf- or il-6 [ 83 , 84 ] . therapies aimed at neutralizing proinflammatory cytokines such as tnf- and il-1 , such as the monoclonal antibody infliximab and canakinumab , respectively , have been under investigation in the treatment of type 2 diabetic patients . considering anti - tnf antibodies , the results are disappointing as many clinical trials in type 2 diabetic patients have failed to demonstrate an effect of tnf neutralization on insulin sensitivity [ 8589 ] . on the other hand , in patients with high grade inflammatory diseases such as rheumatoid arthritis and ankylosing spondylitis , anti - tnf therapy has been successfully associated with reduction in insulin resistance and metabolic syndrome components [ 9095 ] . the molecular mechanisms of tnf- blockade on insulin signaling were related to reduction in irs-1 serine phosphorylation and increase in akt phosphorylation in peripheral mononuclear cells from rheumatoid arthritis patients . the long - term effects of anti - il-1 therapy are now examined in the large phase iii clinical trial cantos ( canakinumab anti - inflammatory thrombosis outcomes study ) . the study included and treated 17,200 patients with various doses of anti - il-1 antibody every 3 months and followed up over 4 years . the primary endpoint of the cantos study will be cardiovascular events , and secondary endpoints include new onset type 2 diabetes and diabetes - specific markers . such large and long - term trials could provide a novel cytokine - based therapy for the secondary prevention of new - onset diabetes as well as confirm the autoinflammatory nature of metabolic disorders . however , identification of pharmacologically potent and selective small molecule jnk inhibitors has been limited . compound a , a reversible atp - competitive aminopyridine inhibitor of jnk , was developed by pfizer . testing this compound in obese mice decreased their body weight as well as blood glucose and triglyceride concentrations and increased insulin sensitivity to levels comparable to those in lean control mice . a substrate competitive inhibitor of jnk , bi-78d3 , has also been shown to restore insulin sensitivity in a murine model of type 2 diabetes after a single dose . a more recent drug discovery is the compound 19 , a potent and selective dual substrate and atp - competitive jnk bidentate inhibitor . glucose intolerant noncnzo10/ltj mice were injected intraperitoneally daily for four days with 25 mg / kg 19 , and this compound was remarkably effective in restoring normoglycemia without inducing hypoglycemia compared to the vehicle control . these studies demonstrate that inhibition of jnk is an effective strategy to ameliorate insulin resistance . however clinical trials are needed to test these compounds in humans and show their efficacy and long - term toxicity . g - protein coupled receptors ( gpcrs ) constitute a family of membrane proteins characterized by a common motif , the seven transmembrane domains . ligand binding to these receptors leads to conformational changes in the receptor and activation of intracellular guanine nucleotide - binding proteins ( g - proteins ) . several enzymes including adenylyl and guanylyl cyclases , phospholipases a2 and c , phosphodiesterases and phosphatidylinositol 3-kinases ( pi3ks ) and other intracellular transduction cascades are triggered downstream to these receptors . these pathways affect both the insulin signaling and the inflammatory proteins expression ( figure 3 ) . several of these receptors have been shown to regulate insulin secretion and tissue sensibility to this hormone , becoming potential targets for intervention in conditions of insulin resistance / deficiency . in this paper , we will briefly discuss a class of gpcrs that share in common the fact of being activated by fatty acids ( fas - gpcrs ) . other gpcrs implicated in the glucose metabolism and regulation of inflammation such as glp-1 , glucose - dependent insulinotropic polypeptide ( gip ) , the bile acid ( tgr5 ) , cholecystokinin , the cannabinoid receptors ( cbs ) , and muscarinic receptors are beyond the focus of the present paper and are discussed elsewhere . proteins that regulate gpcrs signaling such as gpcr kinases and arrestins , which are implicated in the control of food intake , regulation of insulin action , inflammation , adipogenesis , and other processes that are associated with weight gain and development of insulin resistance , are the focus of recent reviews [ 100 , 101 ] , so they are not discussed here . the fas - gpcrs receptors , which include the gprs 40 , 41 , 43 , 84 , 119 , and 120 , present distinct ligand specificity and tissue distribution . regarding their participation in the glucose metabolism , it has been demonstrated that their activation ( at least , gpr40 and gpr119 ) directly stimulates insulin secretion by -cells and protects these cells from gluco- and lipotoxicity ( gpr40 ) [ 103 , 104 ] . activation of fas - gpcrs induces also the release of gut - derived hormones including glucagon - like peptide-1 ( glp-1 ) and glucose - dependent insulinotropic polypeptide ( gip ) [ 105109 ] . these latter two - gut - derived hormones not only modulate the gastrointestinal functions such as motility , but also the insulin secretion and food intake . in addition to these effects , fas - gpcrs , mainly , gpr43 , 84 , and 120 , present relevant effects in the inflammatory cell activation . in this sense , activation of gpr120 and -arrestin 2 by n-3 fatty acids ( docosahexaenoic and -linolenic acids ) attenuates the production of tnf- , il-6 , and macrophage chemoattractant protein-1 ( mcp-1 , also known as ccl2 ) . this anti - inflammatory effect seems to be important for the beneficial action of these fatty acids in the model of obesity induced by high - fat diet , preventing development of glucose intolerance , insulin resistance , and obesity . altogether the findings herein discussed highlight the importance of these receptors for glucose homeostasis , control of inflammatory cells activation , and food intake , processes that are linked through complexes interaction , which are not completely understood . histone deacetylase ( hdac ) is a family of enzymes that together with the histone acetyltransferases ( hats ) controls the degree of protein acetylation . inhibition of hdac activity by different compounds ( e.g. , short chain fatty acids such as butyrate , valproic acid , trichostatin a , and other compounds ) increases the acetylation of histone and nonhistone proteins including nfb , myod , p53 , and n - fat and , consequently , affects gene expression and proteins activities leading to changes in different aspects of cell biology including cell motility , proliferation , differentiation , and apoptosis . in addition to their well - known anti - inflammatory effects , other recent evidence has been obtained , which together strongly indicates hdac as a target for novel therapies in insulin resistance and diabetes.histone hyperacetylation has been associated with an increase in insulin expression and protection of -cells against cytokine - induced apoptosis , as reviewed by christensen et al . .the isoforms 4 , 5 , and 9 of hdac are associated with the development of and cells of the pancreas .oral administration of hdaci ( itf2357 ) reduced -cells toxicity associated with streptozotocin administration . additionally , the authors also showed that this hdaci protected islets from cytokine - induced toxicity and reduced production of no and chemokines in islets ; administration of sodium butyrate ( diet supplementation or oral tributyrin ( a prodrug of butyrate ) ) to high - fat fed mice attenuated body weight gain , improved lipid and glucose metabolism parameters , and inhibited the development of obesity - associated changes including activation of inflammatory pathways and hepatic steatosis [ 115 , 116 ] . recently , it has been suggested that this effect of butyrate involves inhibition of hdac 3 activity , an effect that leads to activation of ppar- and expression of fgf21 , which stimulates lipid oxidation , triglyceride clearance , and energy expenditure ; hdac 6 knockout mice are protected from hyperglycemia , glucose intolerance , and insulin resistance secondary to chronic corticoid administration ; hdaci increases the number of t regulatory cells and their suppressive function , an effect that may be important in the context of adipose tissue inflammation [ 119 , 120 ] . histone hyperacetylation has been associated with an increase in insulin expression and protection of -cells against cytokine - induced apoptosis , as reviewed by christensen et al . . the isoforms 4 , 5 , and 9 of hdac are associated with the development of and cells of the pancreas . additionally , the authors also showed that this hdaci protected islets from cytokine - induced toxicity and reduced production of no and chemokines in islets ; administration of sodium butyrate ( diet supplementation or oral tributyrin ( a prodrug of butyrate ) ) to high - fat fed mice attenuated body weight gain , improved lipid and glucose metabolism parameters , and inhibited the development of obesity - associated changes including activation of inflammatory pathways and hepatic steatosis [ 115 , 116 ] . recently , it has been suggested that this effect of butyrate involves inhibition of hdac 3 activity , an effect that leads to activation of ppar- and expression of fgf21 , which stimulates lipid oxidation , triglyceride clearance , and energy expenditure ; hdac 6 knockout mice are protected from hyperglycemia , glucose intolerance , and insulin resistance secondary to chronic corticoid administration ; hdaci increases the number of t regulatory cells and their suppressive function , an effect that may be important in the context of adipose tissue inflammation [ 119 , 120 ] . ppar- receptor activation has been shown to have significant effects on increasing insulin sensitivity in fat and muscle cells . it improves glucose metabolism and reduces inflammation ( figure 3 ) and has a crucial role in adipocyte differentiation . ppar- is a nuclear receptor that acts as a transcription factor upon activation , by regulating the transcription and expression of specific genes such as adipokines . several studies have demonstrated the anti - inflammatory activities of ppar-. inhibition of ppar- function by inflammatory cytokines may contribute to pathogenesis of many diseases such as insulin resistance , atherosclerosis , inflammation , and cancer cachexia [ 122124 ] . activation of serine kinases including ikk , erk , jnk , and p38 may be involved in the tnf regulation of ppar- ( figure 3 ) ( reviewed in ) . ikk acts through at least two mechanisms : inhibition of ppar- expression and activation of ppar- corepressor . in macrophages , where ppar gamma is also expressed , it inhibits tlr and ifn- mediated inflammatory responses . in obesity , macrophages invade adipose tissue promoting the inflammation characteristic of insulin resistance . therefore , macrophage ppar- function gained considerable pharmacological interest . diet - induced obesity influences the state of adipose tissue macrophages from an m2-polarized state ( that protects adipocytes from inflammation ) to an m1 proinflammatory state . studies have demonstrated that this obesity - induced phenotypic alteration of macrophage polarization is orchestrated by ppar- . these researchers demonstrated that ppar- is required for the maturation of alternatively activated macrophages ( m2 macrophages ) by using mice with specific macrophage deletion of ppar-. prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue and other tissues , leading to metabolic abnormalities such as insulin resistance . . showed that ppar- is involved with adipose tissue - specific lymphocyte accumulation and activation , leading to cell differentiation . t regulatory cells ( tregs ) are a small subset of t lymphocytes , normally constituting only 520% of the cd4 compartment . these cells are thought to be one of the most critical defenses against excessive immune responses , avoiding autoimmunity , allergy , inflammation , infection , and tumorigenesis [ 132 , 133 ] . typically , tregs control other t cell populations , but can also influence the activities of innate immune system cells . treg cells are characterized by high - level expression of the forkhead / winged - helix transcription factor , foxp3 . cipolletta et al . have demonstrated that ppar- collaborates with foxp3 to impose on naive cd4 t cells the characteristic of visceral adipose tissue treg cells . in fact , feuerer et al . demonstrated that treg cells with a unique phenotype were highly enriched in the abdominal fat of normal mice , but were specifically reduced at this tissue in an insulin - resistant model of obesity . a recent study highlighted the negative effect of leptin on the proliferative capacity of treg . in fact , it is well demonstrated that obese subjects present low treg cell number and adiponectin production and high leptin secretion . tlrs are transmembrane receptors that play a critical role in the detection of microbial infection and in the induction of inflammatory and immune responses against conserved microbial structures , called pathogen - associated molecular patterns . each member of tlr family recognizes a specific pathogen component which , upon activation , triggers a signaling cascade leading to cytokine production and adaptive immune response . among the tlrs , tlr2 and tlr4 play a critical role in the pathogenesis of insulin resistance , diabetes , and atherosclerosis in both clinical and experimental conditions [ 138 , 139 ] . c3h / hej mice with a mutation in tlr4 are protected against the development of high fat diet - induced obesity . in addition , these mice demonstrate decreased adiposity , increased oxygen consumption , a decreased respiratory exchange ratio , improved insulin sensitivity , and enhanced insulin - signaling capacity in adipose tissue , muscle , and liver . moreover , in all these tissues , control mice fed a high - fat diet showed an increase in ikappab kinase complex and c - jun nh(2)-terminal kinase activity , which is prevented in c3h / hej mice . studies in mice demonstrate that tlr-2 and tlr-4 activation and cytokine production stimulated by these receptors lead to the development of diabetes ( figure 3 ) [ 140 , 141 ] . more recently , tlr4 has been indicated as a molecular link between free fatty acids , inflammation , and the innate immune system . studied tlr2 and tlr4 mrna and protein expression , their ligands , and intracellular signaling in monocytes of recently diagnosed type 2 diabetic patients and observed that there is significant elevation of tlr2 and tlr4 protein , mrna , endogenous ligands , and cofactors which , together with hyperglycemia , contribute to the proinflammatory state of type 2 diabetes . koopet et al . observed that tlr activation promotes upregulation of il-6 and mcp-1 release in isolated human adipocytes via specific activation of erk . tlr-4 deficient mice had also markedly lower circulating concentrations of mcp-1 and much less nf - kb protein in nuclear extracts prepared from adipose tissue . in contrast , tlr-4 deficiency did not attenuate the induction of tumor necrosis factor - alpha ( tnf- ) or interleukin-6 ( il-6 ) expression in adipose tissue promoted by diet with high saturated fatty acids . nowadays , based on several studies it is clear that tlr4 inhibition is a pharmacologic tool to avoid inflammation in insulin resistance patients . arachidonic acid ( n-6 fatty acid ) serves as precursor of immune - active lipid mediators known as eicosanoids . classes of eicosanoids include lipoxins , leukotrienes , and pgs , and their effects on the immune system have been extensively explored and reviewed . , resolvins are new mediators generated from n-3 fatty acids docosahexaenoic acid ( dha , c22:6n-3 ) and eicosapentaenoic ( epa , c20 : 5n-3 ) identified first in resolving inflammatory exudates and in tissues enriched with dha . the names resolvin ( resolution phase interaction products ) and docosatrienes were given because these bioactive compounds demonstrate potent anti - inflammatory and immunoregulatory actions ( figure 3 ) . these mediators prevented neutrophil entry to inflammation sites and cytokine production and reduced exudates in rats with experimental peritonitis . the compounds derived from epa carrying potent biological actions are named the e series and are denoted as resolvins of the e series . those synthesized from dha are resolvins of the 17s - d series that have immunoregulatory and neuroprotective actions . in addition , aspirin treatment triggers the formation of 15-epimeric lipoxins , termed aspirin - triggered lipoxins , that also play a role in resolution of inflammation through inhibition of neutrophil tissue infiltration and stimulation of macrophage phagocytosis of apoptotic neutrophils . studies demonstrate that n-3 fatty acid feeding promotes endogenous production of resolving d1 and 17-hydroxy - dha , a marker of resolvin biosynthesis , in adipose tissue of obese - diabetic mice . transgenic overexpression of fat-1 , which encodes a desaturase enzyme that is able to convert -6 to -3 fatty acids , partially protects against obesity - induced insulin resistance in mice and is associated with an increase in the resolvin biosynthetic pathway marker 17-hydroxy - dha . these studies demonstrated that high - fat feeding results in a deficient endogenous resolvin and protectin biosynthesis and that these compounds are restored in fat-1 transgenic mice . provided evidence that adipose tissue expresses all the enzymes necessary for the formation of bioactive lipid mediators derived from both omega-6 and omega-3-pufas . in ob / ob mice dha significantly increased adipose tissue levels of adiponectin , which alleviated hepatic steatosis and insulin resistance . recent findings indicate also that dha ( at micromolar concentrations ) and resolvin d1 ( at nanomolar concentrations ) consistently decrease m1 macrophage activation in adipose tissue and increase m2 cells . these effects are related to stimulation of arginase 1 expression and attenuation of ifn/lps - induced th1 cytokine secretion . hellmann et al . suggested that stimulation of the inflammation resolution with the endogenous proresolving mediator resolving d1 provides a novel therapeutic strategy for treating obesity - induced diabetes . the authors observed that in leptin - receptor deficient mouse resolvin d1 prevents the accumulation of macrophages in adipose tissue and restores systemic insulin sensitivity . notably , these inflammation - resolving factors are important tools to decrease adipose tissue inflammation that is common in insulin resistance . micrornas ( mirnas or mirs ) are short noncoding rnas that have been demonstrated to be master regulators of the cellular transcriptome and proteome [ 155157 ] . regulatory mirnas bind to the complementary segments within 3-untranslated region ( 3utr ) of target transcripts through watson - crick base pairing , causing translational inhibition or mrna cleavage and suppression of gene expression . over 1000 mirnas have been identified in the human genome , which are estimated to regulate thousands of protein - coding genes [ 158 , 159 ] . there is also increasing experimental evidence that mirnas are involved in the control of several critical biological processes such as metabolism , cell proliferation , apoptosis , and disease development and progression [ 160163 ] . single - stranded mature mirnas with 2024 nucleotides in length are derived from genomically encoded sequences through transcription and complex mrna processing . change in gene expression or processing in dysfunctional or abnormal cells or tissues leads to an altered mirna expression level . profile of these molecules may be used as biomarkers for classifying human diseases and disease status [ 162 , 164 , 165 ] . recent studies have observed an association between specific mirnas and insulin resistance [ 166 , 167 ] , supporting the fact that mirnas may play a role in the pathological development of type 2 diabetes mellitus and leading also to the hypothesis that mirnas may represent a new class of glucose metabolism regulators with therapeutic potential for improving insulin sensitivity in peripheral tissues . supporting this idea , frost and olson have demonstrated that global and pancreas - specific overexpression of the mirna let-7 in mice results in impaired glucose tolerance and reduced glucose - induced pancreatic insulin secretion . pharmacological inhibition of the mirnalet-7family with specific anti - mir was sufficient to prevent and treat impaired glucose tolerance in diet - induced obesity mice , at least in part , by improving insulin sensitivity in liver and muscle . in addition , mirna let-7was able to block glucose - induced insulin secretion from the pancreas , suggesting that knockdown of this mirnaalso might improve pancreatic -cell function . a role of the heart in systemic metabolic control and the involvement of the heart - specific microrna , mir-208a , as potential therapeutic target for metabolic disorder have been proposed . impaired metabolism of energy - providing substrates and myocardial lipid accumulation are early abnormalities in obese and insulin - resistant individuals . grueter et al . have shown that med13 , a subunit of the mediator complex , controls transcription by thyroid hormone andother functions of nuclear hormone receptors in heart , controlling metabolic homeostasis and energy expenditure in mice . they have also shown that mir-208a , a heart - specific mirna encoded by an intron ofthe cardiac - specific -myosin heavy - chain ( mhc ) gene , negatively regulates med13 expression . elevated cardiac expression of med13 or pharmacologic inhibition of mir-208a in mice led to resistance to high - fat diet - induced obesity and improved systemic insulin sensitivity and glucose tolerance in mice . conversely , genetic deletion of med13 specifically in cardiomyocytes enhanced obesity in response to high - fat diet and exacerbated metabolic syndrome . as discussed previously , inflammation and oxidative stress participate in the propagation and development of obesity and associated metabolic disorders associated to insulin resistance . maladaptive production of various adipokines ( e.g. , adiponectin , resistin , visfatin , and leptin ) , migration of monocytes , and subsequent transformation into macrophages within affected tissues are key factors in the self - perpetuating inflammation associated with metabolic disorders [ 171 , 172 ] . in particular , plasma levels of adiponectin are significantly lower in obese individuals and have been associated with inflammation , insulin resistance , and cardiovascular disease . hulsmans et al . identified mir-146b-5p as a downregulated mir in monocytes of obese subjects with targets in the irak / nfkb - related gene cluster . they identified obesity - associated low levels of globular adiponectin as cause of the decrease in mir-146b-5p . the role of mir-146b-5p in the protection against inflammation was further supported by the finding that , after sequestration of mir-146b-5p , the cells lost their potency to raise their anti - inflammatory action in response to high levels of adiponectin . a possible role of mir-107 in regulating the inflammatory process that might lead to type 2 diabetes mellitus has been proposed by foley and o'neill . because the tlr4 ( lps receptor ) has been shown to downregulate mir-107 in activated macrophages located in adipose tissue , and mir-107 has been demonstrated to be dysregulated in murine and rodent models of obesity and insulin resistance , respectively [ 176178 ] , it has been suggested that mir-107 may be the link between obesity , inflammation , and insulin resistance . the authors hypothesize that decreased mir-107 by tlr4 is required to limit proinflammatory signaling pathways , since this effect will stabilize caveolin-1 , blocking the tlr4 pathway by displacement of myd88 . decreased mir-107 is an attempt to increase insulin sensitivity in the resolution phase of inflammation . a defect in this process , particularly in the ability of tlr4 to decrease mir-107 , an interesting model would be transgenic mice , in which we could manipulate mir-107 in vivo and pharmacologically inhibit mir-107 . the discovery and identification of new biomarkers involved in the pathogenesis of chronic inflammation and insulin resistance have been fundamental to understanding how these processes work and to direct further studies for the prevention or treatment of related disorders . particularly , these discoveries have aided to identify new target genes , lipids , proteins , and other metabolites involved in the development or severity of chronic diseases , for example , obesity , type 2 diabetes mellitus , metabolic syndrome , cardiovascular diseases , cancer , dyslipidemia , and cancer . probably , with the help of new tools and advanced methodologies , we will have a better characterization and understanding of the mechanisms and mediators involved in the different inflammatory and insulin resistance conditions , addressing further biotechnological studies for the development of new potential clinical therapies and interventions .

inflammation and insulin resistance are common in several chronic diseases , such as obesity , type 2 diabetes mellitus , metabolic syndrome , cancer , and cardiovascular diseases . various studies show a relationship between these two factors , although the mechanisms involved are not completely understood yet . here , we discuss the molecular basis of insulin resistance and inflammation and the molecular aspects on inflammatory pathways interfering in insulin action . moreover , we explore interventions based on molecular targets for preventing or treating correlated disorders , advances for a better characterization , and understanding of the mechanisms and mediators involved in the different inflammatory and insulin resistance conditions . finally , we address biotechnological studies for the development of new potential therapies and interventions .

1. Crosstalk between Inflammatory Pathways 2. Identification of New Molecular Targets for Reducing Inflammation in Insulin Resistance Models 3. Concluding Remarks

these proteins activate several signaling pathways involved in the regulation of important cellular events such as glucose uptake and metabolism , protein synthesis , gene expression , cell survival , growth , development , and differentiation [ 46 ] . irs proteins are phosphorylated on various tyrosine residues of the c - terminal region , generating specific sites for binding of proteins containing src homoly-2 ( sh2 ) domains , including phosphatidylinositol-3 kinase ( pi-3k ) , nck , and grb-2 . these kinases are involved in the most important biological effects induced by insulin , such as translocation of glucose transporter-4 ( glut-4 ) from intracellular vesicles to plasma membrane , glycogen and protein synthesis , antiapoptotic effects , and gene expression ( figure 1 ) [ 711 ] . after being activated , mitogen - activated protein kinase ( mapk ) cascade starts with the association of shc to insulin receptor , binding of grb-2 to shc or to irs-1 , and formation of the grb-2/sos ( son of sevenless ) in the plasma membrane [ 1719 ] . mapk pathway is involved in the differentiation , cell growth , and development induced by insulin , as well as some metabolic effects , as glycogen synthesis and glut-4 translocation to plasma membrane ( figure 1 ) [ 2224 ] . disturbances in several proteins involved in the insulin signaling pathways have been found in different conditions of insulin resistance , including obesity , type 2 diabetes mellitus , metabolic syndrome , cardiovascular diseases , inflammatory disorders , and cancer [ 2628 ] . here , we will discuss possible mechanisms involved in the development of insulin resistance related to inflammatory processes . insulin resistance occurs when the insulin - sensitive tissues , mainly skeletal muscle , adipose tissue , and liver , lose the ability to respond properly to the hormone [ 29 , 30 ] . it is associated with several chronic diseases , especially those linked to obesity , such as type 2 diabetes mellitus , metabolic syndrome , dyslipidemias , cardiovascular diseases , cancer , and neurodegenerative diseases [ 3133 ] . however , the precise mechanisms involved in insulin resistance are not fully understood yet [ 3437 ] . several factors have been proposed to participate in the development of insulin resistance , including increased plasma - free fatty acid level , subclinical chronic inflammation , oxidative and nitrative stress , altered gene expression , and mitochondrial dysfunction [ 29 , 37 ] . since free fatty acids are elevated in obesity and related diseases , these metabolites have been proposed to be responsible for the impairment in the insulin action , but the mechanisms are not completely known yet [ 38 , 39 ] . high availability of fatty acids , specially long - chain saturated fatty acids , results in the establishment of insulin resistance in liver , skeletal muscle , and adipose tissue [ 40 , 41 ] . various hypothesis have been proposed to explain the insulin resistance induced by saturated fatty acids , including randle cycle , oxidative stress , modulation of gene transcription , inflammation , accumulation of intracellular lipid derivatives ( diacylglycerol and ceramides ) , and mitochondrial dysfunction [ 4247 ] ( for review , see martins et al . ) a chronic state of inflammation in the insulin responsive tissues is a major contributor to insulin resistance in obesity and related diseases . thus , a crosstalking between inflammation and insulin resistance has been suggested by several authors . however , the precise mechanism as well as the mediators involved in this interaction is not completely defined yet . in this paper , we discuss how inflammatory signaling pathways impair insulin signaling ( see below ) . in insulin resistance , increased ros production and/or decreased ros degradation is observed , leading to an oxidative stress condition and activation of signaling pathways related to stress . there is evidence that oxidative stress is also involved in muscle disorders , contributing to the insulin resistance process . activation of signaling pathways to stress has been suggested to participate in the development of insulin resistance by impairing the signaling by this hormone . for example , expression of genes involved in lipid and glucose metabolism , insulin signaling , inflammation , redox balance , and mitochondrial function is modified , suggesting that these processes participate in the pathophysiology of insulin resistance [ 5557 ] . examples of proteins with this function include the receptors of the toll - like ( tlrs ) family , the c - type lectin receptors , the purinergic and advanced glycation endproductsreceptors ( rage ) , and the intracellular nucleotide oligomerization domain ( nod ) , and retinoic acid - inducible gene ( rig)-i - like receptors ( rlrs ) . after recognizing their ligands , several downstream pathways including c - jun nh(2)-terminal kinase ( jnk ) and ib kinase complex ( ikk ) are activated , resulting in changes on transcription factors activity and expression of proteins such as cytokines , enzymes , chemokines , adhesion molecules , and amplification of the inflammatory response . the activation of the inflammatory pathways described above is a hallmark of obesity , and it has been associated with the development of insulin resistance , atherosclerosis , and other tissue dysfunctions that are secondary to fat accumulation . an increased production of inflammatory mediators and activation of inflammatory pathways in several tissues including adipose tissue ( at ) , liver , pancreas , skeletal muscle , and hypothalamus are present in obese individuals and define a subclinical inflammatory process also known as meta - inflammation , little information is available regarding the inducers and sensors involved in obesity - associated inflammation . the release of damps by necrotic adipose cells , the increase in the flux of nonesterified fatty acids ( increased rates of lipolysis ) , the reduction in the oxygen tension ( hypoxia ) leading to activation of hypoxia - induced factor ( hif)-1 , which controls the expression of proinflammatory proteins , and the production of chemokines by adipose cells have been suggested to play a role in the initiation of inflammatory process . despite several advances in the field , the initial events involved in the beginning of inflammation in the at and the complex interactions between them are not clearly understood , and new components are continuously described and added to the puzzle such as leukotrienes and the apoptosis inhibitor of macrophage ( aim ) [ 62 , 63 ] . the obesity - induced inflammation is associated with activation of resident cells such as kupffer cells in the liver and leukocyte infiltration and shift in the polarized state of tissue / recruited leukocytes . these changes are better characterized in the adipose tissue , in which an increased infiltration by proinflammatory m1-type macrophages ( classical macrophages ) , th1 , th17 and cd8 t cells and a reduction in the content of less inflammatory cells such as m2-macrophage , t reg , and th2 cells were demonstrated [ 6568 ] . several chronic diseases are characterized by increased inflammatory process and insulin resistance , such as obesity , type 2 diabetes mellitus , metabolic syndrome , cardiovascular diseases , and cancer . for example , various studies suggest the involvement of some inflammatory factors in the development of insulin resistance , including cytokines ( tnf- , il-1 , il-6 ) , ros , and rns ( figure 2 ) . in addition , other factors involved in inflammatory processes also can impair insulin sensitivity , particularly lipopolysaccharides ( lpss ) and environmental stress ( hypoxia , nutrients , and ph ) ( figure 2 ) . the second kinase activates members of the signal transducers and activators of transcription ( stat ) family , which are involved in several biological effects , such as expression of genes related to inflammation , apoptosis , differentiation , growth , morphogenesis , migration , and proliferation . moreover , phosphorylation on serine / threonine residues also increases irs protein degradation , contributing to the establishment of insulin resistance [ 7173 ] . nonfunctional tlr-4 expression protects mice from insulin resistance and inflammation induced by high - fat diet , and tlr-4 gene silencing by small interference of rna reduces inflammation in acute lung injury induced by lipopolysaccharide . obese and type 2 diabetic animals are prevented from insulin resistance and inflammation by specific inhibitors or gene mutation ( knockout or nonfunctional gene ) of ikk or jnk [ 7678 ] . for example , fatty acids can directly activate toll - like receptors ( tlrs ) , g - protein coupled receptors ( gpcrs ) , and tumor necrosis factor- ( tnf- ) receptor as well as modulate inflammatory signaling pathways involved in the increase in cytokine secretion ( tnf- , il-1 , and il-6 ) [ 60 , 79 , 80 ] , oxidative and nitrative stress , mitochondrial dysfunction , endoplasmic reticulum ( er ) stress , and proinflammatory gene expression . alterations in the expression of genes and proteins involved in the inflammatory process are clearly associated with insulin resistance and several metabolic abnormalities , including mitochondrial dysfunction , decreased fat oxidation , increased ectopic lipid storage , and impaired insulin signaling pathways ( figure 2 ) . proinflammatory cytokines were also involved in the reduction of mitochondrial function [ 79 , 82 ] . therapies aimed at neutralizing proinflammatory cytokines such as tnf- and il-1 , such as the monoclonal antibody infliximab and canakinumab , respectively , have been under investigation in the treatment of type 2 diabetic patients . on the other hand , in patients with high grade inflammatory diseases such as rheumatoid arthritis and ankylosing spondylitis , anti - tnf therapy has been successfully associated with reduction in insulin resistance and metabolic syndrome components [ 9095 ] . the primary endpoint of the cantos study will be cardiovascular events , and secondary endpoints include new onset type 2 diabetes and diabetes - specific markers . such large and long - term trials could provide a novel cytokine - based therapy for the secondary prevention of new - onset diabetes as well as confirm the autoinflammatory nature of metabolic disorders . a substrate competitive inhibitor of jnk , bi-78d3 , has also been shown to restore insulin sensitivity in a murine model of type 2 diabetes after a single dose . several of these receptors have been shown to regulate insulin secretion and tissue sensibility to this hormone , becoming potential targets for intervention in conditions of insulin resistance / deficiency . other gpcrs implicated in the glucose metabolism and regulation of inflammation such as glp-1 , glucose - dependent insulinotropic polypeptide ( gip ) , the bile acid ( tgr5 ) , cholecystokinin , the cannabinoid receptors ( cbs ) , and muscarinic receptors are beyond the focus of the present paper and are discussed elsewhere . proteins that regulate gpcrs signaling such as gpcr kinases and arrestins , which are implicated in the control of food intake , regulation of insulin action , inflammation , adipogenesis , and other processes that are associated with weight gain and development of insulin resistance , are the focus of recent reviews [ 100 , 101 ] , so they are not discussed here . in addition to these effects , fas - gpcrs , mainly , gpr43 , 84 , and 120 , present relevant effects in the inflammatory cell activation . this anti - inflammatory effect seems to be important for the beneficial action of these fatty acids in the model of obesity induced by high - fat diet , preventing development of glucose intolerance , insulin resistance , and obesity . altogether the findings herein discussed highlight the importance of these receptors for glucose homeostasis , control of inflammatory cells activation , and food intake , processes that are linked through complexes interaction , which are not completely understood . , short chain fatty acids such as butyrate , valproic acid , trichostatin a , and other compounds ) increases the acetylation of histone and nonhistone proteins including nfb , myod , p53 , and n - fat and , consequently , affects gene expression and proteins activities leading to changes in different aspects of cell biology including cell motility , proliferation , differentiation , and apoptosis . in addition to their well - known anti - inflammatory effects , other recent evidence has been obtained , which together strongly indicates hdac as a target for novel therapies in insulin resistance and diabetes.histone hyperacetylation has been associated with an increase in insulin expression and protection of -cells against cytokine - induced apoptosis , as reviewed by christensen et al . .the isoforms 4 , 5 , and 9 of hdac are associated with the development of and cells of the pancreas .oral administration of hdaci ( itf2357 ) reduced -cells toxicity associated with streptozotocin administration . additionally , the authors also showed that this hdaci protected islets from cytokine - induced toxicity and reduced production of no and chemokines in islets ; administration of sodium butyrate ( diet supplementation or oral tributyrin ( a prodrug of butyrate ) ) to high - fat fed mice attenuated body weight gain , improved lipid and glucose metabolism parameters , and inhibited the development of obesity - associated changes including activation of inflammatory pathways and hepatic steatosis [ 115 , 116 ] . recently , it has been suggested that this effect of butyrate involves inhibition of hdac 3 activity , an effect that leads to activation of ppar- and expression of fgf21 , which stimulates lipid oxidation , triglyceride clearance , and energy expenditure ; hdac 6 knockout mice are protected from hyperglycemia , glucose intolerance , and insulin resistance secondary to chronic corticoid administration ; hdaci increases the number of t regulatory cells and their suppressive function , an effect that may be important in the context of adipose tissue inflammation [ 119 , 120 ] . the isoforms 4 , 5 , and 9 of hdac are associated with the development of and cells of the pancreas . additionally , the authors also showed that this hdaci protected islets from cytokine - induced toxicity and reduced production of no and chemokines in islets ; administration of sodium butyrate ( diet supplementation or oral tributyrin ( a prodrug of butyrate ) ) to high - fat fed mice attenuated body weight gain , improved lipid and glucose metabolism parameters , and inhibited the development of obesity - associated changes including activation of inflammatory pathways and hepatic steatosis [ 115 , 116 ] . recently , it has been suggested that this effect of butyrate involves inhibition of hdac 3 activity , an effect that leads to activation of ppar- and expression of fgf21 , which stimulates lipid oxidation , triglyceride clearance , and energy expenditure ; hdac 6 knockout mice are protected from hyperglycemia , glucose intolerance , and insulin resistance secondary to chronic corticoid administration ; hdaci increases the number of t regulatory cells and their suppressive function , an effect that may be important in the context of adipose tissue inflammation [ 119 , 120 ] . inhibition of ppar- function by inflammatory cytokines may contribute to pathogenesis of many diseases such as insulin resistance , atherosclerosis , inflammation , and cancer cachexia [ 122124 ] . activation of serine kinases including ikk , erk , jnk , and p38 may be involved in the tnf regulation of ppar- ( figure 3 ) ( reviewed in ) . in obesity , macrophages invade adipose tissue promoting the inflammation characteristic of insulin resistance . prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue and other tissues , leading to metabolic abnormalities such as insulin resistance . tlrs are transmembrane receptors that play a critical role in the detection of microbial infection and in the induction of inflammatory and immune responses against conserved microbial structures , called pathogen - associated molecular patterns . among the tlrs , tlr2 and tlr4 play a critical role in the pathogenesis of insulin resistance , diabetes , and atherosclerosis in both clinical and experimental conditions [ 138 , 139 ] . c3h / hej mice with a mutation in tlr4 are protected against the development of high fat diet - induced obesity . studies in mice demonstrate that tlr-2 and tlr-4 activation and cytokine production stimulated by these receptors lead to the development of diabetes ( figure 3 ) [ 140 , 141 ] . more recently , tlr4 has been indicated as a molecular link between free fatty acids , inflammation , and the innate immune system . studied tlr2 and tlr4 mrna and protein expression , their ligands , and intracellular signaling in monocytes of recently diagnosed type 2 diabetic patients and observed that there is significant elevation of tlr2 and tlr4 protein , mrna , endogenous ligands , and cofactors which , together with hyperglycemia , contribute to the proinflammatory state of type 2 diabetes . nowadays , based on several studies it is clear that tlr4 inhibition is a pharmacologic tool to avoid inflammation in insulin resistance patients . in ob / ob mice dha significantly increased adipose tissue levels of adiponectin , which alleviated hepatic steatosis and insulin resistance . notably , these inflammation - resolving factors are important tools to decrease adipose tissue inflammation that is common in insulin resistance . there is also increasing experimental evidence that mirnas are involved in the control of several critical biological processes such as metabolism , cell proliferation , apoptosis , and disease development and progression [ 160163 ] . recent studies have observed an association between specific mirnas and insulin resistance [ 166 , 167 ] , supporting the fact that mirnas may play a role in the pathological development of type 2 diabetes mellitus and leading also to the hypothesis that mirnas may represent a new class of glucose metabolism regulators with therapeutic potential for improving insulin sensitivity in peripheral tissues . a role of the heart in systemic metabolic control and the involvement of the heart - specific microrna , mir-208a , as potential therapeutic target for metabolic disorder have been proposed . as discussed previously , inflammation and oxidative stress participate in the propagation and development of obesity and associated metabolic disorders associated to insulin resistance . , adiponectin , resistin , visfatin , and leptin ) , migration of monocytes , and subsequent transformation into macrophages within affected tissues are key factors in the self - perpetuating inflammation associated with metabolic disorders [ 171 , 172 ] . in particular , plasma levels of adiponectin are significantly lower in obese individuals and have been associated with inflammation , insulin resistance , and cardiovascular disease . a possible role of mir-107 in regulating the inflammatory process that might lead to type 2 diabetes mellitus has been proposed by foley and o'neill . because the tlr4 ( lps receptor ) has been shown to downregulate mir-107 in activated macrophages located in adipose tissue , and mir-107 has been demonstrated to be dysregulated in murine and rodent models of obesity and insulin resistance , respectively [ 176178 ] , it has been suggested that mir-107 may be the link between obesity , inflammation , and insulin resistance . the discovery and identification of new biomarkers involved in the pathogenesis of chronic inflammation and insulin resistance have been fundamental to understanding how these processes work and to direct further studies for the prevention or treatment of related disorders . particularly , these discoveries have aided to identify new target genes , lipids , proteins , and other metabolites involved in the development or severity of chronic diseases , for example , obesity , type 2 diabetes mellitus , metabolic syndrome , cardiovascular diseases , cancer , dyslipidemia , and cancer . probably , with the help of new tools and advanced methodologies , we will have a better characterization and understanding of the mechanisms and mediators involved in the different inflammatory and insulin resistance conditions , addressing further biotechnological studies for the development of new potential clinical therapies and interventions .

in recent years , the incidence of surgery among the nation 's aged has increased at a far greater rate than for younger people ( lubitz and deacon , 1982 ) . in 1965 , the number of operations per 1,000 aged persons was 105 ; in 1977 , 166 operations per 1,000 aged were performed , a 58-percent increase . for the population 64 years of age or under , surgery rates were 74 per 1,000 in 1965 and 92 per 1,000 in 1977 , a 24-percent increase . the increase in surgery among the aged no doubt reflects technical advances that offer hope of relief from chronic conditions associated with aging . these advances include improved techniques of lens extraction , the development of coronary artery bypass surgery , and the emergence of new materials in the field of orthopedic surgery . accompanying these advances have been developments in anesthesiology and post - surgical care that allow physicians to operate on aged patients who would not , formerly , have been considered good candidates for surgery . the increase in the rate of surgery raises questions about the risks of surgery among the aged . the purpose of this article is to present patterns of post - surgical mortality , including deaths occurring both in the hospital and after discharge . mortality after eight common surgical procedures is studied for medicare enrollees 65 years of age or over . hospital discharges for these operations constituted 23 percent of the 3.1 million total surgical discharges among the 26.5 million aged persons enrolled under part a of medicare . these procedures were selected because ( with the exception of coronary artery bypass ) they are among the most frequently performed on the aged . coronary artery bypass was chosen because it represents a highly technical procedure performed with increasing frequency , and new information on the risks and benefits has recently appeared ( coronary artery surgery study , 1983a and b ) . another reason for including coronary artery surgery is that relatively little is known about the outcomes of this surgery for aged patients ( chassin et al . , 1985 ) . these procedures , with the exception of femur fracture reduction , are elective to some degree ( cageorge et al . , 1981 ; wennberg et al . ( 1982 ) examined the implications of physician disagreement and uncertainty over indications for surgery and the benefits of different operations . the authors believe this professional disagreement is a major source of variation in surgery rates ( and , by implication , in adverse outcomes of surgery ) for different operations among different populations . wennberg et al . stressed that public dissemination of improved information on surgical outcomes ( such as mortality ) is an important step toward encouraging more informed professional decisionmaking in this area . many studies have found large geographic variations in the incidence of surgery , apparently unrelated to variations in need , within areas of new england ( mcpherson et al . , 1982 ) and within the states of kansas ( lewis , 1969 ) , wisconsin ( detmer and tyson , 1978 ) , and new york ( lembcke , 1952 ) . large variations in surgical rates were also found within the canadian provinces of ontario ( stockwell and vayda , 1979 ) and manitoba ( roos and roos , 1981 ) . if some of the operations performed in areas with high rates of surgery are really not necessary , then the mortality associated with this surgery may also not be necessary . mortality is not limited to in - hospital deaths but is tracked for periods up to a year after surgery . the mortality experience of surgery patients is compared with mortality patterns in the general medicare aged population . variations in risk among age and sex categories are presented , and comparisons are drawn among regions of the united states in terms of mortality rates . because about 95 percent of all people 65 years of age or over are covered by medicare , these data generally represent the surgical outcomes of the aged in the united states and thus expand on studies limited to the surgical outcomes in selected hospitals or groups of hospitals . in addition , these data expand on those studies because they track deaths that occurred up to 12 months after discharge . future studies of surgical outcomes among medicare patients will address the relationship between mortality and surgical volume by hospital and describe the rehospitalization experience of aged medicare beneficiaries undergoing selected operations . data for this study were obtained from two files of the medicare statistical system of the health care financing administration ( hcfa ) : the medicare provider analysis and review ( medpar ) file and the enrollment file . the medpar file contains information on discharges from short - stay hospitals for a 20-percent sample of medicare beneficiaries , selected on the basis of their medicare identification numbers . the information includes demographic data ( e.g. , age , sex ) , hospital data ( e.g. , size , location ) , and information about the stay ( e.g. , principal surgical procedure , principal diagnosis , and patient survival in the hospital ) . using the international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) , in 1979 and 1980 the principal surgical procedure and principal diagnosis were coded for each discharge in the sample . the medpar file contained no information on secondary diagnoses or operations , although such information has been coded since 1984 . the enrollment file maintains current entitlement information for all enrollees , including date of death . the records of hospital stays for the eight selected operations were linked to the enrollment file to identify all deaths , regardless of where the deaths occurred , for a 12-month period following surgery . discharges of aged beneficiaries occurring in 1979 and 1980 were pooled to increase sample size . rehospitalizations for a study procedure were not excluded , i.e. , if an individual underwent more than one study procedure during the period 1979 - 80 , each procedure was included as a separate operation . all patients with a principal diagnosis of cancer for the stay in which the surgery was performed were excluded from the study . it was assumed that mortality rates for cancer patients would be determined , to a large extent , by their disease and would be less influenced by their operations per se . four percent of all surgical discharges were excluded because of a principal diagnosis of cancer ; this included 17 percent of prostatectomy discharges and 0.7 percent of all other discharges . first , the exact date of death is not always identifiable because the date of death in the medicare enrollment file is sometimes coded as the last day of the month of death . for this reason , the exact time between surgery and death had to be estimated . assuming that dates of surgery are distributed uniformly throughout the month , we defined deaths within the first 1.5 months of surgery as those occurring in the month of surgery and the following month . similarly , deaths within 2.5 months were defined as those occurring in the month of surgery and the following 2 months . secondly , there are known to be some problems with the reliability of diagnostic and surgical data in the medicare statistical system . the national academy of sciences ' institute of medicine ( iom ) ( 1977 ) compared medicare diagnostic and surgical information with that obtained by a trained field team that reviewed medical records for a sample of hospital cases . discrepancies were found for many diagnoses and procedures . in 21 percent of the cases reviewed , there was a discrepancy between the iom abstract and the medicare record . the 21-percent disagreement rate included disagreements about whether or not a procedure was performed and also about the nature of the principal procedure . in 43 percent of the cases in which medicare records indicated a procedure was performed , there was a disagreement between the medicare record and the iom record . most of the discrepancies were caused by incomplete narrative information on the medicare claim form from which codes were assigned . other errors included routine misuse of the coding system and the improper designation of one of several procedures as the principal one . some types of errors might result in some undercounting of certain operations , but these errors would not invalidate the information obtained for those cases identified for our study . other types of errors are undoubtedly minor in nature and would also not invalidate the study ( e.g. , errors involving only the fourth digit of the surgical code ) . the iom study did not reveal any errors that would appear to systematically bias our study results . because the study data constitute a 20-percent sample of discharges for selected procedures , there is sampling error associated with the estimates presented in the tables . lastly , the study does not include medicare disabled beneficiaries ( under 65 years of age ) , who constitute approximately 10 percent of the medicare population . average annual numbers of discharges ( noncancer related ) per 10,000 aged enrollees for 1979 - 80 , by age and sex , are presented in table 1 . lens extraction and turp were the most common procedures ; lens extraction accounted for 114.1 discharges annually per 10,000 enrollees , and turp accounted for 121.3 per 10,000 male enrollees . the next most common operations were cholecystectomy ( 36.9 discharges per 10,000 enrollees ) and inguinal hernia repair ( 34.7 discharges per 10,000 enrollees ) . coronary artery bypass was the least common of this group of procedures , with an annual average of 6.5 discharges per 10,000 aged enrollees during 1979 - 80 . ( the annual number of noncancer related discharges among aged enrollees for these procedures is given in the technical note , table b. ) the majority of some of these operations are performed on medicare beneficiaries . in the united states in 1979 , 86 percent of hip arthroplasties ( other ) , 76 percent of lens extractions , 74 percent of turp 's , 69 percent of femur fracture reductions , and 57 percent of hip arthroplasties ( total hip replacements ) were performed on people 65 years of age or over ( national center for health statistics , 1982 ) . however , for cholecystectomy , inguinal hernia repair , and coronary bypass , between 23 and 28 percent of each procedure was performed on aged people . different incidence rates by age are evident for the various operations . the number of discharges per 10,000 enrollees for hip arthroplasty ( other ) and femur fracture reduction increased sharply with age , whereas the incidence of coronary artery bypass decreased ( table 1 ) . these differences probably reflect both underlying patterns of morbidity and medical opinion about the advisability of doing certain operations at different ages . there are also marked differences in discharge rates between males and females , with males exhibiting higher rates at all ages for coronary bypass and hernia repair , whereas females exhibit higher rates for hip arthroplasty of both types and femur fracture reduction . this latter difference in rates is related to a higher incidence of hip fracture among aged females , which is , in turn , related to loss of bone mass ( osteoporosis ) with age ( national institutes of health , 1984 ) . the average annual number of deaths occurring within the first 1.5 months following surgery is shown in table 2 . the largest number of deaths , 4,595 annually , occurred after femur fracture reduction . among hip arthroplasty ( other ) patients , deaths following both operations were greatest among patients 85 years of age or over . among the eight procedures studied , deaths per 10,000 operations in the 1.5 months following surgery were highest following femur fracture reduction ( 878 ) , hip arthroplasty ( other ) ( 750 ) , and coronary artery bypass ( 615 ) ( table 3 ) . deaths among lens extraction patients were by far the least common , with only 49 per 10,000 operations . the death rate for males at all ages was greater than that for females at all ages , with the exception of coronary artery bypass at all ages and inguinal hernia repair among those 65 - 74 years of age . the difference in death rates by sex was especially large for femur fracture reduction , with males experiencing approximately twice the mortality of females in the 1.5 months following surgery . higher in - hospital mortality rates for females undergoing coronary artery bypass surgery have been noted in two other studies ( fisher et al . , 1982 ; and douglas , et al . , 1981 ) , although both of these studies reported that the difference in mortality between males and females was not statistically significant for the group 60 years of age or over . differences in coronary artery bypass mortality by sex may be related to the fact that females have smaller physical stature and smaller coronary arteries ( fisher et al . , 1982 ) . the similarity in mortality rates between patients undergoing femur fracture reduction and those undergoing hip arthroplasty ( other ) is because of the high incidence of hip fracture among the latter group . seventy - one percent of these patients had a principal diagnosis of femur fracture ( data not shown in tables ) . somewhat surprisingly , hip arthroplasty ( other ) patients without a principal diagnosis of femur fracture exhibited mortality patterns similar to patients with a fracture . it is possible that some of the patients without a principal diagnosis of femur fracture may have had that as a secondary diagnosis . ( as noted earlier , only the principal diagnosis is recorded in the medicare statistical system ) . mortality rates for patients undergoing total hip replacement varied considerably according to whether a principal diagnosis of femur fracture was present ( table 4 ) . for each age and sex group , patients with a fracture diagnosis exhibited considerably higher mortality rates than those with other diagnoses . higher mortality rates for patients with a fracture may reflect the effects of the injury , as well as the fact that the surgery is less likely to be elective , and many cases may be poor surgical risks . in fact , those total hip replacement patients with a diagnosis of femur fracture exhibited mortality rates similar to patients undergoing femur fracture reduction and hip arthroplasty ( other ) . this suggests that a fractured femur is a strong determinant of mortality , regardless of the procedure used to repair it . a diagnosis of femur fracture is much more frequent among the oldest arthroplasty ( total replacement ) patients ( table 4 ) . only 5.1 percent of those 65 - 74 years of age exhibited a principal diagnosis of femur fracture , whereas 35.9 percent of those 85 years of age or over showed this diagnosis . this explains why the mortality rate for total hip replacement patients rises so steeply with increasing age . females were generally more likely to exhibit a diagnosis of femur fracture than males , although females ' mortality rates for total hip replacement tended to be lower . the age - specific and sex - specific mortality risk of the study group during the 1.5 months following surgery , relative to the experience of the medicare aged population at large , is shown in table 5 . the comparisons are presented in the form of mortality ratios ( mr ) , i.e. , the ratio of observed deaths to those expected if the sample patients experienced the prevailing mortality pattern of the medicare aged population . the expected mortality pattern is estimated by dividing medicare deaths for 1979 and 1980 by the sum of part a program enrollments ( as of july 1 of both years ) , then prorating them by month ( technical note , table c ) . although observed mortality rates for specified procedures are compared with the prevailing mortality rate in the medicare population , this should not be taken as an indication that post - surgical patients are truly expected to experience prevailing mortality rates . because many surgical patients have poor underlying health , it is not known what their mortality patterns would be if the study procedures had not been performed . they are , rather , presented as a descriptive measure that puts observed death rates into perspective . the highest mr 's among the eight procedures studied are associated with coronary artery bypass , with an overall mr of 12.54 during the 1.5 months following surgery ( table 5 ) . the mr 's for females are much higher than those for males . in the group 65 - 74 years of age , which accounts for most of the bypass operations , males have mr 's of 9.41 and females , 30.70 . as discussed previously , higher mortality for females undergoing coronary artery bypass surgery is also reflected in the age - specific deaths per 10,000 operations ( table 3 ) . patients undergoing femur fracture reduction and hip arthroplasty ( other ) also had high mr 's ; for femur fracture reduction , mr 's exceeded 9 for all groups except females 85 years of age or over , and for hip arthroplasty ( other ) the mr 's were 8 or more for all age - sex groups except females over 85 years of age . mr 's for lens extraction patients were consistently less than 1 , which reflects a variety of factors . technical advances in the procedure have made lens extraction relatively nontraumatic , with only a brief hospital stay involved . it is also likely that only patients considered to be in relatively good overall health are selected for this particular procedure because it is highly elective . contrasting patterns in age - specific mr 's are evident for some of the operations . for females , the mr 's decrease sharply with age for femur fracture reduction and hip arthroplasty ( other ) , whereas mr 's are relatively steady for males . the mr for males with inguinal hernia repair increases with age1.08 for males 65 - 74 years of age , 1.67 for males 75 - 84 years of age , and 2.15 for males 85 years of age or over . this most likely reflects the fact that the proportion of males with a principal diagnosis of obstructed hernia increases with age from 4.3 percent for males 65 - 75 to 14.9 percent for those 85 or over , and the mortality rate associated with these patients is considerably higher than for other patients ( data not shown in tables ) . higher mortality rates for patients with obstructed hernias probably reflect , in part , the fact that inguinal hernia repair for these patients is not elective surgery , and many may be poor surgical risks . mr 's for lens extraction patients decreased with age for both sexes , perhaps reflecting that the selection process for the operation results in older patients being in better health relative to their age - sex cohorts than younger patients . coronary artery bypass patients also exhibited a decreasing mr with age , from 13.12 for patients 65 - 74 years of age to 10.33 for patients 75 - 84 years of age . the mr 's for total hip replacement patients increased sharply in the oldest group for both males and females , reflecting the increase , by age , in the proportion of cases exhibiting a principal diagnosis of fracture . for cholecystectomy and turp , mr 's remained relatively stable across ages . among all operations , males tended to have lower mr 's than did females in the 65 - 74 years of age group and higher mr 's in the 85 years of age or over group ( with the exception of total hip replacement ) . the cumulative percents of death following surgery , unadjusted for age and sex differences , for various time periods are shown in table 6 . mortality after 12.5 months was highest for patients undergoing femur fracture reduction ( 23.9 percent ) , and hip arthroplasty ( other ) ( 21.1 percent ) , reflecting the higher age of this group , as well as the complications associated with a fractured femur . this high mortality rate is similar to that reported by jensen and tondevold ( 1979 ) for hip fracture patients , 26.8 percent after 1 year , and less than that reported by beals ( 1972 ) , 50 percent after 1 year , and evans et al . lens extraction ( 5.3 percent ) , total hip replacement ( 5.9 percent ) , and inguinal hernia repair ( 6.4 percent ) patients had the lowest mortality after 12.5 months . indirectly standardized mr 's ( adjusted for age and sex ) for various time periods following surgery are presented in table 7 . for all operations except lens extraction , the indirectly standardized mortality ratio ( ismr ) drops substantially after the first 1.5 months following surgery . the ismr is highest for coronary artery bypass patients in the first 1.5 months ( 12.54 ) , but declines very quickly thereafter , and is not significantly different from 1 after 3.5 months . femur fracture reduction and hip arthroplasty ( other ) patients exhibit the highest ismr 's after the first 1.5 months and remain the highest throughout the rest of the first year . the ismr for turp is not extremely high initially ( 2.49 in the first 1.5 months ) , but does not decline as rapidly as for other operations , stabilizing in the area of 1.3 to 1.4 after a few months . after the first 1.5 months , the ismr for inguinal hernia repair is not significantly different from 1 . elevated ismr 's , over a several - months period , may be related to these patients ' underlying poor health , which led to surgery in the first place , or the elevated ismr 's may be related to long - term complications following surgery . as mentioned earlier , the initial low mr for lens extraction patients probably reflects a favorable selection factor . lens extraction operations are normally not performed on patients who are very ill , and the operation itself is generally not life - threatening . consequently , lens extraction patients might be expected to have a lower mortality following surgery than the general medicare population . total hip replacement patients exhibit a very unexpected pattern in that their ismr is high initially ( 3.43 in the first 1.5 months and 1.41 between 2.5 and 3.5 months after surgery ) , but it drops significantly below 1 after approximately 6 months . the ismr for these patients also remains substantially below 1 for the months 12.5 to 18.5 ( data not shown in tables ) . we are not sure why total hip replacement patients should exhibit better than average mortality patterns at 6 months and more following surgery . it is possible that the operation itself acts as a screen , or selection process , that produces a surviving group of healthier than average patients . it is also possible that because total hip replacement is usually an elective procedure , the patient population is relatively healthy to begin with . the rate of in - hospital deaths is compared with the rate in the 1.5 months following surgery in table 8 . the number of in - hospital deaths includes only those deaths that occurred during the hospital stay ( regardless of its length ) during which the operation was performed . a number of studies of post - surgical mortality have been limited to data on in - hospital deaths . this study indicates that such studies probably miss a substantial proportion of post - surgical deaths . in - hospital deaths as a percent of all deaths occurring within 1.5 months of surgery are shown in table 8 ; the assumption used is that all in - hospital deaths occur within 1.5 months of surgery . only for coronary artery bypass did the rate of in - hospital deaths , as a percent of all deaths within 1.5 months following surgery , exceed 70 percent . for turp and inguinal hernia repair , fewer than 50 percent of deaths during this time period occurred in the hospital . lens extraction patients are excluded because of their low death rates and brief hospital stays . regional data ( with region defined as region of patient residence ) on age - adjusted and sex - adjusted mortality rates within 1.5 months of surgery are provided in table 9 . the west has the lowest mortality rate of all four regions for four of the seven procedures : turp , femur fracture reduction , coronary artery bypass , and hip arthroplasty ( other ) . these results are similar to luft 's ( 1980 ) findings , which showed generally lower rates of in - hospital post - surgical mortality in the west . for cholecystectomy , repair of inguinal hernia , and total hip replacement , the west has a mortality rate similar to that for the other regions . in particular , mortality following total hip replacement is almost twice as high in the south as in the northeast . mortality following inguinal hernia repair is also higher in the north central region than elsewhere . a possible explanation for the lower mortality rates in the west is that rates of surgery there are higher than the national average for some procedures . a high incidence of surgery could be associated with a higher number of low - risk patients ( not explained by age or sex differences ) undergoing these operations , which would lead to a lower mortality rate . roos and roos ( 1981 ) examined this issue for hospital stays in manitoba and concluded that the proportion of high - risk patients in their study did not vary between areas with high and low surgery rates . to examine the possible relationship between surgery rates and mortality , a spearman 's rank correlation coefficient was computed for the regional mortality rates and age- and sex - adjusted surgery rates ( technical note , table d ) . in order to make mortality and surgery rates comparable across procedures , the regional rates were then pooled across procedures , yielding 28 pairs of mortality and surgery rates . the correlation coefficient for these data was -.11 , which did not achieve statistical significance . thus , there is no evidence that low mortality is systematically associated with high surgery rates on a regional basis across procedures . it is possible , however , that mortality and surgery rates may be related for individual procedures . for example , the surgery rate for coronary artery bypass is much higher in the west than elsewhere ( 10.8 per 10,000 in the west for the 65 - 84 years of age population versus 7.2 per 10,000 nationally ) , and the mortality rate there is much lower . the high mortality rates associated with several study procedures indicate that considerable risk is involved when certain operations are performed on the aged , particularly on the very old . over 6 percent of the coronary artery bypass patients died within 6 weeks of surgery , and approximately 8.8 percent of the patients undergoing femur fracture reduction died within the same time period . hospital use and mortality associated with femur fracture reduction and , to a large extent , hip arthroplasty ( other ) , reflect the widespread problem of osteoporosis among the aged , particularly females . this condition causes the bones to lose mass and become brittle , making them more susceptible to fracture . often an osteoporotic patient will suffer a fractured hip following a fall in the home or elsewhere . the high mortality rate following femur fracture reduction , as well as hip arthroplasty ( other ) in the presence of fracture , indicates how serious the problem of osteoporosis is among the aged . to the extent that the rate of bone mass loss can be slowed ( by diet , exercise , or drugs ) and the risk of falls lowered , hospitalizations and mortality among the aged might be reduced ( national institutes of health , 1984 ) . coronary artery bypass surgery recently came under scrutiny in the coronary artery surgery study ( cass ) sponsored by the national heart , lung , and blood institute and conducted during 1974 - 79 . ( 1983 ) noted a perioperative ( within 30 days ) mortality rate of 5.2 percent for patients over 65 years of age , which is in line with the medicare mortality rate of 6.15 percent within 1.5 months of surgery reported previously . was significantly higher than the 1.9 percent mortality experienced by the cass population under 65 years of age . gersh et al . also reported a trend toward increased mortality with age for the population over 65 years of age , which is also a finding of our study ( cited earlier ) . ( 1985 ) report an overall perioperative mortality rate of 2.7 percent ( based on a literature review of several different studies ) , which is a substantially lower rate than that exhibited by the medicare population over 65 years of age . also report a composite perioperative mortality rate of 6.5 percent for patients over 69 years of age . in a separate series of reports ( coronary artery surgery study , 1983a and b ) , the cass investigators concluded , on the basis of a randomized trial , that coronary artery bypass surgery does not increase life expectancy when compared with medical treatment for certain patients under 65 years of age with moderate levels of stable angina ; however , some evidence was presented that quality of life is improved by the surgery compared with medication only . other controlled trials have shown that certain high - risk patients have better long - term survival with coronary artery bypass surgery ( european coronary surgery study group , 1982 ; and veterans ' administration coronary artery bypass surgery cooperative study group , 1984 ) . because the controlled trials , with the exception of the veterans ' administration study , did not include aged patients , the high mortality reported for the aged in this and other studies raises the question of whether coronary artery bypass for the aged has comparable benefits in longevity . for some study procedures , higher than average mortality rates persisted for many months after surgery for all age groups . in particular , mortality rates associated with turp , cholecystectomy , hip arthroplasty ( other ) , and femur fracture reduction remained higher than the rates in the overall medicare population for a full year following surgery . it is not known how these patients would have fared without surgery , nor are the outcomes known for medicare patients who forego these operations when faced with the choice . ( in the case of femur fracture reduction and hip arthroplasty [ other ] , the procedures are generally not elective . ) the persistence of higher than average mortality rates suggests investigation into other unfavorable outcomes that follow surgery . the mortality rates were the highest for all procedures for the oldest group ( 85 years of age or over ) , e.g. , in the first 1.5 months , 6.3 percent for turp patients and 11.6 percent for cholecystectomy patients . the fact that mortality rates increase sharply with age raises the question of whether certain elective procedures should be performed at younger ages if it appears that surgery may eventually be necessary . wennberg et al . ( 1980 ) address this issue for cholecystectomy for silent gallstones and prostatectomy for prostatic hypertrophy ; fitzpatrick et al . ( 1977 ) also explore this dilemma in the case of cholecystectomy for silent gallstones . they point out that surgery performed at younger ages , when symptoms are minimal , will produce less risk than surgery done later as an emergency . on the other hand , elective surgery does present significant risk , and the patient 's symptoms may not worsen over time , or the patient may die of other causes in the meantime . although conclusive answers on these issues must come from more detailed clinical studies , the information on numbers of deaths and age - specific mortality rates contained in this study may suggest areas for further research . the large number of post - surgical deaths after discharge from the hospital suggests that studies of post - surgical mortality that are limited to in - hospital deaths may be underestimating the risks of surgery or of the conditions leading to surgery . clearly , studies of surgical outcomes should follow patients for a few months after surgery , with the length of time depending on the procedure . age - adjusted and sex - adjusted mortality rates were lower in the west than elsewhere for most study procedures . we examined the hypothesis that enrollees in the west may undergo surgery more often than elsewhere , and that therefore hospitals in the west may operate on more low - risk patients , with lower mortality rates as a result . we concluded that the data do not exhibit any systematic relationship between mortality rates and surgery rates across regions for these study procedures , although a relationship may hold for individual operations . more research needs to be done in this area to determine if patterns of medical practice , criteria for patient selection , or differences in patient risk factors are responsible for the better mortality experience in the west . the kind of data presented in this article should be useful to other researchers for several purposes . for example , the data can provide a baseline for comparison with data describing local experiences . mortality statistics similar to those in this article can also be monitored over time , either nationally or locally , to help assess the impact of new technologies and procedures . many operations are developed and evaluated largely on the population under 65 years of age . but these operations are then performed at an increasing rate on older patients as physician experience grows and operative techniques are perfected . data such as these can monitor the outcomes among the aged of such procedures ( e.g. , coronary artery bypass surgery ) . additionally , because these data represent the total national experience of the aged , they supplement data from clinical trials and prospective studies , which often are based in major medical centers that may have better than average outcomes . thus these data can monitor the outcome experience of new procedures as they diffuse from leading medical centers into other hospitals . data such as these can be used to help evaluate the impact on quality of changes in the organization and financing of medical care . for example , secular trends in mortality can provide insights into the impact of medicare 's new prospective payment system for hospitals . the quality and completeness of medicare diagnostic and procedure data are expected to improve . since 1984 , hcfa has been collecting data on up to five diagnoses and three surgical procedures per stay on 100 percent of all hospitals stays . data quality will be monitored carefully because , under prospective payment , diagnostic and surgical data will be the most important single items in determining payments to hospitals . thus , the usefulness of the kind of data this article presents should be enhanced . in an effort to verify the accuracy of the observed number of deaths occurring within 1.5 months of surgery , age - specific and sex - specific mr 's were recalculated for patients undergoing surgery between the 10th and 20th of the month for femur fracture reduction and coronary artery bypass . by restricting this subsample to surgeries performed in the middle of the month , measurement error in identifying the number of deaths that occured within 6 weeks of surgery the mr 's for these cases were then compared with those calculated on the basis of the entire sample . as shown in table 10 , agreement between the two groups as expected , the mr 's for the larger cells tend to agree more closely than those for the smaller cells . lastly , the distribution of surgeries over days of the month was examined to determine if more surgeries tend to occur early or late in the month . if such a tendency exists , the number of deaths occurring within the same month as surgery or the subsequent month could systematically overestimate ( or underestimate ) the number of deaths occurring within 6 weeks of surgery . the number of operations performed on days 1 - 10 ( pooled across procedures ) was compared with those performed on days 21 - 30 , with an appropriate adjustment to account for february 's fewer days . a total of 92,389 operations were performed on days 1 - 10 , whereas 89,811 ( adjusted ) were performed on days 21 - 30 . although the difference is statistically significant ( p<.001 ) , the numbers of procedures are close enough so that any substantial impact on mortality statistics is unlikely . the slightly skewed distribution of surgeries during the month would tend to positively bias the estimates of observed deaths in the first 6 weeks . this bias would be offset to some extent : because of the rapid decline in death rates following surgery , more deaths will tend to be missed in weeks 5 - 6 than will be incorrectly counted from weeks 7 - 8 .

this study examines post - surgical mortality , up to 1 year after surgery , for eight common operations among aged medicare enrollees . the operations with the highest mortality in the 1.5 months after surgery were femur fracture reduction , hip arthroplasty ( other , i.e. , not total replacement ) , and coronary artery bypass . mortality was still above average for femur fracture reduction , hip arthroplasty ( other ) , and transurethral prostatectomy 1 year after surgery . the highest mortality rates following surgery were for people 85 years of age or over . this raises the following question : should certain elective surgery be performed at younger ages if it appears that surgery may eventually be needed ?

Introduction Methods Findings Conclusions Technical note

for the population 64 years of age or under , surgery rates were 74 per 1,000 in 1965 and 92 per 1,000 in 1977 , a 24-percent increase . these advances include improved techniques of lens extraction , the development of coronary artery bypass surgery , and the emergence of new materials in the field of orthopedic surgery . accompanying these advances have been developments in anesthesiology and post - surgical care that allow physicians to operate on aged patients who would not , formerly , have been considered good candidates for surgery . the purpose of this article is to present patterns of post - surgical mortality , including deaths occurring both in the hospital and after discharge . mortality after eight common surgical procedures is studied for medicare enrollees 65 years of age or over . these procedures were selected because ( with the exception of coronary artery bypass ) they are among the most frequently performed on the aged . coronary artery bypass was chosen because it represents a highly technical procedure performed with increasing frequency , and new information on the risks and benefits has recently appeared ( coronary artery surgery study , 1983a and b ) . these procedures , with the exception of femur fracture reduction , are elective to some degree ( cageorge et al . many studies have found large geographic variations in the incidence of surgery , apparently unrelated to variations in need , within areas of new england ( mcpherson et al . , 1982 ) and within the states of kansas ( lewis , 1969 ) , wisconsin ( detmer and tyson , 1978 ) , and new york ( lembcke , 1952 ) . if some of the operations performed in areas with high rates of surgery are really not necessary , then the mortality associated with this surgery may also not be necessary . mortality is not limited to in - hospital deaths but is tracked for periods up to a year after surgery . variations in risk among age and sex categories are presented , and comparisons are drawn among regions of the united states in terms of mortality rates . because about 95 percent of all people 65 years of age or over are covered by medicare , these data generally represent the surgical outcomes of the aged in the united states and thus expand on studies limited to the surgical outcomes in selected hospitals or groups of hospitals . in addition , these data expand on those studies because they track deaths that occurred up to 12 months after discharge . , size , location ) , and information about the stay ( e.g. , principal surgical procedure , principal diagnosis , and patient survival in the hospital ) . using the international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) , in 1979 and 1980 the principal surgical procedure and principal diagnosis were coded for each discharge in the sample . the records of hospital stays for the eight selected operations were linked to the enrollment file to identify all deaths , regardless of where the deaths occurred , for a 12-month period following surgery . rehospitalizations for a study procedure were not excluded , i.e. assuming that dates of surgery are distributed uniformly throughout the month , we defined deaths within the first 1.5 months of surgery as those occurring in the month of surgery and the following month . similarly , deaths within 2.5 months were defined as those occurring in the month of surgery and the following 2 months . because the study data constitute a 20-percent sample of discharges for selected procedures , there is sampling error associated with the estimates presented in the tables . lastly , the study does not include medicare disabled beneficiaries ( under 65 years of age ) , who constitute approximately 10 percent of the medicare population . ( the annual number of noncancer related discharges among aged enrollees for these procedures is given in the technical note , table b. ) in the united states in 1979 , 86 percent of hip arthroplasties ( other ) , 76 percent of lens extractions , 74 percent of turp 's , 69 percent of femur fracture reductions , and 57 percent of hip arthroplasties ( total hip replacements ) were performed on people 65 years of age or over ( national center for health statistics , 1982 ) . however , for cholecystectomy , inguinal hernia repair , and coronary bypass , between 23 and 28 percent of each procedure was performed on aged people . the number of discharges per 10,000 enrollees for hip arthroplasty ( other ) and femur fracture reduction increased sharply with age , whereas the incidence of coronary artery bypass decreased ( table 1 ) . there are also marked differences in discharge rates between males and females , with males exhibiting higher rates at all ages for coronary bypass and hernia repair , whereas females exhibit higher rates for hip arthroplasty of both types and femur fracture reduction . the average annual number of deaths occurring within the first 1.5 months following surgery is shown in table 2 . the largest number of deaths , 4,595 annually , occurred after femur fracture reduction . among hip arthroplasty ( other ) patients , deaths following both operations were greatest among patients 85 years of age or over . among the eight procedures studied , deaths per 10,000 operations in the 1.5 months following surgery were highest following femur fracture reduction ( 878 ) , hip arthroplasty ( other ) ( 750 ) , and coronary artery bypass ( 615 ) ( table 3 ) . the death rate for males at all ages was greater than that for females at all ages , with the exception of coronary artery bypass at all ages and inguinal hernia repair among those 65 - 74 years of age . the difference in death rates by sex was especially large for femur fracture reduction , with males experiencing approximately twice the mortality of females in the 1.5 months following surgery . higher in - hospital mortality rates for females undergoing coronary artery bypass surgery have been noted in two other studies ( fisher et al . , 1981 ) , although both of these studies reported that the difference in mortality between males and females was not statistically significant for the group 60 years of age or over . differences in coronary artery bypass mortality by sex may be related to the fact that females have smaller physical stature and smaller coronary arteries ( fisher et al . the similarity in mortality rates between patients undergoing femur fracture reduction and those undergoing hip arthroplasty ( other ) is because of the high incidence of hip fracture among the latter group . somewhat surprisingly , hip arthroplasty ( other ) patients without a principal diagnosis of femur fracture exhibited mortality patterns similar to patients with a fracture . mortality rates for patients undergoing total hip replacement varied considerably according to whether a principal diagnosis of femur fracture was present ( table 4 ) . higher mortality rates for patients with a fracture may reflect the effects of the injury , as well as the fact that the surgery is less likely to be elective , and many cases may be poor surgical risks . in fact , those total hip replacement patients with a diagnosis of femur fracture exhibited mortality rates similar to patients undergoing femur fracture reduction and hip arthroplasty ( other ) . a diagnosis of femur fracture is much more frequent among the oldest arthroplasty ( total replacement ) patients ( table 4 ) . only 5.1 percent of those 65 - 74 years of age exhibited a principal diagnosis of femur fracture , whereas 35.9 percent of those 85 years of age or over showed this diagnosis . females were generally more likely to exhibit a diagnosis of femur fracture than males , although females ' mortality rates for total hip replacement tended to be lower . the age - specific and sex - specific mortality risk of the study group during the 1.5 months following surgery , relative to the experience of the medicare aged population at large , is shown in table 5 . the comparisons are presented in the form of mortality ratios ( mr ) , i.e. although observed mortality rates for specified procedures are compared with the prevailing mortality rate in the medicare population , this should not be taken as an indication that post - surgical patients are truly expected to experience prevailing mortality rates . the highest mr 's among the eight procedures studied are associated with coronary artery bypass , with an overall mr of 12.54 during the 1.5 months following surgery ( table 5 ) . in the group 65 - 74 years of age , which accounts for most of the bypass operations , males have mr 's of 9.41 and females , 30.70 . as discussed previously , higher mortality for females undergoing coronary artery bypass surgery is also reflected in the age - specific deaths per 10,000 operations ( table 3 ) . patients undergoing femur fracture reduction and hip arthroplasty ( other ) also had high mr 's ; for femur fracture reduction , mr 's exceeded 9 for all groups except females 85 years of age or over , and for hip arthroplasty ( other ) the mr 's were 8 or more for all age - sex groups except females over 85 years of age . for females , the mr 's decrease sharply with age for femur fracture reduction and hip arthroplasty ( other ) , whereas mr 's are relatively steady for males . the mr for males with inguinal hernia repair increases with age1.08 for males 65 - 74 years of age , 1.67 for males 75 - 84 years of age , and 2.15 for males 85 years of age or over . this most likely reflects the fact that the proportion of males with a principal diagnosis of obstructed hernia increases with age from 4.3 percent for males 65 - 75 to 14.9 percent for those 85 or over , and the mortality rate associated with these patients is considerably higher than for other patients ( data not shown in tables ) . higher mortality rates for patients with obstructed hernias probably reflect , in part , the fact that inguinal hernia repair for these patients is not elective surgery , and many may be poor surgical risks . coronary artery bypass patients also exhibited a decreasing mr with age , from 13.12 for patients 65 - 74 years of age to 10.33 for patients 75 - 84 years of age . among all operations , males tended to have lower mr 's than did females in the 65 - 74 years of age group and higher mr 's in the 85 years of age or over group ( with the exception of total hip replacement ) . the cumulative percents of death following surgery , unadjusted for age and sex differences , for various time periods are shown in table 6 . mortality after 12.5 months was highest for patients undergoing femur fracture reduction ( 23.9 percent ) , and hip arthroplasty ( other ) ( 21.1 percent ) , reflecting the higher age of this group , as well as the complications associated with a fractured femur . this high mortality rate is similar to that reported by jensen and tondevold ( 1979 ) for hip fracture patients , 26.8 percent after 1 year , and less than that reported by beals ( 1972 ) , 50 percent after 1 year , and evans et al . lens extraction ( 5.3 percent ) , total hip replacement ( 5.9 percent ) , and inguinal hernia repair ( 6.4 percent ) patients had the lowest mortality after 12.5 months . for all operations except lens extraction , the indirectly standardized mortality ratio ( ismr ) drops substantially after the first 1.5 months following surgery . the ismr is highest for coronary artery bypass patients in the first 1.5 months ( 12.54 ) , but declines very quickly thereafter , and is not significantly different from 1 after 3.5 months . femur fracture reduction and hip arthroplasty ( other ) patients exhibit the highest ismr 's after the first 1.5 months and remain the highest throughout the rest of the first year . the ismr for turp is not extremely high initially ( 2.49 in the first 1.5 months ) , but does not decline as rapidly as for other operations , stabilizing in the area of 1.3 to 1.4 after a few months . after the first 1.5 months , the ismr for inguinal hernia repair is not significantly different from 1 . elevated ismr 's , over a several - months period , may be related to these patients ' underlying poor health , which led to surgery in the first place , or the elevated ismr 's may be related to long - term complications following surgery . total hip replacement patients exhibit a very unexpected pattern in that their ismr is high initially ( 3.43 in the first 1.5 months and 1.41 between 2.5 and 3.5 months after surgery ) , but it drops significantly below 1 after approximately 6 months . the rate of in - hospital deaths is compared with the rate in the 1.5 months following surgery in table 8 . a number of studies of post - surgical mortality have been limited to data on in - hospital deaths . this study indicates that such studies probably miss a substantial proportion of post - surgical deaths . in - hospital deaths as a percent of all deaths occurring within 1.5 months of surgery are shown in table 8 ; the assumption used is that all in - hospital deaths occur within 1.5 months of surgery . only for coronary artery bypass did the rate of in - hospital deaths , as a percent of all deaths within 1.5 months following surgery , exceed 70 percent . regional data ( with region defined as region of patient residence ) on age - adjusted and sex - adjusted mortality rates within 1.5 months of surgery are provided in table 9 . the west has the lowest mortality rate of all four regions for four of the seven procedures : turp , femur fracture reduction , coronary artery bypass , and hip arthroplasty ( other ) . these results are similar to luft 's ( 1980 ) findings , which showed generally lower rates of in - hospital post - surgical mortality in the west . a possible explanation for the lower mortality rates in the west is that rates of surgery there are higher than the national average for some procedures . to examine the possible relationship between surgery rates and mortality , a spearman 's rank correlation coefficient was computed for the regional mortality rates and age- and sex - adjusted surgery rates ( technical note , table d ) . for example , the surgery rate for coronary artery bypass is much higher in the west than elsewhere ( 10.8 per 10,000 in the west for the 65 - 84 years of age population versus 7.2 per 10,000 nationally ) , and the mortality rate there is much lower . over 6 percent of the coronary artery bypass patients died within 6 weeks of surgery , and approximately 8.8 percent of the patients undergoing femur fracture reduction died within the same time period . hospital use and mortality associated with femur fracture reduction and , to a large extent , hip arthroplasty ( other ) , reflect the widespread problem of osteoporosis among the aged , particularly females . the high mortality rate following femur fracture reduction , as well as hip arthroplasty ( other ) in the presence of fracture , indicates how serious the problem of osteoporosis is among the aged . coronary artery bypass surgery recently came under scrutiny in the coronary artery surgery study ( cass ) sponsored by the national heart , lung , and blood institute and conducted during 1974 - 79 . ( 1983 ) noted a perioperative ( within 30 days ) mortality rate of 5.2 percent for patients over 65 years of age , which is in line with the medicare mortality rate of 6.15 percent within 1.5 months of surgery reported previously . was significantly higher than the 1.9 percent mortality experienced by the cass population under 65 years of age . also reported a trend toward increased mortality with age for the population over 65 years of age , which is also a finding of our study ( cited earlier ) . ( 1985 ) report an overall perioperative mortality rate of 2.7 percent ( based on a literature review of several different studies ) , which is a substantially lower rate than that exhibited by the medicare population over 65 years of age . also report a composite perioperative mortality rate of 6.5 percent for patients over 69 years of age . in a separate series of reports ( coronary artery surgery study , 1983a and b ) , the cass investigators concluded , on the basis of a randomized trial , that coronary artery bypass surgery does not increase life expectancy when compared with medical treatment for certain patients under 65 years of age with moderate levels of stable angina ; however , some evidence was presented that quality of life is improved by the surgery compared with medication only . other controlled trials have shown that certain high - risk patients have better long - term survival with coronary artery bypass surgery ( european coronary surgery study group , 1982 ; and veterans ' administration coronary artery bypass surgery cooperative study group , 1984 ) . because the controlled trials , with the exception of the veterans ' administration study , did not include aged patients , the high mortality reported for the aged in this and other studies raises the question of whether coronary artery bypass for the aged has comparable benefits in longevity . for some study procedures , higher than average mortality rates persisted for many months after surgery for all age groups . in particular , mortality rates associated with turp , cholecystectomy , hip arthroplasty ( other ) , and femur fracture reduction remained higher than the rates in the overall medicare population for a full year following surgery . it is not known how these patients would have fared without surgery , nor are the outcomes known for medicare patients who forego these operations when faced with the choice . ( in the case of femur fracture reduction and hip arthroplasty [ other ] , the procedures are generally not elective . ) the mortality rates were the highest for all procedures for the oldest group ( 85 years of age or over ) , e.g. , in the first 1.5 months , 6.3 percent for turp patients and 11.6 percent for cholecystectomy patients . the fact that mortality rates increase sharply with age raises the question of whether certain elective procedures should be performed at younger ages if it appears that surgery may eventually be necessary . they point out that surgery performed at younger ages , when symptoms are minimal , will produce less risk than surgery done later as an emergency . on the other hand , elective surgery does present significant risk , and the patient 's symptoms may not worsen over time , or the patient may die of other causes in the meantime . although conclusive answers on these issues must come from more detailed clinical studies , the information on numbers of deaths and age - specific mortality rates contained in this study may suggest areas for further research . the large number of post - surgical deaths after discharge from the hospital suggests that studies of post - surgical mortality that are limited to in - hospital deaths may be underestimating the risks of surgery or of the conditions leading to surgery . clearly , studies of surgical outcomes should follow patients for a few months after surgery , with the length of time depending on the procedure . age - adjusted and sex - adjusted mortality rates were lower in the west than elsewhere for most study procedures . we examined the hypothesis that enrollees in the west may undergo surgery more often than elsewhere , and that therefore hospitals in the west may operate on more low - risk patients , with lower mortality rates as a result . , coronary artery bypass surgery ) . in an effort to verify the accuracy of the observed number of deaths occurring within 1.5 months of surgery , age - specific and sex - specific mr 's were recalculated for patients undergoing surgery between the 10th and 20th of the month for femur fracture reduction and coronary artery bypass . by restricting this subsample to surgeries performed in the middle of the month , measurement error in identifying the number of deaths that occured within 6 weeks of surgery the mr 's for these cases were then compared with those calculated on the basis of the entire sample . this bias would be offset to some extent : because of the rapid decline in death rates following surgery , more deaths will tend to be missed in weeks 5 - 6 than will be incorrectly counted from weeks 7 - 8 .

habitual physical activity ( hpa ) is an established determinant of health in children and is required for healthy development , including the growth of bone and muscle mass , improved balance and motor skills , maintaining a healthy weight and improved psychological wellbeing [ 1 , 2 ] . limited evidence suggests young children with motor disorders are less physically active than their typically developing peers . consequently they may be at risk of suboptimal growth and development in addition to the development of secondary conditions such as chronic pain , fatigue , and low bone density which can lead to diminished bone health [ 4 , 5 ] . australian physical activity guidelines state children aged from one to five years should be physically active for three hours throughout the day and should not be sedentary , restrained , or kept inactive , for more than one hour at a time , with the exception of sleeping . studies investigating the link between hpa and health outcomes for children with motor impairments have not been conducted . these studies are urgently needed to ( i ) determine the hpa patterns and intensities of children with motor disabilities , ( ii ) support the importance of physical activity promotion and inactivity prevention , ( iii ) determine the dose - response relationship between physical activity and health outcomes , and ( iv ) allow assessment of the effectiveness of interventions aimed at increasing hpa . physical activity is defined as any bodily movements using skeletal muscles that results in energy expenditure . the international classification of functioning , disability and health ( icf ) further divides physical activity into the concepts of performance and capacity . this is distinct from capacity which refers to a child 's maximal ability to perform an activity in an ideal or standardised environment . performance and capacity to complete a task ( e.g. , jumping ) can be determined by observing the child undertake that specific task within a limited time frame in their natural and ideal environment , respectively . hpa , on the other hand , describes a child 's typical daily activity pattern and hence includes their performance of a multitude of activities which necessitates measurement across multiple days . the variables of frequency ( how often a child does an activity ) , intensity ( how hard a child works to do the activity ) , duration ( how long a child does an activity ) , and mode ( what the child is doing ) are used to characterise hpa patterns , while activity - related energy expenditure ( aee ) is used as a summary variable of all the other indicators [ 9 , 10 ] . direct observation is considered the gold standard for physical activity classification , while doubly labelled water in combination with resting energy expenditure is considered the gold standards for calculating aee . the measurement of hpa in typically developing children ( tdc ) has received a great deal of attention in the last decade , primarily due to a concern over the increasing rate of overweight and obesity in childhood . a systematic review of pedometers and accelerometers has identified moderate - to - good evidence of validity and strong evidence of reliability for the actigraph accelerometer in preschool - aged tdc . children with motor impairments may not be walking but instead cruising , crawling , bottom shuffling , rolling , use walking aids or a wheelchair as means of locomotion , and those who are walking may have different gait patterns and speed of movement than tdc . this variation in movement patterns necessitates validation of motion sensors specifically for this population . systematic reviews of measures of physical activity in children and adolescents with cerebral palsy ( cp ) have been conducted [ 15 , 16 ] . included children with cp from birth to 18 years and concluded that the subjective measures of the activities scale for kids - performance version ( askp ) and the children 's assessment of participation and enjoyment ( cape ) are the only measures with established validity and reliability in children with physical disabilities from the age of six years . the review by clanchy et al . included children with cp aged 1018 years and identified the cape as the measure with the strongest evidence of reliability and validity . it was also noted that no measures have been assessed in children with more severe disability who are wheelchair dependent . this paper did not include the askp as it did not meet inclusion criteria of 60% of items relating to a domain of physical activity performance . this was done to ensure the included measures assess physical activity as traditionally defined by caspersen et al . and not participation which does not take into consideration the sedentary or active nature of the activity . both reviews note the lack of hpa measures which can assess activity intensity , which limits their ability to provide meaningful comparison with physical activity guidelines , as children from the age of six years are advised to accumulate 60 minutes of moderate - to - vigorous physical activity every day . this current paper aims to systematically review the clinimetric properties ( validity , reproducibility , and clinical utility ) of measures of hpa in children less than 6 years of age with a motor disability . a systematic literature search was performed by one reviewer ( so ) of the electronic bibliographic databases pubmed , cinahl , embase , sportdiscus , and web of science from their inception to september , 2011 . databases were searched using medical subject headings ( mesh ) terms and text words for physical activity and disability , limiting the search to the age group < 6.0 years . measures of hpa were included which met the following criteria : ( i ) included children less than six years of age with a motor disability caused by neuromuscular disorders ; ( ii ) defined physical activity as any bodily movement produced by skeletal muscles that results in caloric expenditure ; ( iii ) reported physical activity in terms of duration , frequency , intensity , or energy expenditure ; ( iv ) questionnaires where at least 60% of items related to a domain of hpa ; ( v ) evaluated clinimetric properties for the measurement of hpa . studies were excluded if they were ( i ) not published in english , due to lack of translation services ; ( ii ) primarily measured capacity or participation . the title and abstract of all retrieved references were screened by the first author to exclude any papers which did not include children in the target age group or were not on the topic of physical activity measures . a second screening of abstracts was performed by two authors ( so , lm ) independently to exclude those that did not include habitual physical activity measures but rather assessed capacity or participation measures . for the remaining abstracts , full papers were retrieved and screened by two authors ( so , lm ) . publications which did not assess the clinimetric properties of measures of hpa in children with motor disabilities were excluded . upon disagreement between the two reviewers , a third reviewer ( kb ) reviewed the publication in question . the reference lists of included papers and relevant reviews were screened , and electronic author and citation tracking were performed when available , to identify relevant publications not identified by the initial search strategy . for measures where clinimetric properties were found in children less than six years of age with a motor disability , a further search identified any evidence in typically developing children in the same age group . the cosmin ( consensus - based standards for the selection of health measurement instruments ) checklist was used to rate the methodological quality of the study designs used to evaluate the clinimetric properties validity and reproducibility for measures of hpa . the cosmin was developed through an international delphi study to assess the methodological quality of studies on measurement properties of health - related patient - reported outcomes ( hr - pros ) ; however the measurement properties are also relevant to other health - related measurement instruments . the scoring system was developed based on expert discussion and testing on 46 articles identified by a systematic review and uses a each item within a specific measurement property is rated individually as excellent ( + + + ) , good ( + + ) , fair ( + ) , or poor ( ) and an overall score is given by taking the lowest score of any of the items within the assessment of the measurement property . measurement properties included in this paper were measurement error ( cosmin box c ) , hypothesis testing ( cosmin box f ) , and criterion validity ( cosmin box h ) . the methodological quality rating of the papers was separated into study design and statistical analysis . the study design for the assessment of measurement properties was rated as having excellent quality if all relevant items within a given checklist scored excellent . study design was rated as good if information for some items was not complete and therefore could not be scored excellent , though it could be assumed these were of good quality . a fair quality rating was given if there were minor flaws in the design . if the results were not to be trusted because of major flaws in the design , a study is rated as poor . to assess if there were any important flaws in the design or methods of the study ( cosmin box h , item five ) , items from the systematic review of activity monitors in tdc were used as a guide in addition to whether or not the testing protocol included free - living activities . proposed sample size requirements were applied in the clinimetric properties section instead of the study design section . the decision to separate study design and sample size requirements was made as sample size does not determine study design quality as such but does affect the statistical power available to detect a significant result . for the assessment of criterion validity , the cosmin checklist accepts correlations or the area under the receiver operator characteristic ( auc - roc ) curve as excellent statistical methods for continuous measures . for dichotomous scales sensitivity and specificity calculations are considered excellent measures . for the assessment of concurrent validity ( hypothesis testing ) it is up to the reviewer to assess if the method is appropriate and therefore scores excellent . for the assessment of measurement error ( agreement ) , standard error of measurement ( sem ) , smallest detectable change ( sdc ) , and limits of agreement ( loa ) calculated using the bland - altman method are accepted as excellent . according to the cosmin a sample size of less than 30 participants is considered poor , 5099 participants is considered good , and 100 or more participants is considered excellent . authors were contacted to see if they had used any power calculations when determining their sample size . quality criteria for the clinimetric properties of measures were proposed to give a framework to help distinguish between the quality of the studies and the performance of the measurement tools . agreement , criterion validity , and construct validity were therefore scored as good ( + ) , indeterminate ( unable to assess : u / a ) , or poor ( ) . good if correlation with gold standard was 0.70 , sensitivity and specificity 0.70 , and area under receiver operating characteristic curve 0.70 . . measurement error is considered good if the sdc or the loa are smaller than the minimal important change or if authors provide convincing arguments that the agreement is acceptable in a sample size of at least 50 participants . studies that used a poor statistical method scored indeterminate for performance of clinimetric properties . the assessment of clinimetric properties of hr - pro questionnaires might be of a different nature to those assessing activity measures due to the availability of gold standards , something which is not readily available for hr - pros as these are usually questionnaires regarding patient outcomes such as pain and quality of life . the statistical methods and performance of clinimetric properties were therefore scored according to the cosmin but were also discussed further , and a different score was given if authors presented a convincing argument that the measure was acceptable . the clinical utility of measures of hpa was assessed in terms of the need for individual calibration of the equipment , the interpretability of the data output , and the need for software analysis , cost of equipment , and any required software . the feasibility of the equipment for using children aged less than six years with a motor disability in free living situations was also considered ( size , weight , number of sensors , place worn , ease of correct placement , and battery life ) . the richness of data was also assessed in terms of what aspects of hpa ( frequency , duration , intensity , mode , and energy expenditure ) it was able to assess in children with a motor disability . the overall level of evidence for the clinimetric properties of measures of hpa in young children with motor disabilities was determined as a whole by assessing the methodological quality of study design and statistical methods used in the assessment of hpa measures using the worst score counts algorithm , agreement between studies on the quality of clinimetric properties of these hpa measures , and the number of studies available for each hpa measure . the initial screening by titles and abstracts excluded 1567 titles , and the second screening excluded a further 171 papers . four papers and one abstract from conference proceedings on the clinimetric properties of four measures of hpa in children with motor disabilities were identified which included children aged less than six years in their sample . none of the studies focused exclusively on children aged less than six years , which led to the inclusion of studies with a wide age range ( 418 years ) . evidence of clinimetric properties was found for three accelerometer - based activity monitors [ 2226 ] and one pedometer using inertial sensors . a paper by kuo et al . and an abstract from conference proceedings by brandes et al . the activity monitoring pod-331 pedometer which uses inertial sensors ( amp ; dynastream innovations , alberta , canada ) was also assessed in the paper by kuo et al . . papers by stevens et al . and mcdonald et al . were identified for the stepwatch pedometer ( orthocare innovations , wa , usa ) . a paper by aviram et al . assessed the intelligent device for energy expenditure and activity ( ideea ; minisun , ca , usa ) . only mcdonald et al . assessed children with duchenne muscular dystrophy ( dmd ) ; others assessed children with cp who were able to walk with or without aids ( gross motor function classification system levels i iii ) [ 2224 , 26 ] . none of the study samples included children who did not walk as their main means of locomotion . all of the studies of children with motor disabilities included tdc as a reference group [ 2226 ] . sample sizes ranged from 17 to 27 children with motor disabilities and 727 in the tdc reference group . all authors were contacted to clarify the proportion of children in their study who were under the age of six . the study by brandes et al . on the use of the minimod included one child ( 5% ) with cp aged five years old and four children ( 20% ) in the tdc sample aged three to five years . the study by aviram et al . included nine children ( 43% ) aged four to five years with three children in each gmfcs category ( i iii ) . the authors did not supply the number of tdc aged five years , but the youngest of the seven children in the tdc group was 5 years and 8 months old . in the study by kuo et al . it was estimated that a maximum of two children in the cp ( 11% ) and tdc ( 10% ) group were less than six years old , although authors were not able to readily access information to confirm this . it is not known how many children were under the age of six in the study by stevens et al . and mcdonald et al . . the targeted search for studies reporting on the clinimetric properties of the identified measures in young tdc identified a further three studies assessing the minimod and the stepwatch [ 28 , 29 ] . the sample size in the three studies ranged from 20 to 162 children , and ages ranged from 2 to 16 years . the stepwatch study by bjornson et al . reported results for two - to - three - year olds ( n = 60 ) and four - to - five - year olds ( n = 62 ) tdc separately . the stepwatch study by song et al . used two age bands ( 57 years and 911 years ) with ten children in each group , but the number of five year old children in the youngest group was not specified by the authors upon request . in the paper by brandes et al . , authors report on the same sample of tdc ( n = 20 , four children 6.0 years ) as in the previously mentioned abstract from conference proceedings . none of the authors using manual step count as a criterion method reported on the accuracy of the manual step count ( intra- or interrater agreement ) . although it can be assumed that it is a gold standard , no evidence has been provided , and therefore the maximum attainable score for all studies according to the cosmin checklist was good . [ 22 , 27 ] assessed the criterion validity of the minimod in children with cp and tdc compared to manually counted steps and meters walked was rated authors report complete information in regarding test protocol ( monitor settings , placement , data output , average steps needed for length walked , and range of steps across group ) but only tests of continuous walking that may not adequately reflect free - living walking activity . the study by kuo et al . on the criterion validity of the minimod and amp compared to manually counted steps and measured meters walked was rated authors report complete information on test protocol and use of a variety of structured activities : continuous walking ; activity lap walking which includes walking , stopping , completing a simple task , and walking again ; stair ascent and descent . the studies by stevens et al . in children with cp , mcdonald et al . in children with dmd , and song et al . in tdc all assessed the criterion validity of the stepwatch against manual step count and scored poor for study design due to providing no information on parts of their study protocol ( number of steps , range of steps needed , and length walked ) . this does not necessarily mean the design was genuinely poor , but the lack of reporting does not enable assessment on applicability in measuring habitual walking activity and has implications for the interpretation of any reported statistics . the study by bjornson et al . assessed the stepwatch for criterion validity in tdc against manual step counts and was rated good for study design as they report a complete test protocol , though only assessed performance during continuous walking . the study by aviram et al . assessed the criterion validity of energy expenditure ( ee ) measured by the ideea compared to the gold standard of indirect calorimetry using the cosmed ( k4b2 , rome , italy ) which measured oxygen consumption ( vo2 ) in five - second epochs . authors reported complete study protocol information and assessed a range of everyday activities in addition to comfortable and fast treadmill walking and stair climbing . the study scored good for study design due to not providing any evidence of the cosmed being a gold standard measurement although it can be assumed adequate . authors discuss possible limitations of the use of the cosmed , which include a poor fitting mask , which may lead to inaccurate measurement . this study also assessed the test - retest reliability of the ideea and received an the stepwatch study in children with dmd used a heart rate monitor to assess its concurrent validity over four days of wear . this study was rated fair for study design in the assessment of this construct as an a priori hypothesis of the relationship between the stepwatch and heart rate was not stated , but it was possible to deduce what was expected . all studies apart from the study by bjornson et al . had a sample size of less than 30 children which constitutes a poor score on the cosmin checklist , while the sample size of 6062 children in each age group found in the bjornson et al . three of the four authors who replied had not used a power calculation to determine sample size [ 22 , 23 , 26 , 27 ] , and one author stated in their reply that the recruitment of 60 children in each group ( 30 boys and 30 girls ) was chosen as it was expected this would increase the likelihood of approximating a normal distribution . mcdonald et al . used any power calculations to determine their sample size . as the cosmin works on a worst score counts algorithm , the highest score possible for all but one study is therefore poor , but as several studies report significant results , further discussion about the clinimetric performance of measures is warranted . percent agreement was used to assess the criterion validity of the minimod in children with cp and tdc by brandes et al . [ 22 , 27 ] , and the use of this method leads to a poor rating for statistical methods according to the cosmin as it comes under any other statistical method . percent agreement is a relative measure and therefore depends on reporting of the absolute values for meaningful interpretation . authors present rich information such as the average steps needed to walk the 20-meter track ( cp children ) and 160-meter track ( tdc ) and range of steps taken [ 22 , 27 ] . children with cp ( n = 19 ) walked an average of 79.8 steps ( min : 57 ; max : 126 ) , and on average the minimod over- or underestimated by one step ( agreement = 98.7% ; range : 94.1 to 101.8% ) [ 22 , 27 ] . tdc ( n = 20 ) walked an average of 273.7 steps ( min : 207 ; max : 377 ) , and on average the minimod over or underestimated by one step ( agreement = 99.6% ; range : 98.5 to 101.5% ) [ 22 , 27 ] . children with cp walked only one - third of the steps tdc walked , and a higher likelihood that agreement occurred by chance exists . due to good reporting of study protocol and results , percent agreement was considered an the minimod showed good accuracy and precision for the measurement of continuous walking . a limitation of this study was the assessment of continuous walking only , and therefore the criterion validity for measuring hpa is indeterminate . the issue of use of percent agreement arises in two other studies assessing the stepwatch in tdc . percent agreement is used by song et al . who compare steps measured and manual step count . they found a measurement error of 3 1% but did not report how long children walked or how many steps they took which does not allow the assessment of absolute measurement error or the likelihood that the error was low by chance . for this same study , a pearson correlation coefficient has been reported in a separate paper which by cosmin standards was an excellent statistical method , but the lack of information about the study protocol still applies as correlation could have been estimated based on a small number of steps . the use of percent agreement and pearson correlation coefficient in this study receives a poor rating , and evidence of criterion validity based on this study was indeterminate . total steps taken are reported as 100 steps , and therefore it is possible to assess the absolute value of agreement in step count for two - three - year olds ( 99.2 4.6% ) and 4 - 5-year olds ( 100.0 4.4% ) . in this study , the use of percent agreement therefore rates as excellent , and the evidence of criterion validity for continuous walking is good which is further strengthened by the large sample size . a further two studies assess the use of the stepwatch in children with cp and dmd but do not report any statistical methods or results which was rated bland - altman plots and percent of activity laps which were detected were used to assess criterion validity in the combined study of the minimod and amp by kuo et al . and due to this the lack of a specific index to summarise the degree of agreement is a limitation of the bland - altman technique , and inferences about the estimate can not be performed . a strength of the bland - altman technique is that it produces a meaningful graph and computes the confidence limits from the paired differences between the criterion method ( manual step count or meters walked ) and the same variables measured by the minimod . this provides us with the ability to assess both accuracy and precision of measures and allows a comparison between groups and therefore still allows a thorough assessment of the agreement between methods and can be rated as an the minimod performed well in continuous walking trials compared to measured length and direct observation of steps ( mean difference = 0.4 m/0.4 steps , 95% limits of agreement = 4.7 to 4 m/4.1 to 3.3 steps ) but showed a larger random error for activity lap walking ( mean diff . = 2.3 m/0.4 steps , 95% loa = 27.9 to 23.3 m/87.8 to 10.4 steps ) . the minimod only detected walking activity in 1937% of stair walking trials ( ascent / descent ) . in tdc , the minimod performed well for continuous walking trials and poor in structured activity laps ( see table 2 ) ; however it performed better for detecting stair walking ( 84% ) . the amp showed greater underestimation and random error in continuous walking trials ( mean diff . = 4.8 m/3.5 steps , 95% loa = 20.1 to 10.5 m/16.9 to 10 steps ) which increased with increasing distance walked . the amp performed better than the minimod in the structured activity lap walking trials , but still showed considerable bias difference and large random error in this trial ( mean diff . = 3.6 m/11.2 steps , 95% loa = 19.2 to 12.0 m/40 to 17.7 steps ) , however detected more of the stair climbing trials ( 85% ) . results were similar for tdc ( see table 2 ) . due to the lack of an index , a specific cut - off for what classifies as a good result can not be set ; however the results indicate that the minimod has good accuracy and precision for continuous walking . when it comes to more free - living type activities such as the structured walking trial and stair walking , the amp showed poor precision and accuracy in both walking trials , however detected more stair trials . as the focus of this paper is the ability to measure free - living activities , both monitors score poor for evidence of criterion validity for measuring hpa . a pearson correlation coefficient was used to assess agreement between the ee measured by the ideea and the criterion method of indirect calorimetry using the cosmed . for the daily activities trial , total energy expenditure was used as the outcome measure . for comfortable and fast treadmill walking and stair climbing trials , energy expenditure rate ( kcal / minute ) was used . for children with cp and tdc the correlation between measurement outputs was good by cosmin standards during all activities ( cp : r = 0.700.88 ; tdc : a limitation of the pearson correlation coefficient is that it only measures precision not accuracy and is therefore not a true measure of agreement . this is demonstrated by the authors of this paper as the ideea significantly overestimated energy expenditure during the series of everyday activities and during comfortable treadmill walking both in children with cp and tdc , and during fast treadmill walking in tdc ( paired t - test , p < 0.01 ) . during fast treadmill walking in children with cp and during stair walking in both groups , measured energy expenditure did not differ significantly between methods ( paired t - test , p > 0.01 ) . a limitation of the t - test is that it will only show a significant difference if there is a systematic constant difference between two values , not if there is a systematic proportional difference as the paired difference will end up close to zero . a bland - altman plot or a regression analysis would provide a better identification of the nature of any systematic differences in the ee estimation of the ideea . the present energy expenditure calculations used in the ideea software do not accurately assess energy expenditure , and the clinimetric properties of the ideea are therefore considered poor . authors suggest the good correlation between the ideea and the cosmed indicates it can be used as a clinical follow - up tool for quantitative evaluation of efficacy of treatment interventions . as the ideea appears to perform better at higher intensities , the reliability of ee measurement by the ideea was assessed in children with cp ( n = 12 ) using a pearson 's correlation coefficient and a paired t - test . authors aimed to assess agreement between repeated measures as discussed in de vet et al . . this study therefore scored poor for choice of statistical method as measures can significantly correlate despite being significantly different , and the inability of a t - test to detect systematic proportional differences as discussed previously still applies [ 33 , 34 ] . agreement between repeated measures of ee using the ideea is therefore indeterminate based on this study . authors of the stepwatch study in children with dmd used a heart rate monitor to assess its concurrent validity and received a good rating for statistical method as they used a pearson correlation coefficient but did not report standard deviation . evidence of concurrent validity was rated poor as r < 0.70 ( cp : r = 0.295 ; tdc : r = 0.477 ; p < 0.05 ) . clinical utility was assessed for the four identified measures and is summarised in table 2 . the rich data collected by the stepwatch and minimod allows measurement of intensity , frequency , and duration of walking activity . the units are both small and unobtrusive and have battery lives of seven days for the minimod and up to six weeks for the stepwatch , depending on settings . this makes them both feasible tools for the measurement of habitual walking activity . in the event that data collection is delayed once the device is provided to the child 's parents , having a battery life of more than seven days , and therefore not requiring recharging is a strength of the stepwatch as there is less likelihood of loss of data . the minimod is worn around the waist centred at the lower lumbar spine , while the stepwatch is worn on the ankle in a small cuff . consistency of placement for repeated and all - day wear may be easier to achieve with an ankle placement which would reduce measurement error due to inconsistent placement . the amp is small , worn around the ankle , does not require calibration or software analysis , and has a good battery life ; however it only measures total step count or meters walked per wear period . the ideea consists of a data recorder worn on the waist with 5 individual sensors attached to the chest , thighs , and under each foot connected by wires to allow measurement of postures and energy expenditure , and it only has a battery life of 60 hours . both of these factors limit the ideeas utility in the measurement of hpa in young children . a summary of the quality of evidence for criterion validity is provided in table 3 . two studies assess the minimod in children with cp and tdc in samples which include children under the age of six . one of these studies also used free - living type activities , and the minimod displayed poor accuracy and precision in this setting . the amp was assessed in one study of good study design quality which used an excellent statistical method , though displayed poor accuracy and precision in both continuous walking and free - living type activities . four studies assess the stepwatch in samples which included children under the age of six . reporting of study protocol and statistical method was poor in three of these studies . the fourth study was of good quality and used an excellent statistical method . good evidence of the ability of the stepwatch to accurately measure continuous walking in tdc . the ability of the stepwatch to accurately measure free - living activities can not be determined based on this study . the ability of the ideea to measure ee was assessed in one study of good study design quality which used an excellent statistical method ; however the ideea displayed poor accuracy and precision in both free - living type activities and continuous walking trials . this paper has systematically reviewed the clinimetric properties ( validity , reliability , and clinical utility ) of four measures of hpa in children with a motor disability which included children aged less than six years . a precise and accurate measurement of the daily physical activity levels in this population will allow researchers to investigate the dose - response relationship between hpa and health outcomes . it will also allow the assessment of the efficacy of interventions aimed at increasing hpa in terms of establishing the dose and distribution of treatment necessary to achieve worthwhile results in the long term . for clinicians , an accessible , precise , and accurate measurement tool would allow the identification of children with low levels of hpa and in turn the assessment of the effectiveness of prescribed interventions . promising measures of hpa have been assessed in young children with motor disabilities , and the methodological quality of the papers was good to poor for study design and excellent to poor for statistical methods . a limitation of the findings of this paper is that only two studies of overall good methodological quality assessed children while undertaking free - living type activities . under these conditions two activity monitors displayed good accuracy during continuous walking [ 23 , 28 ] . this relates more to the icf definition of walking capacity rather than habitual walking activity ( i.e. , how many steps does a child need to walk a set distance in an ideal environment compared to how many steps do they take during the day in a variety of settings and intensities ) . another limitation is that studies included in this paper assessed children across a wide age range ( 418 years ) , with no studies of children with motor disabilities exclusively focusing on the under six - year age group which limits this review 's ability to make specific recommendations for this age group . the proportion of children aged less than six in the study samples ranged from 5 to 43% for the studies which provided a breakdown of their sample [ 22 , 23 , 26 , 27 ] . the use of the cosmin checklist for the assessment of methodological quality together with quality rating criteria of measurement properties is also a possible limitation . the cosmin checklist was developed for assessing hr - pros and does not have established psychometric properties in assessing objective physical activity measurement tools , and the quality rating criteria were not developed based on consensus . this issue was minimised by being guided by a systematic review of activity monitors in tdc for the assessment of the somewhat ambiguous flaws in design or methods item used in the cosmin ( box h , item 5 ) and by providing an in - depth appraisal of the statistical methods . the current australian physical activity guidelines recommend children aged from 1 to 5 years engage in active play for at least three hours per day without specific intensity recommendations . this recognises the sporadic and intermittent nature of young children 's activity patterns and places demands on the measurement tools used to assess hpa in this population . firstly they have to be able to record activity as a measure of time , and secondly they need to accurately recognise active play behaviours . the accelerometer - based pedometers stepwatch and minimod have the ability to collect the most complete information on habitual walking activity as they record steps within an epoch of time which can be downloaded onto a computer to visualise an activity pattern . they allow the assessment of intensity ( e.g. , steps / min ) , frequency ( e.g. , number of walking bouts ) , and duration ( e.g. , length of walking bouts ) . the validation protocols of the included studies primarily used structured walking activities which may not accurately represent the way young children move in active play , and therefore it can not be assumed activity monitors that do well in continuous walking trials would have done equally well had the children 's steps been counted during free - living play . in typically developing children , activity monitors are usually validated by direct observation of free play to circumvent this issue , and validation studies typically use a narrower age range to control for differences in motor skills [ 36 , 37 ] . children with a motor disability may not be able to walk but instead use a range of other methods of ambulation such as crawling , cruising , rolling , bottom shuffling , walking aids , or wheelchairs . those who are able to walk may have different gait patterns than typically developing children . this could explain why the accelerometer minimod , which relies on recognising gait patterns to count steps , had a lower rate of activity detection in children with cp ( 1997% ) than in typically developing children ( 84100% ) . other measurement tools such as accelerometers , which report raw activity counts per epoch of time , bypass this limitation but are yet to be validated in young children with a motor disability . a systematic review of measures of hpa in tdc preschoolers identified the actigraph ( shalimar , fl , usa ) accelerometer as having the best clinimetric properties in this population . similarly , the systematic review of hpa measurements in adolescents with cp by clanchy et al . identified accelerometers as the most comprehensive measure of hpa patterns despite the limited evidence available , and the actigraph has since been validated in adolescents with cp [ 38 , 39 ] . studies of doubly labelled water [ 40 , 41 ] , the compendium for physical activity questionnaires and actigraph in infants at risk of neurodevelopmental delay , did not meet inclusion criteria as their clinimetric properties for the measurement of hpa in children with motor disabilities aged less than six years had not been assessed . this systematic review identified four measures of hpa with evidence of clinimetric properties in study samples which included children aged less than six with motor disabilities . only a very small number of studies assessing activity monitors in this population are available , and none of the studies focus exclusively on children aged less than six years . the pedometers stepwatch and minimod are the most comprehensive measures of habitual walking activity utilised in the current literature . while both demonstrate good accuracy for step count during continuous walking , only the minimod has been tested during conditions which included walking trials other than continuous walking , and it performed poorly during these conditions . it is possible the ankle placement of the stepwatch would allow a more accurate assessment of free - living walking activities in children with motor disabilities but this is yet to be demonstrated . pedometers are only suitable as an estimate of hpa for children with high functional capacity as children 's hpa patterns are likely to consist of a progressively smaller proportion of walking as the severity of their impairment increases . in the most severely impaired children walking activity further research is needed to ascertain the clinimetric properties of activity monitors available for measuring hpa in young children with motor disabilities , and testing protocols should include a range of activities and ideally direct observation of free play . this will enable an understanding of the hpa patterns of children with motor disabilities across the spectrum of functional capacity for clinicians and researchers alike .

aim . to identify and systematically review the clinimetric properties of habitual physical activity ( hpa ) measures in young children with a motor disability . method . five databases were searched for measures of hpa including : children aged < 6.0 years with a neuromuscular disorder , physical activity defined as bodily movement produced by skeletal muscles causing caloric expenditure , reported hpa as duration , frequency , intensity , mode or energy expenditure , and evaluated clinimetric properties . the quality of papers was assessed using the cosmin - checklist . a targeted search of identified measures found additional studies of typically developing young children ( tdc ) . results . seven papers assessing four activity monitors met inclusion criteria . four studies were of good methodological quality . the minimod had good ability to measure continuous walking but the demonstrated poor ability to measure steps during free - living activities . the intelligent device for energy expenditure and activity and ambulatory monitoring pod showed poor ability to measure activity during both continuous walking and free - living activities . the stepwatch showed good ability to measure steps during continuous walking in tdc . interpretation . studies assessing the clinimetric properties of measures of hpa in this population are urgently needed to allow assessment of the relationship between hpa and health outcomes in this group .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusion

habitual physical activity ( hpa ) is an established determinant of health in children and is required for healthy development , including the growth of bone and muscle mass , improved balance and motor skills , maintaining a healthy weight and improved psychological wellbeing [ 1 , 2 ] . limited evidence suggests young children with motor disorders are less physically active than their typically developing peers . studies investigating the link between hpa and health outcomes for children with motor impairments have not been conducted . these studies are urgently needed to ( i ) determine the hpa patterns and intensities of children with motor disabilities , ( ii ) support the importance of physical activity promotion and inactivity prevention , ( iii ) determine the dose - response relationship between physical activity and health outcomes , and ( iv ) allow assessment of the effectiveness of interventions aimed at increasing hpa . physical activity is defined as any bodily movements using skeletal muscles that results in energy expenditure . the variables of frequency ( how often a child does an activity ) , intensity ( how hard a child works to do the activity ) , duration ( how long a child does an activity ) , and mode ( what the child is doing ) are used to characterise hpa patterns , while activity - related energy expenditure ( aee ) is used as a summary variable of all the other indicators [ 9 , 10 ] . the measurement of hpa in typically developing children ( tdc ) has received a great deal of attention in the last decade , primarily due to a concern over the increasing rate of overweight and obesity in childhood . children with motor impairments may not be walking but instead cruising , crawling , bottom shuffling , rolling , use walking aids or a wheelchair as means of locomotion , and those who are walking may have different gait patterns and speed of movement than tdc . systematic reviews of measures of physical activity in children and adolescents with cerebral palsy ( cp ) have been conducted [ 15 , 16 ] . included children with cp from birth to 18 years and concluded that the subjective measures of the activities scale for kids - performance version ( askp ) and the children 's assessment of participation and enjoyment ( cape ) are the only measures with established validity and reliability in children with physical disabilities from the age of six years . both reviews note the lack of hpa measures which can assess activity intensity , which limits their ability to provide meaningful comparison with physical activity guidelines , as children from the age of six years are advised to accumulate 60 minutes of moderate - to - vigorous physical activity every day . this current paper aims to systematically review the clinimetric properties ( validity , reproducibility , and clinical utility ) of measures of hpa in children less than 6 years of age with a motor disability . databases were searched using medical subject headings ( mesh ) terms and text words for physical activity and disability , limiting the search to the age group < 6.0 years . measures of hpa were included which met the following criteria : ( i ) included children less than six years of age with a motor disability caused by neuromuscular disorders ; ( ii ) defined physical activity as any bodily movement produced by skeletal muscles that results in caloric expenditure ; ( iii ) reported physical activity in terms of duration , frequency , intensity , or energy expenditure ; ( iv ) questionnaires where at least 60% of items related to a domain of hpa ; ( v ) evaluated clinimetric properties for the measurement of hpa . publications which did not assess the clinimetric properties of measures of hpa in children with motor disabilities were excluded . for measures where clinimetric properties were found in children less than six years of age with a motor disability , a further search identified any evidence in typically developing children in the same age group . the cosmin ( consensus - based standards for the selection of health measurement instruments ) checklist was used to rate the methodological quality of the study designs used to evaluate the clinimetric properties validity and reproducibility for measures of hpa . the cosmin was developed through an international delphi study to assess the methodological quality of studies on measurement properties of health - related patient - reported outcomes ( hr - pros ) ; however the measurement properties are also relevant to other health - related measurement instruments . the scoring system was developed based on expert discussion and testing on 46 articles identified by a systematic review and uses a each item within a specific measurement property is rated individually as excellent ( + + + ) , good ( + + ) , fair ( + ) , or poor ( ) and an overall score is given by taking the lowest score of any of the items within the assessment of the measurement property . the methodological quality rating of the papers was separated into study design and statistical analysis . to assess if there were any important flaws in the design or methods of the study ( cosmin box h , item five ) , items from the systematic review of activity monitors in tdc were used as a guide in addition to whether or not the testing protocol included free - living activities . proposed sample size requirements were applied in the clinimetric properties section instead of the study design section . for the assessment of measurement error ( agreement ) , standard error of measurement ( sem ) , smallest detectable change ( sdc ) , and limits of agreement ( loa ) calculated using the bland - altman method are accepted as excellent . quality criteria for the clinimetric properties of measures were proposed to give a framework to help distinguish between the quality of the studies and the performance of the measurement tools . the assessment of clinimetric properties of hr - pro questionnaires might be of a different nature to those assessing activity measures due to the availability of gold standards , something which is not readily available for hr - pros as these are usually questionnaires regarding patient outcomes such as pain and quality of life . the statistical methods and performance of clinimetric properties were therefore scored according to the cosmin but were also discussed further , and a different score was given if authors presented a convincing argument that the measure was acceptable . the clinical utility of measures of hpa was assessed in terms of the need for individual calibration of the equipment , the interpretability of the data output , and the need for software analysis , cost of equipment , and any required software . the feasibility of the equipment for using children aged less than six years with a motor disability in free living situations was also considered ( size , weight , number of sensors , place worn , ease of correct placement , and battery life ) . the richness of data was also assessed in terms of what aspects of hpa ( frequency , duration , intensity , mode , and energy expenditure ) it was able to assess in children with a motor disability . the overall level of evidence for the clinimetric properties of measures of hpa in young children with motor disabilities was determined as a whole by assessing the methodological quality of study design and statistical methods used in the assessment of hpa measures using the worst score counts algorithm , agreement between studies on the quality of clinimetric properties of these hpa measures , and the number of studies available for each hpa measure . four papers and one abstract from conference proceedings on the clinimetric properties of four measures of hpa in children with motor disabilities were identified which included children aged less than six years in their sample . none of the studies focused exclusively on children aged less than six years , which led to the inclusion of studies with a wide age range ( 418 years ) . assessed the intelligent device for energy expenditure and activity ( ideea ; minisun , ca , usa ) . all of the studies of children with motor disabilities included tdc as a reference group [ 2226 ] . on the use of the minimod included one child ( 5% ) with cp aged five years old and four children ( 20% ) in the tdc sample aged three to five years . the targeted search for studies reporting on the clinimetric properties of the identified measures in young tdc identified a further three studies assessing the minimod and the stepwatch [ 28 , 29 ] . , authors report on the same sample of tdc ( n = 20 , four children 6.0 years ) as in the previously mentioned abstract from conference proceedings . [ 22 , 27 ] assessed the criterion validity of the minimod in children with cp and tdc compared to manually counted steps and meters walked was rated authors report complete information in regarding test protocol ( monitor settings , placement , data output , average steps needed for length walked , and range of steps across group ) but only tests of continuous walking that may not adequately reflect free - living walking activity . on the criterion validity of the minimod and amp compared to manually counted steps and measured meters walked was rated authors report complete information on test protocol and use of a variety of structured activities : continuous walking ; activity lap walking which includes walking , stopping , completing a simple task , and walking again ; stair ascent and descent . in tdc all assessed the criterion validity of the stepwatch against manual step count and scored poor for study design due to providing no information on parts of their study protocol ( number of steps , range of steps needed , and length walked ) . assessed the stepwatch for criterion validity in tdc against manual step counts and was rated good for study design as they report a complete test protocol , though only assessed performance during continuous walking . this study also assessed the test - retest reliability of the ideea and received an the stepwatch study in children with dmd used a heart rate monitor to assess its concurrent validity over four days of wear . this study was rated fair for study design in the assessment of this construct as an a priori hypothesis of the relationship between the stepwatch and heart rate was not stated , but it was possible to deduce what was expected . as the cosmin works on a worst score counts algorithm , the highest score possible for all but one study is therefore poor , but as several studies report significant results , further discussion about the clinimetric performance of measures is warranted . percent agreement was used to assess the criterion validity of the minimod in children with cp and tdc by brandes et al . authors present rich information such as the average steps needed to walk the 20-meter track ( cp children ) and 160-meter track ( tdc ) and range of steps taken [ 22 , 27 ] . children with cp ( n = 19 ) walked an average of 79.8 steps ( min : 57 ; max : 126 ) , and on average the minimod over- or underestimated by one step ( agreement = 98.7% ; range : 94.1 to 101.8% ) [ 22 , 27 ] . children with cp walked only one - third of the steps tdc walked , and a higher likelihood that agreement occurred by chance exists . due to good reporting of study protocol and results , percent agreement was considered an the minimod showed good accuracy and precision for the measurement of continuous walking . a limitation of this study was the assessment of continuous walking only , and therefore the criterion validity for measuring hpa is indeterminate . the issue of use of percent agreement arises in two other studies assessing the stepwatch in tdc . in this study , the use of percent agreement therefore rates as excellent , and the evidence of criterion validity for continuous walking is good which is further strengthened by the large sample size . a further two studies assess the use of the stepwatch in children with cp and dmd but do not report any statistical methods or results which was rated bland - altman plots and percent of activity laps which were detected were used to assess criterion validity in the combined study of the minimod and amp by kuo et al . this provides us with the ability to assess both accuracy and precision of measures and allows a comparison between groups and therefore still allows a thorough assessment of the agreement between methods and can be rated as an the minimod performed well in continuous walking trials compared to measured length and direct observation of steps ( mean difference = 0.4 m/0.4 steps , 95% limits of agreement = 4.7 to 4 m/4.1 to 3.3 steps ) but showed a larger random error for activity lap walking ( mean diff . in tdc , the minimod performed well for continuous walking trials and poor in structured activity laps ( see table 2 ) ; however it performed better for detecting stair walking ( 84% ) . the amp performed better than the minimod in the structured activity lap walking trials , but still showed considerable bias difference and large random error in this trial ( mean diff . when it comes to more free - living type activities such as the structured walking trial and stair walking , the amp showed poor precision and accuracy in both walking trials , however detected more stair trials . as the focus of this paper is the ability to measure free - living activities , both monitors score poor for evidence of criterion validity for measuring hpa . this is demonstrated by the authors of this paper as the ideea significantly overestimated energy expenditure during the series of everyday activities and during comfortable treadmill walking both in children with cp and tdc , and during fast treadmill walking in tdc ( paired t - test , p < 0.01 ) . during fast treadmill walking in children with cp and during stair walking in both groups , measured energy expenditure did not differ significantly between methods ( paired t - test , p > 0.01 ) . the present energy expenditure calculations used in the ideea software do not accurately assess energy expenditure , and the clinimetric properties of the ideea are therefore considered poor . authors of the stepwatch study in children with dmd used a heart rate monitor to assess its concurrent validity and received a good rating for statistical method as they used a pearson correlation coefficient but did not report standard deviation . the rich data collected by the stepwatch and minimod allows measurement of intensity , frequency , and duration of walking activity . the units are both small and unobtrusive and have battery lives of seven days for the minimod and up to six weeks for the stepwatch , depending on settings . in the event that data collection is delayed once the device is provided to the child 's parents , having a battery life of more than seven days , and therefore not requiring recharging is a strength of the stepwatch as there is less likelihood of loss of data . the ideea consists of a data recorder worn on the waist with 5 individual sensors attached to the chest , thighs , and under each foot connected by wires to allow measurement of postures and energy expenditure , and it only has a battery life of 60 hours . both of these factors limit the ideeas utility in the measurement of hpa in young children . a summary of the quality of evidence for criterion validity is provided in table 3 . one of these studies also used free - living type activities , and the minimod displayed poor accuracy and precision in this setting . the amp was assessed in one study of good study design quality which used an excellent statistical method , though displayed poor accuracy and precision in both continuous walking and free - living type activities . good evidence of the ability of the stepwatch to accurately measure continuous walking in tdc . the ability of the stepwatch to accurately measure free - living activities can not be determined based on this study . the ability of the ideea to measure ee was assessed in one study of good study design quality which used an excellent statistical method ; however the ideea displayed poor accuracy and precision in both free - living type activities and continuous walking trials . this paper has systematically reviewed the clinimetric properties ( validity , reliability , and clinical utility ) of four measures of hpa in children with a motor disability which included children aged less than six years . a precise and accurate measurement of the daily physical activity levels in this population will allow researchers to investigate the dose - response relationship between hpa and health outcomes . it will also allow the assessment of the efficacy of interventions aimed at increasing hpa in terms of establishing the dose and distribution of treatment necessary to achieve worthwhile results in the long term . for clinicians , an accessible , precise , and accurate measurement tool would allow the identification of children with low levels of hpa and in turn the assessment of the effectiveness of prescribed interventions . promising measures of hpa have been assessed in young children with motor disabilities , and the methodological quality of the papers was good to poor for study design and excellent to poor for statistical methods . a limitation of the findings of this paper is that only two studies of overall good methodological quality assessed children while undertaking free - living type activities . under these conditions two activity monitors displayed good accuracy during continuous walking [ 23 , 28 ] . another limitation is that studies included in this paper assessed children across a wide age range ( 418 years ) , with no studies of children with motor disabilities exclusively focusing on the under six - year age group which limits this review 's ability to make specific recommendations for this age group . the use of the cosmin checklist for the assessment of methodological quality together with quality rating criteria of measurement properties is also a possible limitation . the cosmin checklist was developed for assessing hr - pros and does not have established psychometric properties in assessing objective physical activity measurement tools , and the quality rating criteria were not developed based on consensus . this issue was minimised by being guided by a systematic review of activity monitors in tdc for the assessment of the somewhat ambiguous flaws in design or methods item used in the cosmin ( box h , item 5 ) and by providing an in - depth appraisal of the statistical methods . this recognises the sporadic and intermittent nature of young children 's activity patterns and places demands on the measurement tools used to assess hpa in this population . the validation protocols of the included studies primarily used structured walking activities which may not accurately represent the way young children move in active play , and therefore it can not be assumed activity monitors that do well in continuous walking trials would have done equally well had the children 's steps been counted during free - living play . in typically developing children , activity monitors are usually validated by direct observation of free play to circumvent this issue , and validation studies typically use a narrower age range to control for differences in motor skills [ 36 , 37 ] . children with a motor disability may not be able to walk but instead use a range of other methods of ambulation such as crawling , cruising , rolling , bottom shuffling , walking aids , or wheelchairs . this could explain why the accelerometer minimod , which relies on recognising gait patterns to count steps , had a lower rate of activity detection in children with cp ( 1997% ) than in typically developing children ( 84100% ) . other measurement tools such as accelerometers , which report raw activity counts per epoch of time , bypass this limitation but are yet to be validated in young children with a motor disability . a systematic review of measures of hpa in tdc preschoolers identified the actigraph ( shalimar , fl , usa ) accelerometer as having the best clinimetric properties in this population . studies of doubly labelled water [ 40 , 41 ] , the compendium for physical activity questionnaires and actigraph in infants at risk of neurodevelopmental delay , did not meet inclusion criteria as their clinimetric properties for the measurement of hpa in children with motor disabilities aged less than six years had not been assessed . this systematic review identified four measures of hpa with evidence of clinimetric properties in study samples which included children aged less than six with motor disabilities . only a very small number of studies assessing activity monitors in this population are available , and none of the studies focus exclusively on children aged less than six years . while both demonstrate good accuracy for step count during continuous walking , only the minimod has been tested during conditions which included walking trials other than continuous walking , and it performed poorly during these conditions . it is possible the ankle placement of the stepwatch would allow a more accurate assessment of free - living walking activities in children with motor disabilities but this is yet to be demonstrated . in the most severely impaired children walking activity further research is needed to ascertain the clinimetric properties of activity monitors available for measuring hpa in young children with motor disabilities , and testing protocols should include a range of activities and ideally direct observation of free play .

recent genome - wide transcriptome studies have revealed that the majority of the mammalian genome is transcribed , thereby giving rise to a range of coding and noncoding rna ( ncrna ) transcripts . the latest human gencode release ( version 24 ) , presented by the encyclopedia of dna elements consortium ( encode ) , showed that the human genome is composed of 60554 genes ( figure 1 ) . among those , 19815 are protein - coding genes , whereas the remaining two - thirds of the genome represents noncoding rna genes ( 15941 long noncoding rna genes , 9882 small noncoding rna genes , and 14505 pseudogenes ) . noncoding rnas are predominant in the genome and are classified as either housekeeping or regulatory noncoding rnas . housekeeping noncoding rnas are constitutively expressed and they function as key regulatory molecules of many cellular processes . this group includes ribosomal ( rrna ) , transfer ( trna ) , small nuclear ( snrna ) , and small nucleolar rnas ( snornas ) . the second group , regulatory noncoding rnas , is divided into short ncrnas ( < 200 nucleotides ) or long noncoding rnas ( lncrnas , > 200 nucleotides ) . short ncrnas are represented by micrornas ( mirnas ) , small interfering rnas ( sirnas ) , and piwi - associated rnas ( pirnas ) , and , in few cases , by antisense rnas and enhancer rnas ( ernas ) . long noncoding rnas group includes antisense rnas and enhancer rnas ( ernas ) [ 13 ] ( figure 2 ) . in this review , this class of ncrnas was first discovered in the mouse during large - scale sequencing of full - length cdna libraries . generally , lncrnas were characterized as transcripts longer than 200 nucleotides with some features typical for protein - coding mrna . they are transcribed by rna - polymerase ( pol ) ii , capped on the 5 end , polyadenylated , and commonly expressed as alternatively spliced variants . moreover , lncrnas were found to have the same trimethylation marks of h3k4 and h3k36 at their promoters and transcribed regions . in contrast to mrna , lncrnas usually contain intron / exon structure , they are expressed at lower levels , often in a tissue - specific manner , and their sequence is poorly evolutionary conserved . additionally , they do not possess open reading frames ( orfs ) , 3 utr , and termination regions ; thus the majority have limited coding potential . nevertheless , recent studies have demonstrated that some lncrnas can be associated with polysomes and ribosomes , suggesting that they may act as coding transcripts and give rise to small peptides . an emerging number of studies related to the discovery of new lncrnas and their position to the nearest protein - coding genes have resulted in the introduction of a new nomenclature for these molecules . long noncoding rnas may be classified into five categories : ( 1 ) sense lncrnas that overlap the nearest protein - coding genes at the same strand ; ( 2 ) antisense lncrnas located across the exons of protein - coding genes from the opposite strand ; ( 3 ) bidirectional lncrnas transcribed on the opposite strand within 1 kb from the nearest protein - coding gene ; ( 4 ) intronic lncrnas that overlap intronic regions of coding genes in either the sense or antisense orientation ; ( 5 ) intergenic lncrnas that represent the largest group of lncrna ; they are located between protein - coding genes but are at least 1 kb away from the nearest protein - coding gene ( figure 3 ) . in recent years , numerous studies have aimed to identify functions of newly discovered long noncoding rnas . although the primary role of lncrnas was their epigenetic regulation of protein - coding gene expression , only a limited number of such long noncoding transcripts have been identified . it was soon discovered that lncrnas play a role in variety of biological process , acting in the nucleus , or in the cytoplasm , or in both . functions of lncrnas are manifested by three types of interaction : rna - rna , rna - dna , and rna - protein , all of which depend on the site of action . in the nucleus , for example , the main role of lncrnas is assumed to be epigenetic imprinting . one of the best - described mechanisms of lncrnas action during the epigenetic regulation is the x chromosome inactivation , in which x chromosome inactive - specific transcript ( xist ) plays a key role . recent studies demonstrated that lncrnas can interact with chromatin remodeling complexes including polycomb repressive complex ( prc2 ) 2 , trithorax / mll , and h3k9 methyltransferase g9a , acting as docking platforms to specific genomic loci . these protein complexes modify dna methylation patterns via trimethylation leading to transcriptional repression ( prc2 ) or activation ( trithorax / mll ) of the lncrna target genes . numerous lncrnas have been recognized as scaffolds for inhibitory prc2 including xist , kcnq1ot1 , and hotair [ 8 , 25 ] . other lncrnas , like hottip , directly bind to protein complexes of trithorax / mll . in these situations , , it has been postulated that these lncrnas are guides for chromatin - modifying complexes , where they first recognize the target localizations at the chromatin , bind to them , and then form docking platforms for protein partners . in addition to regulation of genomic imprinting , lncrnas are also involved in the control of gene expression at the transcriptional level . lncrnas can directly interact with transcription factors , acting as decoys or inhibitors for their binding to the target dna sequence . for example , two lncrna molecules , lethe and p50-associated cox-2 extragenic rna ( pacer ) , have been shown to interact with different subunits of nf-b , thus preventing it from binding to the promoter region of the target gene [ 10 , 27 ] . moreover , lncrnas exert their regulatory function during mrna processing and stability , interacting with the heterogeneous nuclear ribonucleoproteins ( hnrnps ) . an example of this type of rna - protein linkage is demonstrated for several lncrnas including lincrna - p21 , lincrna - cox2 , or thril ; thus they affect either activation or repression of target genes . other actions of lncrna in the nucleus are manifested by alternative splicing regulation and subnuclear compartment formation ( figure 4 ) . in the cytosol , lncrnas exert their function by interacting with target mrnas ( or mirnas ) through base - pairing . in this manner , lncrnas may either stabilize ( e.g. , bace1-as prevents mirna - induced repression of bace1 transcript ) or decay ( e.g. , 1/2-sbsrnas bind to the alu element of smd target mrnas within the 3 utr region ) target transcripts . moreover , lncrnas promote translation of transcripts ( e.g. , antisense uchl1 interacts with uchl1 mrna , resulting in recruitment of ribosomes ) or repress this process ( e.g. , lincrna - p21 binds to target mrna causing recruitment of translation repressors ) ( figure 4 ) . inflammation is a complex process , aiming to defend the body against harmful agents by removing or neutralizing them in order to restore tissue homeostasis . the complexity of this process results from the broad spectrum of inflammatory pathways , various inflammatory inducers , sensors , and mediators . in the classic view , the inflammatory response is induced immediately after stimulation by the inflammatory stimuli , such as a bacterial pathogen . cells , such as neutrophils , dendritic cells ( dcs ) , and macrophages , express evolutionarily conserved toll - like receptors ( tlrs ) on their surface . the tlr family is composed of several classes of transmembrane proteins and recognizes specific structures present on the microbes , known as pathogen - associated molecular patterns ( pamps ) . induction of tlrs triggers a multistep signaling cascade , thereby resulting in the modulation of gene expression involved in the immune response . key processes regulated by tlrs are related to the secretion of various mediators by immune cells , phagocytosis , cell migration , metabolic reprogramming , and tissue repair . in general , based on the length , the inflammatory response can be classified into acute or chronic . acute inflammation usually develops locally , and its role is to eliminate toxic agents , repair damaged tissue , and restore homeostasis . in some circumstances , local inflammation might be greater , therefore called a generalized inflammatory response , which is associated with the response of the whole organism . although the acute phase leads to the beneficial recovery of the injured tissue and healing , this nonspecific immune process could have also negative effects . without the proper resolution phase , it can easily turn into a chronic state . chronic inflammation may appear after the switch from the acute phase , and it is characterized by the specific , long - term cellular , and humoral immune response present at the site of tissue injury . in consequence , it may significantly contribute to dangerous pathophysiological changes and initiate the development of chronic diseases . each inflammatory stage one such factor is nf-b ( nuclear factor kappa - light - chain - enhancer of activated b cells ) , representing a family of transcription factors . nf-b plays an important role in gene expression regulation of multiple factors involved in immune , acute phase , and inflammatory responses . nf-b , in addition to controlling the development and function of the immune system , is required in other physiological processes such as suppression of apoptosis and cell proliferation and differentiation . in mammals , the active form of nf-b is composed of homo- and heterodimers of the nf-b family members , which can be divided into five different subunits : p50 , p52 , p65 ( rela ) , c - rel , and relb . all of these share homology of the specific n - terminal domain responsible for the dimerization and binding to target dna sequences . depending on the local cytokines produced by the immune cells , nf-b also controls macrophage relocalization , activation , and differentiation into two different phenotypes : proinflammatory ( m1 ) or anti - inflammatory ( m2 ) . in response to proinflammatory cytokines , nf-b is also involved in the activation of t lymphocytes , which proliferate and secrete cytokines such as interferon gamma ( ifn ) , interleukin 6 ( il-6 ) , or tnf-. the result of nf-b activation is stimulation of proinflammatory cytokine production by macrophages and t lymphocytes , thereby enhancing the inflammatory response . it is characterized by an excessive fat accumulation and enhanced production of proinflammatory cytokines / chemokines by adipose tissues . it is commonly caused by a combination of excessive food intake , lack of physical activity , and genetic susceptibility . a great majority of obese individuals present features of the metabolic syndrome : ( a ) increased waist circumference , ( b ) insulin resistance , ( c ) hyperglycemia , ( d ) hypertension , and ( e ) hypertriglyceridemia . appearance of these symptoms is associated with a high risk of serious disease development , including type 2 diabetes , cardiovascular diseases , , nonalcoholic fatty liver disease ( nafld ) , and even some cancers . the mechanism of metabolic disorder development is still not fully understood ; nevertheless numerous studies have suggested an adipocyte dysfunction as a main cause of metabolic system failure . in this pathological state , adipose tissue secretes various mediators , including cytokines , inducing a chronic , low - grade inflammatory response that results in recruitment of immune cells into the tissue . the excessive accumulation of tissue fat leads to disturbance both in the secretory and in the storage functions of adipocytes . it is postulated that the excessive fat mass induces the production of reactive oxygen species and the development of chronic oxidative stress in patients with obesity and metabolic dysfunction [ 44 , 45 ] . in adipose tissue of obese patients is observed severe inflammatory response , characterized by high production of cytokines and increased infiltration of immune cells , including monocytes , stimulated to differentiate into macrophages and lymphocytes t , activated to proliferate and secrete cytokines [ 46 , 47 ] . cytokines secreted by activated macrophages act on tlrs present on the surface of adipocytes , stimulating them to produce adipokines and cytokines . based on these observations , it has been found that the reactions of both the immune and metabolic systems are closely connected , and they play an important role in the development of metabolic disorders . adipocytes play a central role in energy homeostasis by fine - tuning the equilibrium between nutrient deposition ( white adipose tissue , wat ) and energy expenditure ( brown adipose tissue , bat ) . additionally , adipose tissue acts as an endocrine organ by secreting factors ( adipokines ) that regulate whole body energy and glucose homeostasis . adipogenesis is a complex process governed by a network of transcription factors , cofactors , and signaling intermediates from numerous pathways . the transcriptional cascade that regulates adipocyte differentiation is predominantly driven by peroxisome proliferator - activated receptor gamma ( ppar ) , shown to be both necessary and sufficient for adipogenesis . importantly , adipogenesis is highly regulated not only by ppar but also by the coordinated effects of other transcription factors including ccaat / enhancer - binding proteins ( c / ebps ) , kruppel - like factors ( klfs ) or wingless proteins ( wnt ) , and transcriptional cofactors [ 5254 ] . in recent years , there has been growing attention paid to noncoding rnas as a novel , cell intrinsic regulatory mechanism . specifically , lncrnas are now emerging as important regulators of gene expression both on the transcriptional and on the posttranscriptional levels . long noncoding rnas are being discovered using modern techniques of microarray or next - generation sequencing . however , only small portions have been fully characterized . in the case of adipogenesis , there is wide expression of lncrnas , controlling a variety of genes involved in the formation , differentiation , and activation of adipocytes ( figure 5 ) . steroid receptor rna activator ( sra ) lncrna was described for the first time as a molecule regulating adipogenesis . it was shown that lncrna sra , initially identified as a coactivator of steroid receptors , also acts as a transcriptional coactivator of ppar [ 55 , 56 ] . lncrna sra promotes adipocyte differentiation by binding to the n - terminal portion of ppar and enhances its transcriptional activity . additionally , its overexpression significantly increased mrna and protein levels of adipocyte master regulators ppar and c / ebp as well as ppar target genes . moreover , gene expression profiling showed that lncrna sra regulates expression of genes related to various cellular processes including cell cycle and insulin transduction pathways . further experiments proved that lncrna sra overexpression inhibits phosphorylation of p38 mitogen activated protein kinase ( mapk ) and c - jun nh2-terminal kinase ( jnk ) in the early differentiation of st2 mesenchymal precursor cells . in contrast , knockdown of lncrna sra increased p38 and jnk phosphorylation while reducing insulin receptor mrna and protein levels , which led to decreased downstream signaling . supporting that data , sra knock - out mice ( sra ) are resistant to high fat diet ( hfd ) induced obesity . after 14 weeks of hfd , sra mice are characterized by reduced wat mass and decreased expression of adipocyte genes such as adiponectin or fatty acid - binding protein 4 . the lean phenotype is also associated with smaller adipocytes in wat compared to wt mice , reduced liver mass , fewer lipid droplets in the liver , and decreased expression of lipogenesis - associated genes . finally , sra animals have improved insulin sensitivity . in a recent paper , xiao and coworkers identified lncrna adinr ( adipogenic differentiation induced noncoding rna ) that , in cis , transcriptionally activates c / ebp. it was upregulated 2030-fold during the course of adipogenesis and was transcribed from a position ~450 bp upstream of the c / ebp gene . knockdown of lncrna adinr with sirna resulted in a dramatic adipogenic defect as shown by a decreased number of oil red o positive cells and reduced adipogenic transcripts c / ebp , ppar , fatty acid - binding protein 4 , and lipoprotein lipase . importantly , inhibition of adipogenesis caused by depletion of lncrna adinr was rescued by overexpression of c / ebp. the lncrna adinr mechanism of action relies on its binding to pa1 followed by recruitment of mll3/4 histone methyltransferase complexes . in turn , that causes an increase in h3k4me3 and a decrease in h3k27me3 histone modification in the c / ebp locus during adipogenesis . expression of the pu.1 gene gives rise to both the mrna encoding pu.1 protein and antisense ( as ) lncrna originating from a promoter in the antisense strand of intron 3 . the protein pu.1 was originally demonstrated to be a key transcription factor regulating hematopoiesis , but recent studies revealed that it is also expressed in 3t3-l1 preadipocytes and murine adipocytes isolated from wat [ 60 , 61 ] . gain and loss of function studies established the pu.1 protein as an inhibitor of 3t3-l1 preadipocyte differentiation , acting via downregulation of ppar . on the other hand , pu.1 as lncrna promotes adipogenesis through preventing pu.1 mrna translation by binding to pu.1 mrna to form an mrna / as lncrna duplex . formation of this sense - antisense rna duplex was also confirmed in porcine adipocytes by an endogenous ribonuclease protection assay combined with rt - pcr . in line with the previous data , expression of ppar and fatty acid synthase ( fasn ) was significantly upregulated in the lncrna pu.1 shrna treated group . in a number of studies , global transcriptome profiling was used to select specific lncrnas either involved in adipogenesis or characteristic for adipocytes specifically isolated from wat or bat . in a seminal study done by sun and coworkers , the transcriptome of primary brown and white adipocytes , preadipocytes , and cultured adipocytes was profiled . scientists identified 175 lncrnas that were specifically regulated during adipogenesis . to further validate lncrnas that are functionally important for adipogenesis , researchers selected the top 20 lncrna genes based on the following criteria : ( i ) significant upregulation in both brown and white fat cultures , ( ii ) binding to ppar or cepb promoters , and ( iii ) independent validation of adipose - specific expression . by using rnai - mediated loss of function , the top 10 most important lncrnas were selected and called regulated in adipogenesis ( lncraps ) in order to highlight their key role in proper differentiation of adipocyte precursors . in comparison to white adipocytes , brown adipocytes have a higher mitochondrial content and express uncoupling protein 1 ( ucp1 ) , which disperses chemical energy through heat production . brown adipose tissue helps to regulate energy expenditure in rodents and newborn babies , but it has been considered to have no physiologic relevance in adults [ 65 , 66 ] . however , recent studies demonstrated that metabolically functional bat is also present in adult humans . as a result , an understanding of bat physiology might provide an effective treatment of obesity or other metabolic disorders . identification of the lncrnas crucial for bat differentiation and function was done using whole transcriptome rna sequencing . next , gain and loss of function studies established lncrna blnc1 as a potent activator of thermogenic adipocyte differentiation . it functions upon formation of rna - protein complex with the early b cell factor 2 ( ebf2 ) transcription factor to stimulate bat formation . additionally , lncrna blnc1 itself is a target of ebf2 , thereby forming a feed - forward regulatory loop . on the other hand , despite its stimulatory effects on brown preadipocyte differentiation , lncrna blnc1 failed to promote differentiation of 3t3-l1 cells into ucp1 positive adipocytes . another global profiling of gene expression during mouse brown fat cell differentiation , followed by candidate lncrnas genomic context analysis , pathway analysis , and gene ontology enrichment of their associated protein - coding genes was recently described . in this study , scientists identified three lncrnas ( gm15051 , tmem189 , and cebpd ) associated with their flanking coding genes ( hoxa1 , c / ebp , and c / ebp ) that participated in adipose commitment . more recently , rna - seq analysis of murine brown , inguinal white , and epididymal white fat allowed for identification of lncrna cluster enriched in bat . among them , authors showed that bate1 binds to the heterogeneous nuclear ribonucleoprotein u , a factor also required for bat formation . finally , interactions of lncrnas with micrornas in the context of adipogenesis were also described . as demonstrated by gernapudi and coworkers , the mir-140/lncrna neat1 signaling network is necessary for adipogenesis . adipocyte derived stem cells isolated from mir-140 knock - out mice had dramatically decreased adipogenic capabilities found to be associated with the downregulation of lncrna neat1 expression . they also found that the physical interaction with mir-140 in nucleus led to increased expression of neat1 . excessive fat accumulation not only causes pathological expansion of adipose tissue but also leads to triglyceride deposition in the liver . presence of lipid droplets in excess of 5% of total hepatocytes is a diagnostic hallmark of nonalcoholic fatty liver disease ( nafld ) . although most patients suffer from a mild course of illness , approximately 25% of cases progress and can subsequently lead to the development of steatohepatitis , hepatic fibrosis , liver cirrhosis , and hepatoma . scientists and physicians estimate that between 20% and 30% of west 's general population suffers from nafld , which makes this affects more than a billion people worldwide . to address questions about the global expression pattern and functional contribution of lncrnas during nafld , sun and coworkers analyzed microarray expression using rna extracted from liver biopsies of healthy patients and those suffering from nafld . in this analysis , 535 lncrnas were found upregulated in nafld samples compared with controls and 1,200 were downregulated in nafld ( figure 5 ) . out of these , seven nafld samples that had highly up- or downregulated gene expression were validated by quantitative real - time pcr ( qpcr ) . three lncrna results obtained by qpcr were different in comparison to the microarray data , showing no change between analyzed groups . however , changes in other selected hits were confirmed , highlighting the importance of microarray data validation . pancreatic islets , through the secretion of endocrine hormones such as insulin and glucagon , play a key role in metabolic homeostasis . however , one of the most common malfunctions of -cells is caused by lack of insulin , leading to the development of diabetes . additionally , diabetes may develop as a result of insulin resistance , a condition in which the body can not effectively use the insulin produced by -cells . as a result , blood sugar levels are deregulated and various tissues are exposed to prolonged hyperglycemia that , over time , causes serious damage to many of the body 's systems . it should be emphasized that hyperglycemia , a hallmark of diabetes , can cause both acute and long - term complications like cardiovascular disease , stroke , foot ulcers , ketoacidosis , or hyperosmolar coma . based on etiology , who classifies diabetes into four categories , but the vast majority of cases fall into two broad etiopathogenetic groups : type 1 diabetes mellitus ( t1 dm ) or type 2 diabetes mellitus ( t2 dm ) . accounting for 510% of all cases , t1 dm is characterized by the loss of insulin producing -cells due to an autoimmune reaction . type 2 diabetes mellitus represents 9095% of all cases and , unlike t1 dm , is strongly associated with patients ' lifestyle . a number of lifestyle factors are known to be important for development of t2 dm including obesity ( bmi above 30 ) , lack of physical activity , poor diet , or stress . it is a chronic disease that begins with insulin resistance , a condition in which cells ( mostly adipocytes , hepatocytes , and muscle cells ) fail to properly absorb and metabolize glucose . as a result , -cells increase insulin production to overcome hyperglycemia , but this compensatory mechanism is transient and generally fails over time . intensified metabolism of -cells leads to a gradual loss of their endocrine function and finally causes -cell apoptosis . who , in 2014 , the global prevalence of diabetes was estimated to be 9% among adults aged over 18 years and it is expected to double before 2030 [ 75 , 80 ] . additionally , experts project that , in 2030 , diabetes will be the 7th leading cause of death . initial studies aimed at identification of specific lncrnas expressed in murine and human pancreatic islets were done in 2012 [ 81 , 82 ] . by using next - generation sequencing , scientists revealed a large collection of 1359 intergenic lncrnas expressed in murine -cells ( figure 5 ) . many of them were highly tissue - specific , but their function was not evaluated , and future experiments are still needed . similar numbers of 1128 intergenic and antisense lncrnas were identified in human -cells and many of them ( e.g. , hi - lnc12 and hi - lnc77 ) were highly tissue - specific . additionally , lncrna - encoding genes were preferentially located near genes encoding important regulators of -cell function , development , and transcription . one of them , lncrna hi - lnc25 , conserved between mouse and human , was shown to positively regulate glis3 mrna . glis3 encodes a pancreatic islet transcription factor that is mutated in monogenic diabetes and contains t2 dm risk variants [ 83 , 84 ] . in the same set of experiments , scientists also showed that selected lncrnas ( e.g. , hi - lnc12 and hi - lnc25 ) are linked to -cell differentiation program as their expression was significantly higher in human islets when compared to the embryonic pancreas . moreover , the addition of glucose to in vitro -cell culture induced expression of some lncrnas , suggesting their importance for mature islet cell physiology . finally , authors showed deregulation of islets ' lncrnas in t2 dm by comparing profiles of lncrnas in islets from 19 nondiabetic and 16 t2 dm donors . two lncrnas , namely , kcnq1ot1 and hi - lnc45 , were significantly increased or decreased in t2 dm islets , respectively . seven lncrnas specific for -cells , as reported by morn and coworkers , were also discussed in another study that analyzed pancreatic islets from 89 individuals with or without diabetes . microarray analysis , rna sequencing , and exome sequencing methods enabled researchers to identify 493 lncrnas that were differentially expressed in the pancreatic islets . out of those , 17 long intergenic noncoding rnas ( lincrnas ) were significantly associated with donor 's hba1c levels , and two ( loc283177 and snhg5 ) were also involved in gene expression regulation . to obtain insight into functional target genes of lncrnas loc283177 and snhg5 , the authors performed a coexpression analysis , linking their expression with all other genes in pancreatic islets . lncrna loc283177 levels correlated with expression of genes that play a key role in islet function , namely , synaptotagmin 11 , map - kinase activating death domain ( madd ) , and paired box 6 ( pax6 ) . synaptotagmin 11 is known to regulate the exocytosis of insulin and madd proinsulin synthesis , and pax6 is involved in development of pancreatic islets [ 85 , 86 ] . supporting these findings , lncrna loc283177 expression was found to be directly associated with insulin exocytosis in the islets . insulin is secreted from -cells in response to glucose , while other nutrients such as free fatty acids and amino acids can augment glucose - induced insulin secretion . in addition , various hormones ( e.g. , leptin , growth hormone , glucagon like peptide-1 , and estrogen ) also regulate insulin secretion . as a result , -cells are called a metabolic hub in the body that coordinate nutrient metabolism with endocrine system . insufficient insulin production , or insulin resistance , is a prime cause of diabetic complications . among the many tissues affected by hyperglycemia , endothelial cells are very important because they are implicated in pathogenesis of diabetes - related microvascular and macrovascular complications . one of these complications , diabetic retinopathy , was recently shown to be influenced by lncrna malat1 ( metastasis - associated lung adenocarcinoma transcript 1 ) ( figure 5 ) . in a mouse model of streptozotocin - induced diabetes microarray analysis , followed by qpcr validation of selected genes , allowed for identification of 303 differentially expressed lncrnas including 214 downregulated and 89 upregulated genes in diabetic versus nondiabetic samples . one gene , lncrna chr19 : 57956895802671 , a murine ortholog of human lncrna malat1 , had a greater than 10-fold upregulation in diabetic retinas . lncrna malat1 was previously described in humans to be deregulated in several solid tumors and was associated with cancer metastasis and recurrence . it was shown to be significantly upregulated in a rf/6a cell model of hyperglycemia , in the aqueous tumor samples and in fibrovascular membranes of diabetic patients . to further investigate the molecular mechanism of lncrna malat1 action , scientists decided to analyze its function in retinal vasculature and endothelial cell dysfunction in diabetes mellitus . lncrna malat1 knockdown in retinas of db / db mice resulted in amelioration of diabetic retinopathy as manifested by reduced apoptosis of retinal cells and pericytes . importantly , after administration of lncrna malat shrna , scientists observed decreased retinal vascular permeability as measured by evans blue leakage . moreover , in vitro tests showed that lncrna malat1 knockdown decreased retinal endothelial cell proliferation , migration , and tube formation . in rf/6a endothelial cells , lncrna malat1 acts via induction of p38 mapk signaling , and its silencing reduced phosphorylated p38 level but had no effect on phosphorylated erk1/2 or jnk1/2 . pretreatment of rf/6a cells with sb203580 , a p38 mapk pathway inhibitor , strongly blocked the effect of lncrna malat1-induced cell proliferation . more recently , the same research group reported that lncrna miat ( myocardial infarction - associated transcript ) also regulates diabetes mellitus microvascular dysfunction , acting as a competing endogenous rna ( figure 5 ) . similar to a previous report , lncrna miat expression was increased in diabetic retinas and endothelial cells cultured in high glucose medium . as shown by in vivo tests , lncrna miat knockdown ameliorated diabetes mellitus induced retinal microvascular dysfunction . in line with in vivo data , silencing of lncrna miat in endothelial cells cultured in vitro led to an inhibition of proliferation , migration , and tube formation . lncrnas may act as an mrna molecular sponge and regulate mirnas available for binding their target mrnas . bioinformatics suggested that the lncrna miat sequence contains 4 putative mirna binding sites including mir-29a-3p , mir-29b-3p , mir-29c-3p , and mir-150 - 5p . one of these , mir-150 - 5p , was proven to directly target lncrna miat in endothelial cell both in vitro and in vivo . this particular mirna is of great importance for angiogenesis because it regulates the expression of vegf , a key angiogenic factor involved in physiological and pathological angiogenesis . yan and coworkers proved that miat regulates the expression of mir-150 - 5p target gene vegf creating a critical regulatory loop for endothelial cell function . during angiogenesis , lncrna miat is significantly upregulated , which in turn alleviates the mir-150 - 5p repression effect , thereby upregulating the level of mir-150 - 5p target gene , vegf . in contrast to lncrna miat , lncrna meg3 ( maternally expressed gene 3 ) expression was significantly downregulated in the retinas and endothelial cells of stz - induced diabetic mice upon high glucose and oxidative stress conditions . its knockdown regulated retinal endothelial cell proliferation , migration , and tube formation in vitro . as demonstrated in rf/6a endothelial cells , inhibition of lncrna meg3 by sirna significantly increased levels of phosphorylated pi3k and phosphorylated akt at thr and ser . addition of pi3k inhibitors to cell cultures abrogated the observed phenotype , proving that lncrna meg3 regulates hyperproliferation of retinal endothelial cells through pi3k / akt signaling . additionally , its activation influences endothelial cell biology by modulating angiogenesis , proliferation , and microvascular permeability .

ribonucleic acids ( rnas ) are very complex and their all functions have yet to be fully clarified . noncoding genes ( noncoding rna , sequences , and pseudogenes ) comprise 67% of all genes and they are represented by housekeeping noncoding rnas ( transfer rna ( trna ) , ribosomal rna ( rrna ) , small nuclear rna ( snrna ) , and small nucleolar rna ( snorna ) ) that are engaged in basic cellular processes and by regulatory noncoding rna ( short and long noncoding rna ( ncrna ) ) that are important for gene expression / transcript stability . in this review , we summarize data concerning the significance of long noncoding rnas ( lncrnas ) in metabolic syndrome related disorders , focusing on adipose tissue and pancreatic islands .

1. Introduction 2. Biological Function of Long Noncoding RNAs 3. Inflammation and Its Significance in Metabolic Syndrome 4. lncRNA in Obesity and Adipogenesis Control 5. lncRNA in Diabetes and Pancreatic Function/Glucose Homeostasis

recent genome - wide transcriptome studies have revealed that the majority of the mammalian genome is transcribed , thereby giving rise to a range of coding and noncoding rna ( ncrna ) transcripts . the latest human gencode release ( version 24 ) , presented by the encyclopedia of dna elements consortium ( encode ) , showed that the human genome is composed of 60554 genes ( figure 1 ) . among those , 19815 are protein - coding genes , whereas the remaining two - thirds of the genome represents noncoding rna genes ( 15941 long noncoding rna genes , 9882 small noncoding rna genes , and 14505 pseudogenes ) . noncoding rnas are predominant in the genome and are classified as either housekeeping or regulatory noncoding rnas . housekeeping noncoding rnas are constitutively expressed and they function as key regulatory molecules of many cellular processes . this group includes ribosomal ( rrna ) , transfer ( trna ) , small nuclear ( snrna ) , and small nucleolar rnas ( snornas ) . the second group , regulatory noncoding rnas , is divided into short ncrnas ( < 200 nucleotides ) or long noncoding rnas ( lncrnas , > 200 nucleotides ) . short ncrnas are represented by micrornas ( mirnas ) , small interfering rnas ( sirnas ) , and piwi - associated rnas ( pirnas ) , and , in few cases , by antisense rnas and enhancer rnas ( ernas ) . long noncoding rnas group includes antisense rnas and enhancer rnas ( ernas ) [ 13 ] ( figure 2 ) . in this review , this class of ncrnas was first discovered in the mouse during large - scale sequencing of full - length cdna libraries . generally , lncrnas were characterized as transcripts longer than 200 nucleotides with some features typical for protein - coding mrna . they are transcribed by rna - polymerase ( pol ) ii , capped on the 5 end , polyadenylated , and commonly expressed as alternatively spliced variants . in contrast to mrna , lncrnas usually contain intron / exon structure , they are expressed at lower levels , often in a tissue - specific manner , and their sequence is poorly evolutionary conserved . additionally , they do not possess open reading frames ( orfs ) , 3 utr , and termination regions ; thus the majority have limited coding potential . an emerging number of studies related to the discovery of new lncrnas and their position to the nearest protein - coding genes have resulted in the introduction of a new nomenclature for these molecules . long noncoding rnas may be classified into five categories : ( 1 ) sense lncrnas that overlap the nearest protein - coding genes at the same strand ; ( 2 ) antisense lncrnas located across the exons of protein - coding genes from the opposite strand ; ( 3 ) bidirectional lncrnas transcribed on the opposite strand within 1 kb from the nearest protein - coding gene ; ( 4 ) intronic lncrnas that overlap intronic regions of coding genes in either the sense or antisense orientation ; ( 5 ) intergenic lncrnas that represent the largest group of lncrna ; they are located between protein - coding genes but are at least 1 kb away from the nearest protein - coding gene ( figure 3 ) . in recent years , numerous studies have aimed to identify functions of newly discovered long noncoding rnas . although the primary role of lncrnas was their epigenetic regulation of protein - coding gene expression , only a limited number of such long noncoding transcripts have been identified . functions of lncrnas are manifested by three types of interaction : rna - rna , rna - dna , and rna - protein , all of which depend on the site of action . in the nucleus , for example , the main role of lncrnas is assumed to be epigenetic imprinting . recent studies demonstrated that lncrnas can interact with chromatin remodeling complexes including polycomb repressive complex ( prc2 ) 2 , trithorax / mll , and h3k9 methyltransferase g9a , acting as docking platforms to specific genomic loci . numerous lncrnas have been recognized as scaffolds for inhibitory prc2 including xist , kcnq1ot1 , and hotair [ 8 , 25 ] . in these situations , , it has been postulated that these lncrnas are guides for chromatin - modifying complexes , where they first recognize the target localizations at the chromatin , bind to them , and then form docking platforms for protein partners . in addition to regulation of genomic imprinting , lncrnas are also involved in the control of gene expression at the transcriptional level . for example , two lncrna molecules , lethe and p50-associated cox-2 extragenic rna ( pacer ) , have been shown to interact with different subunits of nf-b , thus preventing it from binding to the promoter region of the target gene [ 10 , 27 ] . in this manner , lncrnas may either stabilize ( e.g. the complexity of this process results from the broad spectrum of inflammatory pathways , various inflammatory inducers , sensors , and mediators . cells , such as neutrophils , dendritic cells ( dcs ) , and macrophages , express evolutionarily conserved toll - like receptors ( tlrs ) on their surface . induction of tlrs triggers a multistep signaling cascade , thereby resulting in the modulation of gene expression involved in the immune response . key processes regulated by tlrs are related to the secretion of various mediators by immune cells , phagocytosis , cell migration , metabolic reprogramming , and tissue repair . acute inflammation usually develops locally , and its role is to eliminate toxic agents , repair damaged tissue , and restore homeostasis . although the acute phase leads to the beneficial recovery of the injured tissue and healing , this nonspecific immune process could have also negative effects . chronic inflammation may appear after the switch from the acute phase , and it is characterized by the specific , long - term cellular , and humoral immune response present at the site of tissue injury . each inflammatory stage one such factor is nf-b ( nuclear factor kappa - light - chain - enhancer of activated b cells ) , representing a family of transcription factors . nf-b plays an important role in gene expression regulation of multiple factors involved in immune , acute phase , and inflammatory responses . in mammals , the active form of nf-b is composed of homo- and heterodimers of the nf-b family members , which can be divided into five different subunits : p50 , p52 , p65 ( rela ) , c - rel , and relb . depending on the local cytokines produced by the immune cells , nf-b also controls macrophage relocalization , activation , and differentiation into two different phenotypes : proinflammatory ( m1 ) or anti - inflammatory ( m2 ) . in response to proinflammatory cytokines , nf-b is also involved in the activation of t lymphocytes , which proliferate and secrete cytokines such as interferon gamma ( ifn ) , interleukin 6 ( il-6 ) , or tnf-. it is commonly caused by a combination of excessive food intake , lack of physical activity , and genetic susceptibility . a great majority of obese individuals present features of the metabolic syndrome : ( a ) increased waist circumference , ( b ) insulin resistance , ( c ) hyperglycemia , ( d ) hypertension , and ( e ) hypertriglyceridemia . appearance of these symptoms is associated with a high risk of serious disease development , including type 2 diabetes , cardiovascular diseases , , nonalcoholic fatty liver disease ( nafld ) , and even some cancers . in this pathological state , adipose tissue secretes various mediators , including cytokines , inducing a chronic , low - grade inflammatory response that results in recruitment of immune cells into the tissue . in adipose tissue of obese patients is observed severe inflammatory response , characterized by high production of cytokines and increased infiltration of immune cells , including monocytes , stimulated to differentiate into macrophages and lymphocytes t , activated to proliferate and secrete cytokines [ 46 , 47 ] . based on these observations , it has been found that the reactions of both the immune and metabolic systems are closely connected , and they play an important role in the development of metabolic disorders . adipocytes play a central role in energy homeostasis by fine - tuning the equilibrium between nutrient deposition ( white adipose tissue , wat ) and energy expenditure ( brown adipose tissue , bat ) . additionally , adipose tissue acts as an endocrine organ by secreting factors ( adipokines ) that regulate whole body energy and glucose homeostasis . adipogenesis is a complex process governed by a network of transcription factors , cofactors , and signaling intermediates from numerous pathways . the transcriptional cascade that regulates adipocyte differentiation is predominantly driven by peroxisome proliferator - activated receptor gamma ( ppar ) , shown to be both necessary and sufficient for adipogenesis . importantly , adipogenesis is highly regulated not only by ppar but also by the coordinated effects of other transcription factors including ccaat / enhancer - binding proteins ( c / ebps ) , kruppel - like factors ( klfs ) or wingless proteins ( wnt ) , and transcriptional cofactors [ 5254 ] . in recent years , there has been growing attention paid to noncoding rnas as a novel , cell intrinsic regulatory mechanism . specifically , lncrnas are now emerging as important regulators of gene expression both on the transcriptional and on the posttranscriptional levels . long noncoding rnas are being discovered using modern techniques of microarray or next - generation sequencing . in the case of adipogenesis , there is wide expression of lncrnas , controlling a variety of genes involved in the formation , differentiation , and activation of adipocytes ( figure 5 ) . moreover , gene expression profiling showed that lncrna sra regulates expression of genes related to various cellular processes including cell cycle and insulin transduction pathways . further experiments proved that lncrna sra overexpression inhibits phosphorylation of p38 mitogen activated protein kinase ( mapk ) and c - jun nh2-terminal kinase ( jnk ) in the early differentiation of st2 mesenchymal precursor cells . supporting that data , sra knock - out mice ( sra ) are resistant to high fat diet ( hfd ) induced obesity . the lean phenotype is also associated with smaller adipocytes in wat compared to wt mice , reduced liver mass , fewer lipid droplets in the liver , and decreased expression of lipogenesis - associated genes . in a recent paper , xiao and coworkers identified lncrna adinr ( adipogenic differentiation induced noncoding rna ) that , in cis , transcriptionally activates c / ebp. knockdown of lncrna adinr with sirna resulted in a dramatic adipogenic defect as shown by a decreased number of oil red o positive cells and reduced adipogenic transcripts c / ebp , ppar , fatty acid - binding protein 4 , and lipoprotein lipase . the protein pu.1 was originally demonstrated to be a key transcription factor regulating hematopoiesis , but recent studies revealed that it is also expressed in 3t3-l1 preadipocytes and murine adipocytes isolated from wat [ 60 , 61 ] . in a seminal study done by sun and coworkers , the transcriptome of primary brown and white adipocytes , preadipocytes , and cultured adipocytes was profiled . to further validate lncrnas that are functionally important for adipogenesis , researchers selected the top 20 lncrna genes based on the following criteria : ( i ) significant upregulation in both brown and white fat cultures , ( ii ) binding to ppar or cepb promoters , and ( iii ) independent validation of adipose - specific expression . by using rnai - mediated loss of function , the top 10 most important lncrnas were selected and called regulated in adipogenesis ( lncraps ) in order to highlight their key role in proper differentiation of adipocyte precursors . in comparison to white adipocytes , brown adipocytes have a higher mitochondrial content and express uncoupling protein 1 ( ucp1 ) , which disperses chemical energy through heat production . brown adipose tissue helps to regulate energy expenditure in rodents and newborn babies , but it has been considered to have no physiologic relevance in adults [ 65 , 66 ] . as a result , an understanding of bat physiology might provide an effective treatment of obesity or other metabolic disorders . another global profiling of gene expression during mouse brown fat cell differentiation , followed by candidate lncrnas genomic context analysis , pathway analysis , and gene ontology enrichment of their associated protein - coding genes was recently described . in this study , scientists identified three lncrnas ( gm15051 , tmem189 , and cebpd ) associated with their flanking coding genes ( hoxa1 , c / ebp , and c / ebp ) that participated in adipose commitment . more recently , rna - seq analysis of murine brown , inguinal white , and epididymal white fat allowed for identification of lncrna cluster enriched in bat . adipocyte derived stem cells isolated from mir-140 knock - out mice had dramatically decreased adipogenic capabilities found to be associated with the downregulation of lncrna neat1 expression . excessive fat accumulation not only causes pathological expansion of adipose tissue but also leads to triglyceride deposition in the liver . although most patients suffer from a mild course of illness , approximately 25% of cases progress and can subsequently lead to the development of steatohepatitis , hepatic fibrosis , liver cirrhosis , and hepatoma . to address questions about the global expression pattern and functional contribution of lncrnas during nafld , sun and coworkers analyzed microarray expression using rna extracted from liver biopsies of healthy patients and those suffering from nafld . in this analysis , 535 lncrnas were found upregulated in nafld samples compared with controls and 1,200 were downregulated in nafld ( figure 5 ) . out of these , seven nafld samples that had highly up- or downregulated gene expression were validated by quantitative real - time pcr ( qpcr ) . pancreatic islets , through the secretion of endocrine hormones such as insulin and glucagon , play a key role in metabolic homeostasis . it should be emphasized that hyperglycemia , a hallmark of diabetes , can cause both acute and long - term complications like cardiovascular disease , stroke , foot ulcers , ketoacidosis , or hyperosmolar coma . accounting for 510% of all cases , t1 dm is characterized by the loss of insulin producing -cells due to an autoimmune reaction . type 2 diabetes mellitus represents 9095% of all cases and , unlike t1 dm , is strongly associated with patients ' lifestyle . a number of lifestyle factors are known to be important for development of t2 dm including obesity ( bmi above 30 ) , lack of physical activity , poor diet , or stress . it is a chronic disease that begins with insulin resistance , a condition in which cells ( mostly adipocytes , hepatocytes , and muscle cells ) fail to properly absorb and metabolize glucose . who , in 2014 , the global prevalence of diabetes was estimated to be 9% among adults aged over 18 years and it is expected to double before 2030 [ 75 , 80 ] . many of them were highly tissue - specific , but their function was not evaluated , and future experiments are still needed . additionally , lncrna - encoding genes were preferentially located near genes encoding important regulators of -cell function , development , and transcription . , hi - lnc12 and hi - lnc25 ) are linked to -cell differentiation program as their expression was significantly higher in human islets when compared to the embryonic pancreas . microarray analysis , rna sequencing , and exome sequencing methods enabled researchers to identify 493 lncrnas that were differentially expressed in the pancreatic islets . out of those , 17 long intergenic noncoding rnas ( lincrnas ) were significantly associated with donor 's hba1c levels , and two ( loc283177 and snhg5 ) were also involved in gene expression regulation . lncrna loc283177 levels correlated with expression of genes that play a key role in islet function , namely , synaptotagmin 11 , map - kinase activating death domain ( madd ) , and paired box 6 ( pax6 ) . synaptotagmin 11 is known to regulate the exocytosis of insulin and madd proinsulin synthesis , and pax6 is involved in development of pancreatic islets [ 85 , 86 ] . supporting these findings , lncrna loc283177 expression was found to be directly associated with insulin exocytosis in the islets . , leptin , growth hormone , glucagon like peptide-1 , and estrogen ) also regulate insulin secretion . among the many tissues affected by hyperglycemia , endothelial cells are very important because they are implicated in pathogenesis of diabetes - related microvascular and macrovascular complications . one of these complications , diabetic retinopathy , was recently shown to be influenced by lncrna malat1 ( metastasis - associated lung adenocarcinoma transcript 1 ) ( figure 5 ) . lncrna malat1 was previously described in humans to be deregulated in several solid tumors and was associated with cancer metastasis and recurrence . it was shown to be significantly upregulated in a rf/6a cell model of hyperglycemia , in the aqueous tumor samples and in fibrovascular membranes of diabetic patients . moreover , in vitro tests showed that lncrna malat1 knockdown decreased retinal endothelial cell proliferation , migration , and tube formation . in rf/6a endothelial cells , lncrna malat1 acts via induction of p38 mapk signaling , and its silencing reduced phosphorylated p38 level but had no effect on phosphorylated erk1/2 or jnk1/2 . more recently , the same research group reported that lncrna miat ( myocardial infarction - associated transcript ) also regulates diabetes mellitus microvascular dysfunction , acting as a competing endogenous rna ( figure 5 ) . in line with in vivo data , silencing of lncrna miat in endothelial cells cultured in vitro led to an inhibition of proliferation , migration , and tube formation . bioinformatics suggested that the lncrna miat sequence contains 4 putative mirna binding sites including mir-29a-3p , mir-29b-3p , mir-29c-3p , and mir-150 - 5p . its knockdown regulated retinal endothelial cell proliferation , migration , and tube formation in vitro . additionally , its activation influences endothelial cell biology by modulating angiogenesis , proliferation , and microvascular permeability .

for the reconstruction of endodontically treated teeth , intraradicular posts are recommended to retain the restoration when there is less than 50% of the coronal structure remaining . among other intraradicular anchorage options , adhesively cemented fiber posts reduce the risk of root fracture because they have an elastic modulus ( e=40 gpa ) similar to dentin ( e=18 gpa ) , unlike ceramic ( e=200 gpa ) and metallic posts ( e=200 gpa ) . consequently , fiber posts provide more uniform stress distribution on root dentin compared to other retainers . the clinical success of restorative treatments depends on many factors , including the amount of dental substrate , adequate coronal reconstruction , cavity configuration factor , masticatory load support by the teeth and stress distribution over the bone and periodontal ligament . the periodontal ligament , ranging between 0.1 and 0.3 mm thick , absorbs occlusal loads ; the periodontal fiber compression provides important support when the tooth is submitted to external forces . when a force is applied , the resistance of the periodontal membrane is initially low , but as the load increases , the resistance also increases gradually until tooth dislodgment becomes minimal . the periodontal ligament is compressive to a limited extent and partially elastic . for masticatory loads up to 70 when the fibers reach their maximum load resistance , the periodontal ligament becomes rigid , the load is transmitted to the bone , and tensions are distributed over the root surface . therefore , the properties of masticatory force absorption through the bone and periodontal ligament should be reproduced in laboratorial tests in order to simulate the clinical reality more accurately . however , various studies that assess bond strength between root canal posts and dentin after mechanical fatigue do not simulate periodontal ligament . similarly , most studies fail to simulate the periodontal ligament in fracture resistance tests , although a few studies simulate the periodontal ligament using polyether and polivynilsiloxane based elastomers . a few in vitro studies assessed the effects of periodontal ligament simulation and showed that an artificial periodontal ligament reduced the fracture strength and modified the failure modes of sound in bovine teeth . also , simulating the periodontal ligament significantly reduced fracture resistance of ceramic - fixed partial dentures . considering that root posts reduce the area available for stress distribution , concentrating it on the remaining dental structure and the dentin - post interface ninety single - root bovine teeth were cleansed with periodontal curettes and stored in distilled water . coronal portions were sectioned under water cooling to obtain a standardized 16 mm root length . the most coronal diameter of the root canal was measured with a digital caliper ( starrett 727 , starret , itu , sp , brazil ) , and any tooth with a canal diameter larger than the fiber post diameter ( =1.8 mm , white post # 3 , fgm , joinville , sc , brazil ) was discarded and replaced with a different specimen . the apical foramens were sealed using a photo activated adhesive ( single bond 2 , 3 m espe , saint paul , mn , usa ) associated with a composite resin ( oppalis , fgm ) . the canals were prepared with the same drill used in the posts system ( white post dc # 3 , fgm ) to a 12 mm length . the roots were numbered from 1 to 90 and randomly distributed into 6 groups ( n=15 ) , which varied by the artificial periodontal ligament composition and the test performed ( figure 1 ) . the number sequences were generated by random allocation software ( developed by m. saghaei , isfahan university of medical sciences , isfahan , iran ) . the surfaces of 60 roots were covered with baseplate wax ( lysanda , so paulo , sp , brazil ) that was liquefied at 60c . to simulate the biological space , each root was immersed in wax for 2 seconds up to 3 mm below the most coronal portion of the root , which created an approximately 0.3 mm thick wax layer , considered adequate in prior literature10 , . the thickness of the wax layer was confirmed by measuring the thickness of the root with a digital caliper before and after immersion . all 90 roots were embedded in pvc cylinders ( height : 14 mm ; diameter : 25 mm ) as described in other study . the bur used to prepare the conduits was positioned inside the canal , and while supporting the root , attached to a surveyor . consequently , the long axes of the bur , the root , and the cylinder were parallel to each other and perpendicular to the horizontal axis . self - curing acrylic resin dencrilay ( dencril , caieiras , sp , brazil ) was mixed at 3:1 ( powder : liquid ) and poured inside the pvc cylinder . the most coronal 3 mm section of the root was exposed to simulate the bone limit . after resin polymerization , the teeth covered bywax were separated from the cylinders , and the wax was removed from the root surface and acrylic resin interior using hot water and manual instruments . thirty roots had periodontal ligament simulated with polyether ( impregum soft medium body , 3 m espe ) and 30 roots with addition silicone ( express medium body , 3 m espe ) , according to the randomization . to create the artificial periodontal ligament , the corresponding elastomeric material was inserted along the artificial alveolus created on the acrylic resin . then , the root was repositioned into the alveolar space and the elastomer polymerized ; the elastomer in excess was then removed using a scalpel blade . the glass fiber posts were sectioned to a length of 17 mm ( 5 mm coronal portion and 12 mm intracanal portion ) . each post was cleansed with 70% ethanol ; silane coupling agent ( prosil , fgm ) was applied onto the surface using a microbrush ( cavibrush , fgm ) , and the posts were left undisturbed for 5 minutes , according to the recommendations of the manufacturer . the root canals were conditioned with 37% phosphoric acid ( condac 37 , fgm ) for 15 seconds and washed with 10 ml of distilled water . excess water was removed with absorbent paper points ( tanari , manacapuru , am , brazil ) . the activator of the adhesive system ( adper scotchbond multipurpose , 3 m espe ) was applied into the canal using a microbrush ( cavibrush , fgm ) ; the excess material was removed with paper points ( tanari ) and put to dry for 5 seconds . then , the resin cement relyx arc ( 3 m espe ) was applied on the post with a spatula and in the root canal with a lentulo drill # 40 ( injecta , diadema , sp , brazil ) . the posts were positioned inside the root canal , the cement in excess was removed , and photo - activation was performed for 40 seconds on the occlusal surface with a led ( radii - cal , sdi , melbourne , vic , australia ) . after the posts were cemented , the crown was reconstructed . the coronal dentin was etched with 37% phosphoric acid ( condac 37 , fgm ) for 15 seconds , washed with 10 ml of distilled water , and the excess removed with absorbent paper . the adhesive system ( single bond 2 , 3 m espe ) was applied using a microbrush ( cavibrush , fgm ) ; the core was built up with composite resin ( oppallis , fgm ) using matrices at standardized dimensions and anatomical confguration of an upper central incisor prepared for a full crown . the groups ( n=10 ) were divided according to the artificial periodontal ligament composition ( figure 1 ) . all the specimens were immersed in distilled water at 37c and submitted to 210 cycles in a mechanical fatigue simulator ( er 11000 , erios , so paulo , sp , brazil ) , which is equivalent to 2 years of clinical service at an 88 n load , 2.2 hz frequency and 45 incline from the horizontal axis . twenty - four hours after the mechanical cycling was fnished , the specimens were fixed on the metallic base of a cutting machine ( labcut 1010 , extec corp . , enfeld , ct , usa ) and sectioned perpendicularly to the root long axis . the first slice of approximately 1 mm was discarded , and 3 slices ( 2 mm ) were obtained from each tooth . each slice was positioned with the most coronal portion downward on a metallic device while a cylinder applied a load on the post directed from the apical to the coronal portion at a crosshead speed of 1 mm / min ( emic dl 2000 , emic , so jos dos pinhais , pr , brazil ) . the height and canal diameters of the coronal and apical portions were measured with a digital caliper . the bond strength ( mpa ) was calculated using the formula r = f / a , where f was the force of rupture ( n ) , and a was the bonded area . the bonded area was calculated using the formula to determine the lateral area of a circular straight cone with parallel bases : a=g.(r1+r2 ) , where =3.14 , g = slant height , r1=smaller base radius , and r2=larger base radius . to determine the slant height , the following formula was used : g = h+(r2-r1 ) , where h = specimen thickness and r1 and r2 were the internal diameters of the smaller and larger bases , respectively , divided by 2 . afterwards , the specimens were examined under a stereo discovery v20 optical microscope ( carl zeiss , gbttingen , nsac , germany ) up to 40x to determine the predominant failure pattern . failures were classified as : adhesive failure between dentin and cement ( ad dc ) ; adhesive failure between cement and post ( ad cp ) ; cohesive failure of the dentin ( co d ) ; cohesive failure of the cement ( co c ) ; cohesive failure of the post ( co p ) ; mixed adhesive failure of dentin / adhesive failure between post and cement ( m ad dc / pc ) ; and mixed adhesive / cohesive failure ( m ad / co ) . specimens with representative failures were analyzed using scanning electron microscopy ( jeoljsm-5400 , jeol , tachikawa , ty , japan ) . the groups ( n=10 ) were divided according to the periodontal ligament composition and application of mechanical cycling ( figure 1 ) . specimens of groups c - mc , p - mc and as - mc were submitted to mechanical cycling according to the same protocol used for the push - out test . the specimens that were not mechanically cycled were stored in distilled water at a temperature of 37c during the same time period of the mechanical cycling . twenty - four hours after the mechanical cycling was fnished , all specimens of each group were submitted to a compressive strength test in a universal testing machine ( emic dl 2000 , emic ) . each specimen was positioned on the device aligned at 45 to the tooth long axis , and a constant static load was applied at a crosshead speed of 1 mm / min until failure . failure was defned as the point when the load ( n ) reached its maximum value during the test . the failure modes were classified as favorable and unfavorable under an optical microscope ( stereo discovery v20 , carl zeiss ) . fractures at or above the simulated bone level , fractures of the coronal portion of the core , and dislodgement of the core and/or post coronal portion were considered favorable failures . vertical or oblique radicular fractures below the simulated bone level were classified as unfavorable failures . bond strength values were analyzed by one - way anova , with periodontal ligament material as the variable , and fracture strength values were analyzed by two - way anova , with the periodontal ligament composition and application of mechanical cycling as variables . in both cases , the significance level was 5% . cohesive ( figure 2a ) or mixed adhesive / cohesive ( figure 2b ) failure slices were excluded from the bond strength statistical analysis because they did not represent adhesive failures . ninety single - root bovine teeth were cleansed with periodontal curettes and stored in distilled water . coronal portions were sectioned under water cooling to obtain a standardized 16 mm root length . the most coronal diameter of the root canal was measured with a digital caliper ( starrett 727 , starret , itu , sp , brazil ) , and any tooth with a canal diameter larger than the fiber post diameter ( =1.8 mm , white post # 3 , fgm , joinville , sc , brazil ) was discarded and replaced with a different specimen . the apical foramens were sealed using a photo activated adhesive ( single bond 2 , 3 m espe , saint paul , mn , usa ) associated with a composite resin ( oppalis , fgm ) . the canals were prepared with the same drill used in the posts system ( white post dc # 3 , fgm ) to a 12 mm length . the roots were numbered from 1 to 90 and randomly distributed into 6 groups ( n=15 ) , which varied by the artificial periodontal ligament composition and the test performed ( figure 1 ) . the number sequences were generated by random allocation software ( developed by m. saghaei , isfahan university of medical sciences , isfahan , iran ) . the surfaces of 60 roots were covered with baseplate wax ( lysanda , so paulo , sp , brazil ) that was liquefied at 60c . to simulate the biological space , each root was immersed in wax for 2 seconds up to 3 mm below the most coronal portion of the root , which created an approximately 0.3 mm thick wax layer , considered adequate in prior literature10 , . the thickness of the wax layer was confirmed by measuring the thickness of the root with a digital caliper before and after immersion . all 90 roots were embedded in pvc cylinders ( height : 14 mm ; diameter : 25 mm ) as described in other study . the bur used to prepare the conduits was positioned inside the canal , and while supporting the root , attached to a surveyor . consequently , the long axes of the bur , the root , and the cylinder were parallel to each other and perpendicular to the horizontal axis . self - curing acrylic resin dencrilay ( dencril , caieiras , sp , brazil ) was mixed at 3:1 ( powder : liquid ) and poured inside the pvc cylinder . the most coronal 3 mm section of the root was exposed to simulate the bone limit . after resin polymerization , the teeth covered bywax were separated from the cylinders , and the wax was removed from the root surface and acrylic resin interior using hot water and manual instruments . thirty roots had periodontal ligament simulated with polyether ( impregum soft medium body , 3 m espe ) and 30 roots with addition silicone ( express medium body , 3 m espe ) , according to the randomization . to create the artificial periodontal ligament , the corresponding elastomeric material was inserted along the artificial alveolus created on the acrylic resin . then , the root was repositioned into the alveolar space and the elastomer polymerized ; the elastomer in excess was then removed using a scalpel blade . the glass fiber posts were sectioned to a length of 17 mm ( 5 mm coronal portion and 12 mm intracanal portion ) . each post was cleansed with 70% ethanol ; silane coupling agent ( prosil , fgm ) was applied onto the surface using a microbrush ( cavibrush , fgm ) , and the posts were left undisturbed for 5 minutes , according to the recommendations of the manufacturer . the root canals were conditioned with 37% phosphoric acid ( condac 37 , fgm ) for 15 seconds and washed with 10 ml of distilled water . excess water was removed with absorbent paper points ( tanari , manacapuru , am , brazil ) . the activator of the adhesive system ( adper scotchbond multipurpose , 3 m espe ) was applied into the canal using a microbrush ( cavibrush , fgm ) ; the excess material was removed with paper points ( tanari ) and put to dry for 5 seconds . then , the resin cement relyx arc ( 3 m espe ) was applied on the post with a spatula and in the root canal with a lentulo drill # 40 ( injecta , diadema , sp , brazil ) . the posts were positioned inside the root canal , the cement in excess was removed , and photo - activation was performed for 40 seconds on the occlusal surface with a led ( radii - cal , sdi , melbourne , vic , australia ) . after the posts were cemented , the crown was reconstructed . the coronal dentin was etched with 37% phosphoric acid ( condac 37 , fgm ) for 15 seconds , washed with 10 ml of distilled water , and the excess removed with absorbent paper . the adhesive system ( single bond 2 , 3 m espe ) was applied using a microbrush ( cavibrush , fgm ) ; the core was built up with composite resin ( oppallis , fgm ) using matrices at standardized dimensions and anatomical confguration of an upper central incisor prepared for a full crown . the groups ( n=10 ) were divided according to the artificial periodontal ligament composition ( figure 1 ) . all the specimens were immersed in distilled water at 37c and submitted to 210 cycles in a mechanical fatigue simulator ( er 11000 , erios , so paulo , sp , brazil ) , which is equivalent to 2 years of clinical service at an 88 n load , 2.2 hz frequency and 45 incline from the horizontal axis . twenty - four hours after the mechanical cycling was fnished , the specimens were fixed on the metallic base of a cutting machine ( labcut 1010 , extec corp . , the first slice of approximately 1 mm was discarded , and 3 slices ( 2 mm ) were obtained from each tooth . each slice was positioned with the most coronal portion downward on a metallic device while a cylinder applied a load on the post directed from the apical to the coronal portion at a crosshead speed of 1 mm / min ( emic dl 2000 , emic , so jos dos pinhais , pr , brazil ) . the height and canal diameters of the coronal and apical portions were measured with a digital caliper . the bond strength ( mpa ) was calculated using the formula r = f / a , where f was the force of rupture ( n ) , and a was the bonded area . the bonded area was calculated using the formula to determine the lateral area of a circular straight cone with parallel bases : a=g.(r1+r2 ) , where =3.14 , g = slant height , r1=smaller base radius , and r2=larger base radius . to determine the slant height , the following formula was used : g = h+(r2-r1 ) , where h = specimen thickness and r1 and r2 were the internal diameters of the smaller and larger bases , respectively , divided by 2 . afterwards , the specimens were examined under a stereo discovery v20 optical microscope ( carl zeiss , gbttingen , nsac , germany ) up to 40x to determine the predominant failure pattern . failures were classified as : adhesive failure between dentin and cement ( ad dc ) ; adhesive failure between cement and post ( ad cp ) ; cohesive failure of the dentin ( co d ) ; cohesive failure of the cement ( co c ) ; cohesive failure of the post ( co p ) ; mixed adhesive failure of dentin / adhesive failure between post and cement ( m ad dc / pc ) ; and mixed adhesive / cohesive failure ( m ad / co ) . specimens with representative failures were analyzed using scanning electron microscopy ( jeoljsm-5400 , jeol , tachikawa , ty , japan ) . the groups ( n=10 ) were divided according to the periodontal ligament composition and application of mechanical cycling ( figure 1 ) . specimens of groups c - mc , p - mc and as - mc were submitted to mechanical cycling according to the same protocol used for the push - out test . the specimens that were not mechanically cycled were stored in distilled water at a temperature of 37c during the same time period of the mechanical cycling . twenty - four hours after the mechanical cycling was fnished , all specimens of each group were submitted to a compressive strength test in a universal testing machine ( emic dl 2000 , emic ) . each specimen was positioned on the device aligned at 45 to the tooth long axis , and a constant static load was applied at a crosshead speed of 1 mm / min until failure . failure was defned as the point when the load ( n ) reached its maximum value during the test . the failure modes were classified as favorable and unfavorable under an optical microscope ( stereo discovery v20 , carl zeiss ) . fractures at or above the simulated bone level , fractures of the coronal portion of the core , and dislodgement of the core and/or post coronal portion were considered favorable failures . vertical or oblique radicular fractures below the simulated bone level were classified as unfavorable failures . bond strength values were analyzed by one - way anova , with periodontal ligament material as the variable , and fracture strength values were analyzed by two - way anova , with the periodontal ligament composition and application of mechanical cycling as variables . in both cases , the significance level was 5% . cohesive ( figure 2a ) or mixed adhesive / cohesive ( figure 2b ) failure slices were excluded from the bond strength statistical analysis because they did not represent adhesive failures . one - way anova revealed that the periodontal ligament simulation did not affect the bond strength ( p=0.244 ) . most of the failures were adhesive and occurred between the dentin and resin cement ( figure 2c ) , followed by adhesive failures between the post and resin cement ( table 2 ) . push - out bond strength ( mpa ) means standard deviations c : control ; p : polyether ; as : addition silicone distribution of failure modes after the push - out test all groups were submitted to mechanical cycling . ad dc : adhesive failure between dentin and resin cement ; ad cp : adhesive failure between dentin and fiber post ; co d : cohesive failure of dentin ; co p : cohesive failure of fiber post ; m ad dc / pc : mixed failure with adhesive failure between dentin / resin cement and fiber post / resin cement ; m ad / co : mixed failure with adhesive and cohesive failures means and standard deviations are presented in table 3 . two - way anova showed that the simulation of the periodontal ligament ( p=0.153 ) , the mechanical cycling ( p=0.97 ) , and the interaction between the two factors ( p=0.665 ) did not significantly affect fracture resistance . means standard deviation of the fracture load ( n ) data c , p and as were not mechanically cycled ; c : control ; p : polyether ; as : addition silicone ; c - mc : control submitted to mechanical cycling ; p - mc : polyether submitted to mechanical cycling ; as - mc : addition silicone submitted to mechanical cycling qualitative analysis of failure patterns after the fracture resistance test no post fracture was observed , however , a mesiodistal crack was observed in the posts of several specimens ( figure 4 ) c : control ; p : polyether ; as : addition silicone one - way anova revealed that the periodontal ligament simulation did not affect the bond strength ( p=0.244 ) . most of the failures were adhesive and occurred between the dentin and resin cement ( figure 2c ) , followed by adhesive failures between the post and resin cement ( table 2 ) . push - out bond strength ( mpa ) means standard deviations c : control ; p : polyether ; as : addition silicone distribution of failure modes after the push - out test all groups were submitted to mechanical cycling . ad dc : adhesive failure between dentin and resin cement ; ad cp : adhesive failure between dentin and fiber post ; co d : cohesive failure of dentin ; co p : cohesive failure of fiber post ; m ad dc / pc : mixed failure with adhesive failure between dentin / resin cement and fiber post / resin cement ; m ad / co : mixed failure with adhesive and cohesive failures two - way anova showed that the simulation of the periodontal ligament ( p=0.153 ) , the mechanical cycling ( p=0.97 ) , and the interaction between the two factors ( p=0.665 ) did not significantly affect fracture resistance . means standard deviation of the fracture load ( n ) data c , p and as were not mechanically cycled ; c : control ; p : polyether ; as : addition silicone ; c - mc : control submitted to mechanical cycling ; p - mc : polyether submitted to mechanical cycling ; as - mc : addition silicone submitted to mechanical cycling qualitative analysis of failure patterns after the fracture resistance test no post fracture was observed , however , a mesiodistal crack was observed in the posts of several specimens ( figure 4 ) c : control ; p : polyether ; as : addition silicone periodontal ligament simulation may enable a more accurate in vitro reproduction of the oral environment . in the present study , polyether and addition silicone artificially reproduced the periodontal ligament during mechanical fatigue , after the bond strength between the post and dentin was assessed , as well as the fracture resistance of bovine teeth restored using glass fiber posts , both with and without mechanical cycling . due to the difficulty of obtaining intact single - rooted human teeth and the ethical aspects implicated in their use for research , bovine teeth were used as substitutes for human teeth . bovine and human teeth have macro and microscopic structural similarities , and for that reason bovine teeth have been suggested as a possible alternative . the results of the present study showed similar bond strength among the three groups during the push - out test , and thus , the first null hypothesis was accepted . the intraradicular retainers used in the current study were glass fiber posts , which , based on fnite element analysis , generated stress distribution on the root similar to non - restored teeth , as a consequence of its elastic modulus . based on those studies , the lack of differences between the tested groups may result from a tendency towards homogeneous force distribution during mechanical cycling , reducing stress applied on the post / dentin interface so that the load absorbed by the periodontal ligament may not have been relevant in the study conditions . by contrast , when metallic or ceramic intraradicular posts are used , concentrated tension zones are created because the reconstruction material is more rigid than dentin . the present results may have been different if metallic or ceramic posts were used instead . in endodontically treated teeth restored with posts , shear forces are present in the central region of the specimens , which can lead to adhesive failure at the dentin / post interface . although the finding was not statistically significant , the group lacking an artificial periodontal ligament simulation had lower bond strength than the other groups . if a higher number of cycles had been applied on the specimens during aging , perhaps a significant reduction of the bond strength could have been induced . the push - out test generated predominantly adhesive failures at the dentin / cement interface , similarly to previous studies . that failure pattern is probably due to the high c factor inside the canal and the difficulty visualizing and controlling the moisture of the radicular dentin . furthermore , 9% of adhesive failures between cement and post may have been caused by the strong adhesion between dentin and cement promoted by the three - step total - etch adhesive system . the second null hypothesis was also accepted because there was no statistical difference between the groups in fracture resistance regardless of mechanical cycling . this result contrasts with those of a previous study that observed a significant difference between the groups with and without a periodontal ligament after fracture strength testing ; however , the study did not perform mechanical cycling , and intact bovine teeth were used , which have longer and more voluminous crowns than the specimens utilized in the present study . bearing in mind the aforementioned finite elements analysis studies , this finding may be associated with the primarily uniform stress distribution on teeth restored with glass fiber posts . when fiber posts are used , stress concentration is lower compared to rigid retainers , and thus , the cushioning effect provided by the impression material did not alter fracture resistance . however , if metallic or ceramic posts are used , high stress concentration is generated on the root portion due to the rigidity of the material . although the present study did not evaluate those intraradicular retainers , it is speculated that the periodontal ligament simulation may modify stress distribution during mechanical cycling and fracture resistance test if rigid posts are used . more studies must be performed investigating the influence of periodontal ligament simulation on fracture strength of teeth restored with rigid posts and also comparing different post systems . the absence of difference between experimental and control groups may also be , in part , due to the design of the fracture resistance test . during the test , a load is increasingly applied by an indenter until the restoration fails , which compresses the artificial periodontal ligament between two solid structures . as the elastomer is compressed beyond its elastic limit , its cushioning function is lost , and the load applied by the piston only has effect on the specimen after the elastomer is completely squeezed between the specimen and the acrylic resin . figure 3 shows the relation between the force applied and the deformation through graphics of fracture resistance tests for groups as , p , and c , generated by the universal testing machine . graphics of groups as ( figure 3a ) and p ( figure 3b ) present premature peaks before failure threshold , which do not indicate fracture , but an accommodation of the specimen in the artificial alveolus by the simulated periodontal ligament . this phenomenon does not occur in the specimens of the group c ( figure 3c ) , where the only peak illustrated is specimen fracture . therefore , studies of fracture resistance tests simulating periodontal ligament require attention from the operator to obtain the real value of the fracture . graphics of groups addition silicone ( a ) , polyether ( b ) , and control ( c ) illustrating the force ( n ) and deformation ( mm ) of each specimen during the fracture strength test . in the groups with an artificial periodontal ligament this is not observed in group c because of the rigidity of the acrylic resin , and the graphic is a crescent line until the failure threshold of the restorative assembly is reached mechanical cycling did not affect fracture resistance in any group . presumably , the protocol used to mechanically cycle the specimens was not sufficient to decrease fracture resistance . these results are in accordance with other studies that also did not find a difference in the fracture resistance of teeth restored with fiber posts before and after mechanical aging . the failures occurring in the fracture strength test were mainly favorable ( 88% ) and likely related to the glass fiber posts and composite resin , which have rigidity similar to that of dentin and provide a relatively uniform stress distribution to the root . in addition , the adhesion between the post and restorative materials ( adhesive , cement and composite resin ) of similar properties tends to generate a homogeneous stress distribution . failures were predominantly in the core in a mesiodistal direction , which can be explained by the loading applied in a 45 angle to the palatal surface . the specimens bend and experience tensile , and compressive stresses that are maximal externally and minimum centrally . shear stress also acts on the specimens ; howewer , it is maximal in the center and minimal in the external portions . coronal failures were fractures occurring in the buccal region of the cores with post exposition mesiodistally because of the interaction of tensile , compressive and shear forces . the coronal reconstruction presents a minimal thickness zone of composite resin ( figure 4a ) , and therefore , the failure tends to initiate in that area ( figure 5a ) where tensile stresses are more evident . after failure due to tensile stress , the buccal surface is displaced as a result of the compressive and shear stresses acting on the composite / post interface , and hence , the post becomes exposed ( figure 4b and figure 5b ) . mesiodistal root fractures are also explained by the same failure mechanism ( figure 4c ) . the beginning and propagation of the fracture are shown in figure 5b and 5c , respectively . ( a ) schematic diagram of the specimen positioned for the test , indicating the minimal thickness area ( lingual portion ) of the core where the fracture initiates . ( b ) stereomicroscope representative image of specimen fractured in the mesiodistal direction ( group c ) . ( c ) root fracture associated with core fracture ( group addition silicone ) after the fracture resistance test scanning electron microscopy image of control group . there is displacement of the buccal surface of the core , exposing the post . the fiber post suffers a crack in the mesiodistal direction , which is a consequence of shear stress above the post . ( a ) minimal thickness area and hackle lines ( black arrows ) indicating the start of the fracture . ( b ) hackle lines ( white arrows ) initiating at the composite / post interface . ( c ) hackle lines ( white arrows ) indicated in the buccal surface showing the fracture propagation for the control group without mechanical cycling , there was a higher number of unfavorable failures than in the other groups . when the roots are embedded directly in acrylic resin , there seems to be a stress concentration on the cervical region of the tooth . on the other side , when periodontal ligament simulation is performed , the stress distribution is altered and , instead of occurring stress concentration on a specific region , tension is distributed along the entire root surface . in the present study , polyether and addition silicone were used to reproduce the periodontal ligament , consistent with other studies performing simulation and using similar elastomeric materials . however , current literature lacks a standardized protocol , and as a result , a wide range of materials , such as condensation silicone , addition silicone , polysulfide , polyurethane and an industrial rubber material have been used , as well as other consistencies of the elastomers used in the present study . furthermore , several studies did not indicate the viscosity of the material used , while others failed to clearly identify the material comprising the artificial periodontal ligament . bearing in mind the limitations of the present study , the teeth were restored with composite resin cores instead of crowns , which are not consistent with clinical performance ; however , without core protection , the condition generated by load application in the adhesive interface is more hostile , which is an interesting aspect since the present study assessed the bond strength . also , survival studies , investigation under a longer period of mechanical cycling and fnite element analysis could be interesting options for future studies to assess the long - term behavior of endodontically treated teeth restored with intraradicular posts using different simulated periodontal ligaments . studies evaluating other types of posts are also recommended to validate the influence of an artificial periodontal ligament on the behavior of endodontically treated teeth . the simulation of the periodontal ligament using elastomeric materials does not affect the bond strength and the fracture resistance of the teeth restored with fiber posts and composite cores , and therefore it may not be necessary to perform periodontal ligament simulation in those tests in the experimental conditions of the study .

objectiveconsidering that periodontal ligament simulation may influence the stress distribution over teeth restored with intraradicular retainers , this study aimed to assess the combined effect of mechanical cycling and periodontal ligament simulation on both the bond strength between fiber posts and root dentin and the fracture resistance of teeth restored using glass fiber posts.material and methodsninety roots were randomly distributed into 3 groups ( n=10 ) ( c - mc : control ; p - mc : polyether ; as - mc : addition silicone ) to test bond strength and 6 groups ( n=10 ) ( c : control ; p : polyether ; as : addition silicone , without mechanical cycling , and c - mc , p - mc and as - mc with mechanical cycling ) to test fracture strength , according to the material used to simulate the periodontal ligament . for the bond strength test , fiber posts were cemented , cores were built , mechanical cycling was applied ( 2106 cycles , 88 n , 2.2 hz , and 45 incline ) , and the teeth cut into 3 slices ( 2 mm ) , which were then subjected to the push - out test at 1 mm / min . for the fracture strength test , fiber posts were cemented , cores were built , and half of the groups received mechanical cycling , followed by the compressive strength ( 45 to the long axis and 1 mm / min ) performed on all groups.resultsperiodontal ligament simulation did not affect the bond strength ( p=0.244 ) between post and dentin . simulation of periodontal ligament ( p=0.153 ) and application of mechanical cycling ( p=0.97 ) did not affect fracture resistance.conclusionsthe materials used to simulate the periodontal ligament did not affect fracture or bond strength , therefore periodontal ligament simulation using the tested materials could be considered optional in the conditions of the study .

INTRODUCTION MATERIAL AND METHODS Specimen selection and adequacy Experimental design: root embedment and periodontal ligament simulation Posts cementation Push-out bond strength test Fracture resistance test Statistical analysis RESULTS Push-out bond strength test Fracture resistance test DISCUSSION CONCLUSION

consequently , fiber posts provide more uniform stress distribution on root dentin compared to other retainers . the clinical success of restorative treatments depends on many factors , including the amount of dental substrate , adequate coronal reconstruction , cavity configuration factor , masticatory load support by the teeth and stress distribution over the bone and periodontal ligament . for masticatory loads up to 70 when the fibers reach their maximum load resistance , the periodontal ligament becomes rigid , the load is transmitted to the bone , and tensions are distributed over the root surface . therefore , the properties of masticatory force absorption through the bone and periodontal ligament should be reproduced in laboratorial tests in order to simulate the clinical reality more accurately . however , various studies that assess bond strength between root canal posts and dentin after mechanical fatigue do not simulate periodontal ligament . similarly , most studies fail to simulate the periodontal ligament in fracture resistance tests , although a few studies simulate the periodontal ligament using polyether and polivynilsiloxane based elastomers . a few in vitro studies assessed the effects of periodontal ligament simulation and showed that an artificial periodontal ligament reduced the fracture strength and modified the failure modes of sound in bovine teeth . also , simulating the periodontal ligament significantly reduced fracture resistance of ceramic - fixed partial dentures . the roots were numbered from 1 to 90 and randomly distributed into 6 groups ( n=15 ) , which varied by the artificial periodontal ligament composition and the test performed ( figure 1 ) . to simulate the biological space , each root was immersed in wax for 2 seconds up to 3 mm below the most coronal portion of the root , which created an approximately 0.3 mm thick wax layer , considered adequate in prior literature10 , . consequently , the long axes of the bur , the root , and the cylinder were parallel to each other and perpendicular to the horizontal axis . the most coronal 3 mm section of the root was exposed to simulate the bone limit . thirty roots had periodontal ligament simulated with polyether ( impregum soft medium body , 3 m espe ) and 30 roots with addition silicone ( express medium body , 3 m espe ) , according to the randomization . each post was cleansed with 70% ethanol ; silane coupling agent ( prosil , fgm ) was applied onto the surface using a microbrush ( cavibrush , fgm ) , and the posts were left undisturbed for 5 minutes , according to the recommendations of the manufacturer . the groups ( n=10 ) were divided according to the artificial periodontal ligament composition ( figure 1 ) . all the specimens were immersed in distilled water at 37c and submitted to 210 cycles in a mechanical fatigue simulator ( er 11000 , erios , so paulo , sp , brazil ) , which is equivalent to 2 years of clinical service at an 88 n load , 2.2 hz frequency and 45 incline from the horizontal axis . the first slice of approximately 1 mm was discarded , and 3 slices ( 2 mm ) were obtained from each tooth . each slice was positioned with the most coronal portion downward on a metallic device while a cylinder applied a load on the post directed from the apical to the coronal portion at a crosshead speed of 1 mm / min ( emic dl 2000 , emic , so jos dos pinhais , pr , brazil ) . the bond strength ( mpa ) was calculated using the formula r = f / a , where f was the force of rupture ( n ) , and a was the bonded area . failures were classified as : adhesive failure between dentin and cement ( ad dc ) ; adhesive failure between cement and post ( ad cp ) ; cohesive failure of the dentin ( co d ) ; cohesive failure of the cement ( co c ) ; cohesive failure of the post ( co p ) ; mixed adhesive failure of dentin / adhesive failure between post and cement ( m ad dc / pc ) ; and mixed adhesive / cohesive failure ( m ad / co ) . the groups ( n=10 ) were divided according to the periodontal ligament composition and application of mechanical cycling ( figure 1 ) . specimens of groups c - mc , p - mc and as - mc were submitted to mechanical cycling according to the same protocol used for the push - out test . twenty - four hours after the mechanical cycling was fnished , all specimens of each group were submitted to a compressive strength test in a universal testing machine ( emic dl 2000 , emic ) . each specimen was positioned on the device aligned at 45 to the tooth long axis , and a constant static load was applied at a crosshead speed of 1 mm / min until failure . bond strength values were analyzed by one - way anova , with periodontal ligament material as the variable , and fracture strength values were analyzed by two - way anova , with the periodontal ligament composition and application of mechanical cycling as variables . the roots were numbered from 1 to 90 and randomly distributed into 6 groups ( n=15 ) , which varied by the artificial periodontal ligament composition and the test performed ( figure 1 ) . to simulate the biological space , each root was immersed in wax for 2 seconds up to 3 mm below the most coronal portion of the root , which created an approximately 0.3 mm thick wax layer , considered adequate in prior literature10 , . consequently , the long axes of the bur , the root , and the cylinder were parallel to each other and perpendicular to the horizontal axis . thirty roots had periodontal ligament simulated with polyether ( impregum soft medium body , 3 m espe ) and 30 roots with addition silicone ( express medium body , 3 m espe ) , according to the randomization . each post was cleansed with 70% ethanol ; silane coupling agent ( prosil , fgm ) was applied onto the surface using a microbrush ( cavibrush , fgm ) , and the posts were left undisturbed for 5 minutes , according to the recommendations of the manufacturer . the groups ( n=10 ) were divided according to the artificial periodontal ligament composition ( figure 1 ) . all the specimens were immersed in distilled water at 37c and submitted to 210 cycles in a mechanical fatigue simulator ( er 11000 , erios , so paulo , sp , brazil ) , which is equivalent to 2 years of clinical service at an 88 n load , 2.2 hz frequency and 45 incline from the horizontal axis . , the first slice of approximately 1 mm was discarded , and 3 slices ( 2 mm ) were obtained from each tooth . each slice was positioned with the most coronal portion downward on a metallic device while a cylinder applied a load on the post directed from the apical to the coronal portion at a crosshead speed of 1 mm / min ( emic dl 2000 , emic , so jos dos pinhais , pr , brazil ) . the bond strength ( mpa ) was calculated using the formula r = f / a , where f was the force of rupture ( n ) , and a was the bonded area . failures were classified as : adhesive failure between dentin and cement ( ad dc ) ; adhesive failure between cement and post ( ad cp ) ; cohesive failure of the dentin ( co d ) ; cohesive failure of the cement ( co c ) ; cohesive failure of the post ( co p ) ; mixed adhesive failure of dentin / adhesive failure between post and cement ( m ad dc / pc ) ; and mixed adhesive / cohesive failure ( m ad / co ) . the groups ( n=10 ) were divided according to the periodontal ligament composition and application of mechanical cycling ( figure 1 ) . specimens of groups c - mc , p - mc and as - mc were submitted to mechanical cycling according to the same protocol used for the push - out test . twenty - four hours after the mechanical cycling was fnished , all specimens of each group were submitted to a compressive strength test in a universal testing machine ( emic dl 2000 , emic ) . each specimen was positioned on the device aligned at 45 to the tooth long axis , and a constant static load was applied at a crosshead speed of 1 mm / min until failure . bond strength values were analyzed by one - way anova , with periodontal ligament material as the variable , and fracture strength values were analyzed by two - way anova , with the periodontal ligament composition and application of mechanical cycling as variables . one - way anova revealed that the periodontal ligament simulation did not affect the bond strength ( p=0.244 ) . most of the failures were adhesive and occurred between the dentin and resin cement ( figure 2c ) , followed by adhesive failures between the post and resin cement ( table 2 ) . push - out bond strength ( mpa ) means standard deviations c : control ; p : polyether ; as : addition silicone distribution of failure modes after the push - out test all groups were submitted to mechanical cycling . two - way anova showed that the simulation of the periodontal ligament ( p=0.153 ) , the mechanical cycling ( p=0.97 ) , and the interaction between the two factors ( p=0.665 ) did not significantly affect fracture resistance . means standard deviation of the fracture load ( n ) data c , p and as were not mechanically cycled ; c : control ; p : polyether ; as : addition silicone ; c - mc : control submitted to mechanical cycling ; p - mc : polyether submitted to mechanical cycling ; as - mc : addition silicone submitted to mechanical cycling qualitative analysis of failure patterns after the fracture resistance test no post fracture was observed , however , a mesiodistal crack was observed in the posts of several specimens ( figure 4 ) c : control ; p : polyether ; as : addition silicone one - way anova revealed that the periodontal ligament simulation did not affect the bond strength ( p=0.244 ) . most of the failures were adhesive and occurred between the dentin and resin cement ( figure 2c ) , followed by adhesive failures between the post and resin cement ( table 2 ) . push - out bond strength ( mpa ) means standard deviations c : control ; p : polyether ; as : addition silicone distribution of failure modes after the push - out test all groups were submitted to mechanical cycling . ad dc : adhesive failure between dentin and resin cement ; ad cp : adhesive failure between dentin and fiber post ; co d : cohesive failure of dentin ; co p : cohesive failure of fiber post ; m ad dc / pc : mixed failure with adhesive failure between dentin / resin cement and fiber post / resin cement ; m ad / co : mixed failure with adhesive and cohesive failures two - way anova showed that the simulation of the periodontal ligament ( p=0.153 ) , the mechanical cycling ( p=0.97 ) , and the interaction between the two factors ( p=0.665 ) did not significantly affect fracture resistance . means standard deviation of the fracture load ( n ) data c , p and as were not mechanically cycled ; c : control ; p : polyether ; as : addition silicone ; c - mc : control submitted to mechanical cycling ; p - mc : polyether submitted to mechanical cycling ; as - mc : addition silicone submitted to mechanical cycling qualitative analysis of failure patterns after the fracture resistance test no post fracture was observed , however , a mesiodistal crack was observed in the posts of several specimens ( figure 4 ) c : control ; p : polyether ; as : addition silicone periodontal ligament simulation may enable a more accurate in vitro reproduction of the oral environment . in the present study , polyether and addition silicone artificially reproduced the periodontal ligament during mechanical fatigue , after the bond strength between the post and dentin was assessed , as well as the fracture resistance of bovine teeth restored using glass fiber posts , both with and without mechanical cycling . the results of the present study showed similar bond strength among the three groups during the push - out test , and thus , the first null hypothesis was accepted . the intraradicular retainers used in the current study were glass fiber posts , which , based on fnite element analysis , generated stress distribution on the root similar to non - restored teeth , as a consequence of its elastic modulus . based on those studies , the lack of differences between the tested groups may result from a tendency towards homogeneous force distribution during mechanical cycling , reducing stress applied on the post / dentin interface so that the load absorbed by the periodontal ligament may not have been relevant in the study conditions . in endodontically treated teeth restored with posts , shear forces are present in the central region of the specimens , which can lead to adhesive failure at the dentin / post interface . the push - out test generated predominantly adhesive failures at the dentin / cement interface , similarly to previous studies . the second null hypothesis was also accepted because there was no statistical difference between the groups in fracture resistance regardless of mechanical cycling . this result contrasts with those of a previous study that observed a significant difference between the groups with and without a periodontal ligament after fracture strength testing ; however , the study did not perform mechanical cycling , and intact bovine teeth were used , which have longer and more voluminous crowns than the specimens utilized in the present study . bearing in mind the aforementioned finite elements analysis studies , this finding may be associated with the primarily uniform stress distribution on teeth restored with glass fiber posts . when fiber posts are used , stress concentration is lower compared to rigid retainers , and thus , the cushioning effect provided by the impression material did not alter fracture resistance . although the present study did not evaluate those intraradicular retainers , it is speculated that the periodontal ligament simulation may modify stress distribution during mechanical cycling and fracture resistance test if rigid posts are used . more studies must be performed investigating the influence of periodontal ligament simulation on fracture strength of teeth restored with rigid posts and also comparing different post systems . the absence of difference between experimental and control groups may also be , in part , due to the design of the fracture resistance test . figure 3 shows the relation between the force applied and the deformation through graphics of fracture resistance tests for groups as , p , and c , generated by the universal testing machine . graphics of groups as ( figure 3a ) and p ( figure 3b ) present premature peaks before failure threshold , which do not indicate fracture , but an accommodation of the specimen in the artificial alveolus by the simulated periodontal ligament . therefore , studies of fracture resistance tests simulating periodontal ligament require attention from the operator to obtain the real value of the fracture . graphics of groups addition silicone ( a ) , polyether ( b ) , and control ( c ) illustrating the force ( n ) and deformation ( mm ) of each specimen during the fracture strength test . in the groups with an artificial periodontal ligament this is not observed in group c because of the rigidity of the acrylic resin , and the graphic is a crescent line until the failure threshold of the restorative assembly is reached mechanical cycling did not affect fracture resistance in any group . these results are in accordance with other studies that also did not find a difference in the fracture resistance of teeth restored with fiber posts before and after mechanical aging . the failures occurring in the fracture strength test were mainly favorable ( 88% ) and likely related to the glass fiber posts and composite resin , which have rigidity similar to that of dentin and provide a relatively uniform stress distribution to the root . failures were predominantly in the core in a mesiodistal direction , which can be explained by the loading applied in a 45 angle to the palatal surface . ( a ) schematic diagram of the specimen positioned for the test , indicating the minimal thickness area ( lingual portion ) of the core where the fracture initiates . ( c ) root fracture associated with core fracture ( group addition silicone ) after the fracture resistance test scanning electron microscopy image of control group . ( c ) hackle lines ( white arrows ) indicated in the buccal surface showing the fracture propagation for the control group without mechanical cycling , there was a higher number of unfavorable failures than in the other groups . on the other side , when periodontal ligament simulation is performed , the stress distribution is altered and , instead of occurring stress concentration on a specific region , tension is distributed along the entire root surface . in the present study , polyether and addition silicone were used to reproduce the periodontal ligament , consistent with other studies performing simulation and using similar elastomeric materials . however , current literature lacks a standardized protocol , and as a result , a wide range of materials , such as condensation silicone , addition silicone , polysulfide , polyurethane and an industrial rubber material have been used , as well as other consistencies of the elastomers used in the present study . furthermore , several studies did not indicate the viscosity of the material used , while others failed to clearly identify the material comprising the artificial periodontal ligament . bearing in mind the limitations of the present study , the teeth were restored with composite resin cores instead of crowns , which are not consistent with clinical performance ; however , without core protection , the condition generated by load application in the adhesive interface is more hostile , which is an interesting aspect since the present study assessed the bond strength . also , survival studies , investigation under a longer period of mechanical cycling and fnite element analysis could be interesting options for future studies to assess the long - term behavior of endodontically treated teeth restored with intraradicular posts using different simulated periodontal ligaments . the simulation of the periodontal ligament using elastomeric materials does not affect the bond strength and the fracture resistance of the teeth restored with fiber posts and composite cores , and therefore it may not be necessary to perform periodontal ligament simulation in those tests in the experimental conditions of the study .

advances in percutaneous transcatheter device closure of secundum atrial septal defect ( asd ) have resulted in high success rates.1)2 ) nowadays , it is becoming a primary option for treating secundum asd because transcatheter closures may avoid the complications associated with open - heart surgery and can reduce the complication rates and the length of the hospital stay , compared with surgical repairs.3)4 ) however , certain patients still require surgical repairs , even when the defect size indicates that transcatheter closures are feasible . it has been reported that the risk factors for the failure of transcatheter closure included the patient being a small child , having a large - sized single defect , and having a deficient surrounding the rims.5 ) for such difficult cases , modified transcatheter techniques have been recently introduced . for example , balloon assisted ( ba ) method and methods using pulmonary vein ( pv ) have improved the success rates and overcame the anatomical disadvantages , but those methods still have limitations with varying success rates.3)4)6 ) to find the risk factors for the complicated conventional method of the transcatheter device closure , we investigated the echocardiographic parameters for the anatomy of asd and for the size of the left atrium ( la ) . this study analyzed 92 consecutive patients who had undergone percutaneous transcatheter closure of the secundum asd at the congenital heart diseases center at the asan medical center , seoul , korea between october 2010 and december 2012 . the median age of the study group was 3.5 years ( range , 0.9 - 57.4 years).we excluded cases of combined cardiac anomalies , except for mild valvar pulmonary stenosis , multiple asds except cribriform asd , or associated syndromes . age at intervention , sex , weight , height , and 2d images of the pre - interventional transthoracic echocardiography ( tte ) were analyzed with a post - processing program by image arena ( tomtech imaging systems , munich , germany ) . the defect sizes were measured by intraoperative transesophageal echocardiography ( tee ) and the sizes of device used were also collected from the catheterization reports . prior to each intervention , each patient had been admitted and examined by a single physician through a tte using an ie33 with a s5 - 1 transducer ( philips medical systems , andover , ma , usa ) after resting at least 15 minutes in the supine position . children under 3 years old were sedated with chloral hydrate ( 0.5 cc / kg ) while monitoring the vital signs and oxygen saturation , at room air . the rims around the secundum asd were measured in 3 planes and were defined as follows : superior vena cava and inferior vena cava rim from a subcostal view , posterosuperior ( ps ) and mitral valve rim from an apical four - chamber ( a4c ) view , and posteroinferior and retroaortic ( rao ) rim from a parasternal short axis view ( fig . the la dimensions were measured at the end of the systolic phase ; the anteroposterior diameter ( ap ) was measured in a parasternal longaxis view . the mean value of the length was calculated from the lengths measured in a4c and apical two chamber ( a2c ) views . the la volume was calculated by the length and areas of the la in the a4c and a2c views { biplane area - length method : 0.85(areaa2c)(areaa4c)/dl}. the transcatheter device closure was performed under general anesthesia , and the secundum asd size was repeatedly measured by an intraoperative tee by a single physician who was different from the tte - examiner with an ie33 machine ( philips medical systems , andover , ma , usa ) . prior to the cardiac catheterization , anticoagulation was initiated with heparin ( 100 units / kg , intravenously ) and a standard right cardiac catheterization was performed for a hemodynamic study . we did not measure the balloon that routinely stretched the defect size except when a large aneurysmal interatrial septum was present . all the patients underwent transcatheter closure with the amplatzer septal occluder ( aga medical corporation , golden valley , mn , usa ) and device implantation was initially tried by the conventional method that had already been extensively described.3)4 ) devices measuring1 - 2 mm larger than the defect sizes were measured by an intraoperative tee and were employed in the transcatheter closure . if the device implantation was not successful after multiple attempts using the conventional method , the so - called ' pv method ' was attempted as papa et al.4 ) reported . the pv method is described as follows : after the sheath was located in the right upper pv , the la disc was deployed with the remaining unspread and stretched from the orifice of pv through the la cavity until the right atrial disc was delivered and contacted to the vicinity of interatrial septum from the right atrial side . by pushing the cable with the device , the la disc fell out of the pv and spread automatically and fully with an approximation to the interatrial septum4 ) ( fig . 2a and b ) . if neither the conventional nor the pv method was successful , we tried the ba method next , as kammache et al.6 ) reported through another transvenous approach ( usually in the opposite side femoral vein ) for peripheral balloon insertion . the supporting wire for the balloon was located into either the right or left pv depending upon the locations of the deficient rims . when a balloon was introduced through the wire and positioned at the interatrial septum , the la disc that was ' assisted ' or supported by the inflating balloon was delivered first . this procedure prevented the prolapse of the la disc by keeping the plane of the la disc parallel to the interatrial septum . the right atrial disc was deployed in this position , leaving the device in a dumbbell shape . the subsequent deflation of the balloon approximated the disc towards the interatrial septum . by carefully steadying the device position , after the successful removal of the balloon and the wire , we confirmed the device position and the lack of the atrial shunt using the tee , prior to the final release of the device ( fig . 2c and d ) . if the device implantation was unsuccessful after all three methods , we aborted further attempts at the percutaneous transcatheter closure . devices were not deployed and extracted in cases of unstable positioning with either a minnesota wiggling , a compression of the mitral valve or aortic wall , or significant residual leakage during the tee evaluation . patients , whose percutaneous transcatheter closure was unsuccessful , underwent surgical closure at a later time . an antiplatelet agent ( aspirin 3.5 - 5.0 mg / kg once daily ) was prescribed for six months following all successful closures . device stability , residual shunt , or compression of the cardiac valve and aortic wall were evaluated at post - interventional day 1 , 6 months , and 12 months , using the tte . we also evaluated any clinical symptoms and signs of the patients in the outpatient clinic . we used the statistical package for social science ( spss ) for windows , version 18 ( spss inc . , student ttest and mann - whitney test for continuous variables and chi - square test for categorical variables were used for comparing the groups , as appropriate . the logistic regression test was used and receiver - operating curves were plotted for the cut - off values . this study analyzed 92 consecutive patients who had undergone percutaneous transcatheter closure of the secundum asd at the congenital heart diseases center at the asan medical center , seoul , korea between october 2010 and december 2012 . the median age of the study group was 3.5 years ( range , 0.9 - 57.4 years).we excluded cases of combined cardiac anomalies , except for mild valvar pulmonary stenosis , multiple asds except cribriform asd , or associated syndromes . age at intervention , sex , weight , height , and 2d images of the pre - interventional transthoracic echocardiography ( tte ) were analyzed with a post - processing program by image arena ( tomtech imaging systems , munich , germany ) . the defect sizes were measured by intraoperative transesophageal echocardiography ( tee ) and the sizes of device used were also collected from the catheterization reports . prior to each intervention , each patient had been admitted and examined by a single physician through a tte using an ie33 with a s5 - 1 transducer ( philips medical systems , andover , ma , usa ) after resting at least 15 minutes in the supine position . children under 3 years old were sedated with chloral hydrate ( 0.5 cc / kg ) while monitoring the vital signs and oxygen saturation , at room air . the rims around the secundum asd were measured in 3 planes and were defined as follows : superior vena cava and inferior vena cava rim from a subcostal view , posterosuperior ( ps ) and mitral valve rim from an apical four - chamber ( a4c ) view , and posteroinferior and retroaortic ( rao ) rim from a parasternal short axis view ( fig . the la dimensions were measured at the end of the systolic phase ; the anteroposterior diameter ( ap ) was measured in a parasternal longaxis view . the mean value of the length was calculated from the lengths measured in a4c and apical two chamber ( a2c ) views . the la volume was calculated by the length and areas of the la in the a4c and a2c views { biplane area - length method : 0.85(areaa2c)(areaa4c)/dl}. the transcatheter device closure was performed under general anesthesia , and the secundum asd size was repeatedly measured by an intraoperative tee by a single physician who was different from the tte - examiner with an ie33 machine ( philips medical systems , andover , ma , usa ) . prior to the cardiac catheterization , anticoagulation was initiated with heparin ( 100 units / kg , intravenously ) and a standard right cardiac catheterization was performed for a hemodynamic study . we did not measure the balloon that routinely stretched the defect size except when a large aneurysmal interatrial septum was present . all the patients underwent transcatheter closure with the amplatzer septal occluder ( aga medical corporation , golden valley , mn , usa ) and device implantation was initially tried by the conventional method that had already been extensively described.3)4 ) devices measuring1 - 2 mm larger than the defect sizes were measured by an intraoperative tee and were employed in the transcatheter closure . if the device implantation was not successful after multiple attempts using the conventional method , the so - called ' pv method ' was attempted as papa et al.4 ) reported . the pv method is described as follows : after the sheath was located in the right upper pv , the la disc was deployed with the remaining unspread and stretched from the orifice of pv through the la cavity until the right atrial disc was delivered and contacted to the vicinity of interatrial septum from the right atrial side . by pushing the cable with the device , the la disc fell out of the pv and spread automatically and fully with an approximation to the interatrial septum4 ) ( fig . 2a and b ) . if neither the conventional nor the pv method was successful , we tried the ba method next , as kammache et al.6 ) reported through another transvenous approach ( usually in the opposite side femoral vein ) for peripheral balloon insertion . the supporting wire for the balloon was located into either the right or left pv depending upon the locations of the deficient rims . when a balloon was introduced through the wire and positioned at the interatrial septum , the la disc that was ' assisted ' or supported by the inflating balloon was delivered first . this procedure prevented the prolapse of the la disc by keeping the plane of the la disc parallel to the interatrial septum . the right atrial disc was deployed in this position , leaving the device in a dumbbell shape . the subsequent deflation of the balloon approximated the disc towards the interatrial septum . by carefully steadying the device position , after the successful removal of the balloon and the wire , we confirmed the device position and the lack of the atrial shunt using the tee , prior to the final release of the device ( fig . 2c and d ) . if the device implantation was unsuccessful after all three methods , we aborted further attempts at the percutaneous transcatheter closure . devices were not deployed and extracted in cases of unstable positioning with either a minnesota wiggling , a compression of the mitral valve or aortic wall , or significant residual leakage during the tee evaluation . patients , whose percutaneous transcatheter closure was unsuccessful , underwent surgical closure at a later time . an antiplatelet agent ( aspirin 3.5 - 5.0 mg / kg once daily ) was prescribed for six months following all successful closures . device stability , residual shunt , or compression of the cardiac valve and aortic wall were evaluated at post - interventional day 1 , 6 months , and 12 months , using the tte . we also evaluated any clinical symptoms and signs of the patients in the outpatient clinic . we used the statistical package for social science ( spss ) for windows , version 18 ( spss inc . , student ttest and mann - whitney test for continuous variables and chi - square test for categorical variables were used for comparing the groups , as appropriate . the logistic regression test was used and receiver - operating curves were plotted for the cut - off values . a percutaneous transcatheter closure had been attempted in a total of 92 patients during this study 's period . 3 ) . sixty - six patients ( 71.7% ) underwent successful closures by the conventional method , and 22 patients ( 23.9% ) underwent successful closures by the pv ( n=12 , 13.0% ) and ba method ( n=10 , 10.9% ) . a percutaneous closure was unsuccessful in four patients ( 4.3% ) , who subsequently underwent surgical repair . we divided the successful patients into the conventional method group and the modified methods group , including the pv and ba method . the demographic data of the conventional and modified methods groups are characterized in table 1 . there were no significant differences in age , height , weight , and body surface area between the groups . the mean values of the two groups ' anatomical parameters of asds by echocardiography are shown in table 2 . we found that the modified methods group had the smaller ps rim ( 6.1 mm vs. 7.7 mm ) and larger asd ( 18.1 mm vs. 13.8 mm ) . the mean sizes of the rao rim of both groups were deficient ( < 5 mm ) and were not significantly different . the la diameters and la volumes indexed by body surface area were also similar in the two groups . table 3 shows the anatomical risk factors for using the modified risk factors from the result of the logistic regression test . larger asd and smaller rao rim were found to be significant factors for the modified methods group . we examined the la dimensions / device size because we postulated that the relatively large size of the la disc might technically hinder the device implantation with conventional method . the ratios of the la dimensions ( ap , width , and length ) to the device sizes were significantly smaller in the modified group compared to the conventional method group ( p=0.001 ) . 4 illustrates the statistical differences , in the ratios of the la dimensions to the employed device size , between the conventional and modified methods groups . the mean values of the la ap diameter / device size were 1.56 in the conventional method group and 1.20 in the modified methods group , which was significantly smaller ( p=0.001 ) . the mean la width / device size and la length / device size were also smaller in the modified methods group ( 1.32 vs. 1.71 and 1.61 vs. 2.07 , respectively ) . among the three ratios , the la ap diameter / device size were the most important parameter based on the result of the logistic regression test ( odds ratio 0.059 , 95% confidence interval 0.008 - 0.448 , p=0.006 ) . comparing the two modified methods ( pv and ba ) groups , the ba group showed significantly larger sizes of asd and smaller la diameters / amplatzer septal occluder sizes ( p=0.031 and p=0.027 , respectively ) . the receiver - operating characteristic curve showed the cut - off values for the three ratios ( fig . 5 ) . for the cut - off values for using the conventional methods with the la ap diameter / device size , the la width / device size and la length / device size might be 1.69 , 1.64 and 1.78 , respectively ( 95.0% of specificity and 46.6% of sensitivity ) . when we reviewed the four patients who failed to have the percutaneous closure , fig . 6 illustrates the only la ap diameter / device that was significantly smaller than those of the successful cases ( p=0.031 ) while the la width / device size and la length / device size were not significantly smaller than those of the successful cases ( p=0.066 and 0.443 , respectively ) . the 3 of 4 failed cases showed that the la ap diameters / device sizes were below 1 and that the la lengths were smaller than the la disc of the device . there was no peri - procedural complication , such as device displacement , sustained arrhythmia , or vascular complications , within the initial 24-hours follow - up during the study period . no early complication , such as aortic erosion or thrombosis , ( within the first 6 months ) after the hospital discharge was observed in any of the patients during the outpatient follow - up period . there was no peri - procedural complication , such as device displacement , sustained arrhythmia , or vascular complications , within the initial 24-hours follow - up during the study period . no early complication , such as aortic erosion or thrombosis , ( within the first 6 months ) after the hospital discharge was observed in any of the patients during the outpatient follow - up period . this study showed an overall success rate of 95.7% for the transcatheter device closure of the secundum asd and these results were comparable to other reports.2)3)5 ) among the successful closures , the conventional method accounted for 85% , with the remaining 15% being closed with the modified methods , such as the pv or ba method . because the latter group represented a considerable portion of patients , regarded as technically challenging cases , we tried to identify the predictors for these technical challenges prior to an intervention , possibly helping to prepare and to shorten the procedure time.7 ) this study found two main predictors . one was the anatomical characteristic of a larger defect and smaller retroaortic rim , agreeing with previous reports.4)8),9),10 ) large asds and the locations and the extent of deficient surrounding the rims were previously known to be risk factors of the modified methods.8),9),10 ) papa et al.4 ) reported that the method using pv was helpful in patients with a deficient ps rim . this study showed that a larger defect and smaller rao rim were the most powerful risk factors , although the modified methods group had a smaller ps rim . the rao rim size , which was known to be unrelated to the success rate,8 ) was one of the risk factors for using the modified methods . these anatomical parameters should be precisely measured ; however , we sometimes had difficulty in applying them to the pediatric population because the pre - interventional tee is not a routine procedure in every center . the clinical cut - off values of the defect and surrounding rim size were not reported in children whose body surface area widely varied according to age and hemodynamic status . the second predictor was the ratio of the la dimensions to the employed device sizes . at times , we experienced a deformation of the left atrial disc into an abnormal shape in a small la and it was frequently prolapsed into the right atrium . this resulted not from the la size but from the relationship with the la size and device size ; this conclusion was supported by the fact that the indexed la dimensions and la volumes were not significantly different between the two groups . because we postulated that the instability of the la disc in a relatively small la might be a risk factor for using the modified methods , resulting in the right atrium disc to be settled down first , we found that the la dimensions / device size were significantly smaller in patients who failed with the conventional method . those ratios that were not influenced by the patient 's body surface area could be easily applicable to the pediatric population . because we measured the la dimensions from the three standard tte planes , the ratios can be calculated prior to the intervention , possibly helping to predict the success rate or to screen for the surgical cases . the cut - off values of the la diameters ( ap , width , and length)/device size for using conventional methods were 1.64 , 1.69 , and 1.78 , respectively . high specificity ( 95.0% ) and low sensitivity ( 46.6% ) were important limitations in our study ; however , because this study was designed retrospectively , these cut - off values with very high specificity could be one of the screening parameters for using the modified methods or for the risk of failure . because these values showed good specificity ( 95% ) and low sensitivity ( 46.6% ) , we should prepare modified methods pre - interventionally when the ratios are below the cutoff levels . for example , if the la diameter / device size ratio is under 1.6 , we might prepare for the technical challenges in case of the failure of the conventional method and we could explain the risk . when we analyzed the four failed cases , 3 of 4 patients ( 75% ) showed the la ap diameter / device size was very small ( < 1.0 ) and we could consider surgical correction after much more experience with combining a meticulous intraoperative tee data for the surrounding rims . we could not find a possible explanation for one case , who had a 24 mm - defect size and 1 deficient rao rim . for the screening of the surgical cases , the interatrial septal length might be considered because 75% of the failed cases had an interatrial septal length that was shorter than the la disc length . however , we needed more data and we did not reach a statistical significance in the success rate when we compared the la disc length and la length parallel to the interatrial septal length . our strategy of the transcatheter device closure was that the conventional method was attempted first in all of the cases , and , if unsuccessful , we used the pv and ba methods . the ba method was reserved as the last procedure , as it required an additional vascular access . according to this study , we sometimes secured another vascular access prior to anticoagulation and this procedure might reduce the chance of vascular and bleeding complications . additionally , we modified the ba method described by dalvi and his colleagues3 ) by using smaller peripheral balloons with smaller sheath because we did not use the usual occlusion balloon measuring size of asd.11),12),13 ) this could reduce vascular complications related to the use of larger sheaths.14 ) our study had some limitations . first of all , the number of patients in this study was quite small and additional data should be collected for future studies . the case control study could be better explained for analyzing the impact of the small la dimension as a single risk factor for using the modified methods and also needs a larger study population . because of the study design and procedure strategy , the cut - off values for using the modified methods could only discriminate the apparent case because of low sensitivity . we do not recommend using the modified methods for the initial attempt based only on the la dimension . however , our procedure strategy could make up for it . while evaluating cases from a single operator potentially reduced an inter - operator bias , this limited the generalization of the study results to only one center . although the peri - procedural and acute complication were absent during the short term of the study period , we did not evaluate the long - term complications reported in other studies.15),16),17),18 ) besides the anatomical factors , such as smaller retroaortic rim and larger asds , the ratios of the la diameters to device size were additional predictors for using the modified methods in the percutaneous device closure of the secundum asds . first of all , the number of patients in this study was quite small and additional data should be collected for future studies . the case control study could be better explained for analyzing the impact of the small la dimension as a single risk factor for using the modified methods and also needs a larger study population . because of the study design and procedure strategy , the cut - off values for using the modified methods could only discriminate the apparent case because of low sensitivity . we do not recommend using the modified methods for the initial attempt based only on the la dimension . however , our procedure strategy could make up for it . while evaluating cases from a single operator potentially reduced an inter - operator bias , this limited the generalization of the study results to only one center . although the peri - procedural and acute complication were absent during the short term of the study period , we did not evaluate the long - term complications reported in other studies.15),16),17),18 ) besides the anatomical factors , such as smaller retroaortic rim and larger asds , the ratios of the la diameters to device size were additional predictors for using the modified methods in the percutaneous device closure of the secundum asds .

background and objectivestranscatheter device closure becomes the first option for treating secundum atrial septal defect ( asd ) , but the conventional method is sometimes unsuccessful even when the defect size indicates the closure to be feasible . to increase the success rate , modified methods have been introduced and used . this study aimed to find predictors for using the modified methods in the device closure of secundum asds.subjects and methodsbetween october 2010 and december 2012 , 92 patients with asds underwent the transcatheter device closure . we analyzed the sizes of the defect , the surrounding rims , and the ratios of the left atrium ( la ) dimensions to the device size in the patients who underwent the procedure either using the conventional or modified methods.resultsamong the 88 successful cases ( 95.7% ) , 22 patients ( 25% ) required modified methods ( 12 using pulmonary vein and 10 using balloon ) . the modified method group had the larger size of asds and smaller posterosuperior rim . the mean ratios of the la anteroposterior diameter , width , and length to the device size were all significantly smaller in the modified methods group than in the conventional group ( 1.20 vs. 1.56 , 1.32 vs. 1.71 , and 1.61 vs. 2.07 , respectively ) . we found that the risk factors for the modified methods were smaller retroaortic rim , larger asd , and smaller la dimension / device size.conclusionin addition to larger defects and smaller retroaortic rim , the smaller ratios of the la dimensions to the device size influenced the need for the application of modified methods in the transcatheter device closure of asds .

Introduction Subjects and Methods Patient selection and collecting data Pre-interventional echocardiographic parameters Percutaneous transcatheter intervention Post-interventional follow-up protocols Statistical analysis Results Complications Discussion Limitations Conclusion

advances in percutaneous transcatheter device closure of secundum atrial septal defect ( asd ) have resulted in high success rates.1)2 ) nowadays , it is becoming a primary option for treating secundum asd because transcatheter closures may avoid the complications associated with open - heart surgery and can reduce the complication rates and the length of the hospital stay , compared with surgical repairs.3)4 ) however , certain patients still require surgical repairs , even when the defect size indicates that transcatheter closures are feasible . it has been reported that the risk factors for the failure of transcatheter closure included the patient being a small child , having a large - sized single defect , and having a deficient surrounding the rims.5 ) for such difficult cases , modified transcatheter techniques have been recently introduced . for example , balloon assisted ( ba ) method and methods using pulmonary vein ( pv ) have improved the success rates and overcame the anatomical disadvantages , but those methods still have limitations with varying success rates.3)4)6 ) to find the risk factors for the complicated conventional method of the transcatheter device closure , we investigated the echocardiographic parameters for the anatomy of asd and for the size of the left atrium ( la ) . this study analyzed 92 consecutive patients who had undergone percutaneous transcatheter closure of the secundum asd at the congenital heart diseases center at the asan medical center , seoul , korea between october 2010 and december 2012 . the median age of the study group was 3.5 years ( range , 0.9 - 57.4 years).we excluded cases of combined cardiac anomalies , except for mild valvar pulmonary stenosis , multiple asds except cribriform asd , or associated syndromes . age at intervention , sex , weight , height , and 2d images of the pre - interventional transthoracic echocardiography ( tte ) were analyzed with a post - processing program by image arena ( tomtech imaging systems , munich , germany ) . the defect sizes were measured by intraoperative transesophageal echocardiography ( tee ) and the sizes of device used were also collected from the catheterization reports . the la dimensions were measured at the end of the systolic phase ; the anteroposterior diameter ( ap ) was measured in a parasternal longaxis view . the la volume was calculated by the length and areas of the la in the a4c and a2c views { biplane area - length method : 0.85(areaa2c)(areaa4c)/dl}. the transcatheter device closure was performed under general anesthesia , and the secundum asd size was repeatedly measured by an intraoperative tee by a single physician who was different from the tte - examiner with an ie33 machine ( philips medical systems , andover , ma , usa ) . we did not measure the balloon that routinely stretched the defect size except when a large aneurysmal interatrial septum was present . all the patients underwent transcatheter closure with the amplatzer septal occluder ( aga medical corporation , golden valley , mn , usa ) and device implantation was initially tried by the conventional method that had already been extensively described.3)4 ) devices measuring1 - 2 mm larger than the defect sizes were measured by an intraoperative tee and were employed in the transcatheter closure . if the device implantation was not successful after multiple attempts using the conventional method , the so - called ' pv method ' was attempted as papa et al.4 ) reported . the pv method is described as follows : after the sheath was located in the right upper pv , the la disc was deployed with the remaining unspread and stretched from the orifice of pv through the la cavity until the right atrial disc was delivered and contacted to the vicinity of interatrial septum from the right atrial side . by pushing the cable with the device , the la disc fell out of the pv and spread automatically and fully with an approximation to the interatrial septum4 ) ( fig . if neither the conventional nor the pv method was successful , we tried the ba method next , as kammache et al.6 ) reported through another transvenous approach ( usually in the opposite side femoral vein ) for peripheral balloon insertion . the supporting wire for the balloon was located into either the right or left pv depending upon the locations of the deficient rims . when a balloon was introduced through the wire and positioned at the interatrial septum , the la disc that was ' assisted ' or supported by the inflating balloon was delivered first . this procedure prevented the prolapse of the la disc by keeping the plane of the la disc parallel to the interatrial septum . by carefully steadying the device position , after the successful removal of the balloon and the wire , we confirmed the device position and the lack of the atrial shunt using the tee , prior to the final release of the device ( fig . device stability , residual shunt , or compression of the cardiac valve and aortic wall were evaluated at post - interventional day 1 , 6 months , and 12 months , using the tte . we also evaluated any clinical symptoms and signs of the patients in the outpatient clinic . this study analyzed 92 consecutive patients who had undergone percutaneous transcatheter closure of the secundum asd at the congenital heart diseases center at the asan medical center , seoul , korea between october 2010 and december 2012 . the median age of the study group was 3.5 years ( range , 0.9 - 57.4 years).we excluded cases of combined cardiac anomalies , except for mild valvar pulmonary stenosis , multiple asds except cribriform asd , or associated syndromes . age at intervention , sex , weight , height , and 2d images of the pre - interventional transthoracic echocardiography ( tte ) were analyzed with a post - processing program by image arena ( tomtech imaging systems , munich , germany ) . the defect sizes were measured by intraoperative transesophageal echocardiography ( tee ) and the sizes of device used were also collected from the catheterization reports . the la dimensions were measured at the end of the systolic phase ; the anteroposterior diameter ( ap ) was measured in a parasternal longaxis view . the mean value of the length was calculated from the lengths measured in a4c and apical two chamber ( a2c ) views . the la volume was calculated by the length and areas of the la in the a4c and a2c views { biplane area - length method : 0.85(areaa2c)(areaa4c)/dl}. the transcatheter device closure was performed under general anesthesia , and the secundum asd size was repeatedly measured by an intraoperative tee by a single physician who was different from the tte - examiner with an ie33 machine ( philips medical systems , andover , ma , usa ) . we did not measure the balloon that routinely stretched the defect size except when a large aneurysmal interatrial septum was present . all the patients underwent transcatheter closure with the amplatzer septal occluder ( aga medical corporation , golden valley , mn , usa ) and device implantation was initially tried by the conventional method that had already been extensively described.3)4 ) devices measuring1 - 2 mm larger than the defect sizes were measured by an intraoperative tee and were employed in the transcatheter closure . if the device implantation was not successful after multiple attempts using the conventional method , the so - called ' pv method ' was attempted as papa et al.4 ) reported . the pv method is described as follows : after the sheath was located in the right upper pv , the la disc was deployed with the remaining unspread and stretched from the orifice of pv through the la cavity until the right atrial disc was delivered and contacted to the vicinity of interatrial septum from the right atrial side . by pushing the cable with the device , the la disc fell out of the pv and spread automatically and fully with an approximation to the interatrial septum4 ) ( fig . if neither the conventional nor the pv method was successful , we tried the ba method next , as kammache et al.6 ) reported through another transvenous approach ( usually in the opposite side femoral vein ) for peripheral balloon insertion . the supporting wire for the balloon was located into either the right or left pv depending upon the locations of the deficient rims . when a balloon was introduced through the wire and positioned at the interatrial septum , the la disc that was ' assisted ' or supported by the inflating balloon was delivered first . this procedure prevented the prolapse of the la disc by keeping the plane of the la disc parallel to the interatrial septum . by carefully steadying the device position , after the successful removal of the balloon and the wire , we confirmed the device position and the lack of the atrial shunt using the tee , prior to the final release of the device ( fig . device stability , residual shunt , or compression of the cardiac valve and aortic wall were evaluated at post - interventional day 1 , 6 months , and 12 months , using the tte . we also evaluated any clinical symptoms and signs of the patients in the outpatient clinic . a percutaneous transcatheter closure had been attempted in a total of 92 patients during this study 's period . sixty - six patients ( 71.7% ) underwent successful closures by the conventional method , and 22 patients ( 23.9% ) underwent successful closures by the pv ( n=12 , 13.0% ) and ba method ( n=10 , 10.9% ) . a percutaneous closure was unsuccessful in four patients ( 4.3% ) , who subsequently underwent surgical repair . we divided the successful patients into the conventional method group and the modified methods group , including the pv and ba method . the demographic data of the conventional and modified methods groups are characterized in table 1 . the mean values of the two groups ' anatomical parameters of asds by echocardiography are shown in table 2 . we found that the modified methods group had the smaller ps rim ( 6.1 mm vs. 7.7 mm ) and larger asd ( 18.1 mm vs. 13.8 mm ) . the mean sizes of the rao rim of both groups were deficient ( < 5 mm ) and were not significantly different . table 3 shows the anatomical risk factors for using the modified risk factors from the result of the logistic regression test . larger asd and smaller rao rim were found to be significant factors for the modified methods group . we examined the la dimensions / device size because we postulated that the relatively large size of the la disc might technically hinder the device implantation with conventional method . the ratios of the la dimensions ( ap , width , and length ) to the device sizes were significantly smaller in the modified group compared to the conventional method group ( p=0.001 ) . 4 illustrates the statistical differences , in the ratios of the la dimensions to the employed device size , between the conventional and modified methods groups . the mean values of the la ap diameter / device size were 1.56 in the conventional method group and 1.20 in the modified methods group , which was significantly smaller ( p=0.001 ) . the mean la width / device size and la length / device size were also smaller in the modified methods group ( 1.32 vs. 1.71 and 1.61 vs. 2.07 , respectively ) . among the three ratios , the la ap diameter / device size were the most important parameter based on the result of the logistic regression test ( odds ratio 0.059 , 95% confidence interval 0.008 - 0.448 , p=0.006 ) . comparing the two modified methods ( pv and ba ) groups , the ba group showed significantly larger sizes of asd and smaller la diameters / amplatzer septal occluder sizes ( p=0.031 and p=0.027 , respectively ) . for the cut - off values for using the conventional methods with the la ap diameter / device size , the la width / device size and la length / device size might be 1.69 , 1.64 and 1.78 , respectively ( 95.0% of specificity and 46.6% of sensitivity ) . 6 illustrates the only la ap diameter / device that was significantly smaller than those of the successful cases ( p=0.031 ) while the la width / device size and la length / device size were not significantly smaller than those of the successful cases ( p=0.066 and 0.443 , respectively ) . the 3 of 4 failed cases showed that the la ap diameters / device sizes were below 1 and that the la lengths were smaller than the la disc of the device . no early complication , such as aortic erosion or thrombosis , ( within the first 6 months ) after the hospital discharge was observed in any of the patients during the outpatient follow - up period . no early complication , such as aortic erosion or thrombosis , ( within the first 6 months ) after the hospital discharge was observed in any of the patients during the outpatient follow - up period . this study showed an overall success rate of 95.7% for the transcatheter device closure of the secundum asd and these results were comparable to other reports.2)3)5 ) among the successful closures , the conventional method accounted for 85% , with the remaining 15% being closed with the modified methods , such as the pv or ba method . because the latter group represented a considerable portion of patients , regarded as technically challenging cases , we tried to identify the predictors for these technical challenges prior to an intervention , possibly helping to prepare and to shorten the procedure time.7 ) this study found two main predictors . one was the anatomical characteristic of a larger defect and smaller retroaortic rim , agreeing with previous reports.4)8),9),10 ) large asds and the locations and the extent of deficient surrounding the rims were previously known to be risk factors of the modified methods.8),9),10 ) papa et al.4 ) reported that the method using pv was helpful in patients with a deficient ps rim . this study showed that a larger defect and smaller rao rim were the most powerful risk factors , although the modified methods group had a smaller ps rim . the rao rim size , which was known to be unrelated to the success rate,8 ) was one of the risk factors for using the modified methods . the clinical cut - off values of the defect and surrounding rim size were not reported in children whose body surface area widely varied according to age and hemodynamic status . the second predictor was the ratio of the la dimensions to the employed device sizes . at times , we experienced a deformation of the left atrial disc into an abnormal shape in a small la and it was frequently prolapsed into the right atrium . this resulted not from the la size but from the relationship with the la size and device size ; this conclusion was supported by the fact that the indexed la dimensions and la volumes were not significantly different between the two groups . because we postulated that the instability of the la disc in a relatively small la might be a risk factor for using the modified methods , resulting in the right atrium disc to be settled down first , we found that the la dimensions / device size were significantly smaller in patients who failed with the conventional method . because we measured the la dimensions from the three standard tte planes , the ratios can be calculated prior to the intervention , possibly helping to predict the success rate or to screen for the surgical cases . the cut - off values of the la diameters ( ap , width , and length)/device size for using conventional methods were 1.64 , 1.69 , and 1.78 , respectively . high specificity ( 95.0% ) and low sensitivity ( 46.6% ) were important limitations in our study ; however , because this study was designed retrospectively , these cut - off values with very high specificity could be one of the screening parameters for using the modified methods or for the risk of failure . because these values showed good specificity ( 95% ) and low sensitivity ( 46.6% ) , we should prepare modified methods pre - interventionally when the ratios are below the cutoff levels . for example , if the la diameter / device size ratio is under 1.6 , we might prepare for the technical challenges in case of the failure of the conventional method and we could explain the risk . when we analyzed the four failed cases , 3 of 4 patients ( 75% ) showed the la ap diameter / device size was very small ( < 1.0 ) and we could consider surgical correction after much more experience with combining a meticulous intraoperative tee data for the surrounding rims . we could not find a possible explanation for one case , who had a 24 mm - defect size and 1 deficient rao rim . for the screening of the surgical cases , the interatrial septal length might be considered because 75% of the failed cases had an interatrial septal length that was shorter than the la disc length . however , we needed more data and we did not reach a statistical significance in the success rate when we compared the la disc length and la length parallel to the interatrial septal length . our strategy of the transcatheter device closure was that the conventional method was attempted first in all of the cases , and , if unsuccessful , we used the pv and ba methods . first of all , the number of patients in this study was quite small and additional data should be collected for future studies . the case control study could be better explained for analyzing the impact of the small la dimension as a single risk factor for using the modified methods and also needs a larger study population . because of the study design and procedure strategy , the cut - off values for using the modified methods could only discriminate the apparent case because of low sensitivity . we do not recommend using the modified methods for the initial attempt based only on the la dimension . although the peri - procedural and acute complication were absent during the short term of the study period , we did not evaluate the long - term complications reported in other studies.15),16),17),18 ) besides the anatomical factors , such as smaller retroaortic rim and larger asds , the ratios of the la diameters to device size were additional predictors for using the modified methods in the percutaneous device closure of the secundum asds . first of all , the number of patients in this study was quite small and additional data should be collected for future studies . the case control study could be better explained for analyzing the impact of the small la dimension as a single risk factor for using the modified methods and also needs a larger study population . because of the study design and procedure strategy , the cut - off values for using the modified methods could only discriminate the apparent case because of low sensitivity . we do not recommend using the modified methods for the initial attempt based only on the la dimension . although the peri - procedural and acute complication were absent during the short term of the study period , we did not evaluate the long - term complications reported in other studies.15),16),17),18 ) besides the anatomical factors , such as smaller retroaortic rim and larger asds , the ratios of the la diameters to device size were additional predictors for using the modified methods in the percutaneous device closure of the secundum asds .

peroxisome proliferator - activated receptor gamma ( ppar ) , a steroid / retinoid nuclear receptor family of ligand activated transcription factors , is expressed in vascular and inflammatory cells . it may regulate the expression of genes networks encoding proteins involved in all aspects of adipogenesis and lipid metabolism ( 1 ) . evidences from cell culture models show that blocking ppar activity reduces eets/ soluble epoxide hydrolase ( seh ) and inhibitor - mediated anti - inflammatory effect , indicating ppar is an effector of eets ( 2 ) . activation of ppar may inhibit tumour progression via induction of differentiation and apoptosis in several lung cancers and lung cancer - derived cell lines ( 3 ) . in lung cancers , the expression of ppar is found to be significantly lower in tumour than in adjacent non - tumour tissues , and there is a correlation between a lower survival rate and decreased ppar expression ( 3 , 4 ) . these studies will help to understand the anti - proliferative functions of ppar in tumour cells . epoxyeicosatrienoic acids ( eets ) , are synthesized from arachidonic acid via cytochrome p450 enzymemediated pathway to four regioisomers , 5,6-eet , 8,9-eet , 11,12-eet , and 14,15-eet ( 5 ) . eets are also reported to have diverse physiological and pathophysiological functions , such as vasodilation by activating membrane maxi - ca - activated k ( bkca ) channels , anti - inflammatory effects , ion channel activation , promotion of the proliferation of vascular cells and angiogenesis , modulation of the proliferation , and migration of vessel smooth muscle cells ( 6 , 7 ) . recently , it has been found that eets in particular 11,12- and 14,15-eet , activate several intracellular signaling molecules including tyrosine kinases and phosphatases , p38 map kinase , extracellular regulated protein kinases 1/2 ( erk1/2 ) and map kinase phosphatases ( 8 , 9 ) . chen et al have reported that exogenous eet in cultured human - derived malignant hematologic cell lines may promote proliferation and attenuated apoptosis ( 10 ) . these findings led us to further investigate the effects and mechanisms of eets on proliferation , apoptosis and metastasis processes in tumour cells . in the present study , it was intended to verify the proliferative and anti - apoptotic effects of exogenous 14,15-eet , gw9662 and auda on carcinoma cancer cells . furthermore , the expression of ppar- , as well as regulation of egfr , erk1/2 , pi3k and akt proteins in carcinoma cancer cells was investigated . dulbecco 's modified eagle 's medium ( dmem ) , trypsin and fetal bovine serum ( fbs ) were obtained from hyclone ( hyclone , logan , usa ) . antibodies against epidermal growth factor receptor ( egfr ) , phosphorous egfr ( p - egfr ) , phosphatidylinositol 3-kinase ( pi3k ) , akt , seh and p - akt were purchased from cell signaling technology ( beverly , ma , usa ) . 2-chloro-5-nitro - n - phenyl - benzamide ( gw9662 ) was purchased from sigma ( st . louis , usa ) . 14 , 15-epoxyeicosatrienoic acid ( 14,15- eet ) and adamantyl - ureido - dodecanoic acid ( auda ) were obtained from cayman chemical co. ( ann arbor , usa ) , and ciglitazone was from calbiochem ( san diego , ca , usa ) . all other reagents were purchased from standard commercial suppliers . human lingual squamous cell carcinoma ( tca-8113 ) and umbilical vein endothelial ( huvec , ecv304 cells ) cell lines were obtained from shanghai institute of cell biology ( introduced from american type culture collection ) . when the cells were grown to 60% confluence , they were incubated with fbs - free dmem at 37 c for 12 hrs to allow for synchronization . then , auda and/or gw9662 ( 10 m ) were added into medium in the presence or absence of 14,15-eet ( 100 nm ) . after 12 or 24 hrs treatment , the medium was removed and replace with new medium containing 5 mg / ml mtt , and incubated for 4 hrs . the cells ( 1.510 cells ) were cultured in the presence of 14,15-eet , 14,15-eet / auda and/or gw9662 for 12 hrs in 24-wells plates , and then collected and fixed with 70% ethanol . the cells were resuspended and incubated in phosphate citrate buffer ( 4 mm citric acid and 192 mm na2hpo4 ) for 30 min at room temperature . the cells were collected by centrifugation at 1000 g for 10 min , and were resuspended in pbs containing propidium iodide / rnase ( 10 g / ml ) . the ratio of sub - g1 dna content were detected and analyzed using cellquest program on a facstar - plus flow cytometer ( bd biosciences , san jose , ca , usa ) . luciferase assays were performed using a dual- glo luciferase assay system ( promega , madison , wi , usa ) with phrl - tk vector as an internal control for normalization of transfection efficiency . transfection was performed in 24-well culture plates , and luciferase activities were measured using a luminometer ( bio - lumat lb9507 , berthold , wildbad , germany ) . briefly , cells were seeded into 24-well plates at a density of 810 cells / well , and cultured for 24 hrs . after removal of medium , the cells were co - transfected with 0.4 g of ppre - tk- luc , 0.4 g of pcmx / ppar and 0.1 g of control plasmid ( phrl - tk ) using lipofectamine plus reagents ( invitrogen , carlsbad , ca ) . after 4 hrs , 500 l of full medium was added into each well and then after 24 hrs , the cells were incubated with 100 nm 14,15-eet , 10m gw9662 and/or 20 m ciglitazone for 12 hrs , and then luciferase activities were measured . total rna from cell cultures was isolated with trizol reagent , and the amount of rna was measured spectrophotometrically . the obtained cdnas were then used as the templates for quantitative rt - pcr with the use of the brilliant sybr green qpcr master mix ( stratagene , la jolla , ca , usa ) . after treatments of 14,15-eet , 14,15-eet / auda and/or gw9662 , the cells were lysed using lysate buffer , and cell lysate supernatants were harvested by centrifugation at l0000 rpm for 10 min at 4c . proteins were separated in a sds- polyacrylamide gel ( 10% ) with 5% stacking gel in sds - tris - glycine running buffer . the membranes were incubated in ttbs buffer containing specific antibodies for 12 hrs , and then incubated with horseradish peroxidase - conjugated goat anti - rabbit igg ( 1:10000 , boehringer mannheim ) for 60 min . the immunodetection was accomplished by using an enhanced chemiluminescence detection system ( pierce chemical co. , usa ) and exposure to x - ray film . comparisons between groups were performed by a student 's paired two - tailed t test . one - way analysis of variance was used to examine differences in response to treatments and between groups . dulbecco 's modified eagle 's medium ( dmem ) , trypsin and fetal bovine serum ( fbs ) were obtained from hyclone ( hyclone , logan , usa ) . antibodies against epidermal growth factor receptor ( egfr ) , phosphorous egfr ( p - egfr ) , phosphatidylinositol 3-kinase ( pi3k ) , akt , seh and p - akt were purchased from cell signaling technology ( beverly , ma , usa ) . 2-chloro-5-nitro - n - phenyl - benzamide ( gw9662 ) was purchased from sigma ( st . louis , usa ) . 14 , 15-epoxyeicosatrienoic acid ( 14,15- eet ) and adamantyl - ureido - dodecanoic acid ( auda ) were obtained from cayman chemical co. ( ann arbor , usa ) , and ciglitazone was from calbiochem ( san diego , ca , usa ) . all other reagents were purchased from standard commercial suppliers . human lingual squamous cell carcinoma ( tca-8113 ) and umbilical vein endothelial ( huvec , ecv304 cells ) cell lines were obtained from shanghai institute of cell biology ( introduced from american type culture collection ) . when the cells were grown to 60% confluence , they were incubated with fbs - free dmem at 37 c for 12 hrs to allow for synchronization . then , auda and/or gw9662 ( 10 m ) were added into medium in the presence or absence of 14,15-eet ( 100 nm ) . after 12 or 24 hrs treatment , the medium was removed and replace with new medium containing 5 mg / ml mtt , and incubated for 4 hrs . the cells ( 1.510 cells ) were cultured in the presence of 14,15-eet , 14,15-eet / auda and/or gw9662 for 12 hrs in 24-wells plates , and then collected and fixed with 70% ethanol . the cells were resuspended and incubated in phosphate citrate buffer ( 4 mm citric acid and 192 mm na2hpo4 ) for 30 min at room temperature . the cells were collected by centrifugation at 1000 g for 10 min , and were resuspended in pbs containing propidium iodide / rnase ( 10 g / ml ) . the ratio of sub - g1 dna content were detected and analyzed using cellquest program on a facstar - plus flow cytometer ( bd biosciences , san jose , ca , usa ) . luciferase assays were performed using a dual- glo luciferase assay system ( promega , madison , wi , usa ) with phrl - tk vector as an internal control for normalization of transfection efficiency . transfection was performed in 24-well culture plates , and luciferase activities were measured using a luminometer ( bio - lumat lb9507 , berthold , wildbad , germany ) . briefly , cells were seeded into 24-well plates at a density of 810 cells / well , and cultured for 24 hrs . after removal of medium , the cells were co - transfected with 0.4 g of ppre - tk- luc , 0.4 g of pcmx / ppar and 0.1 g of control plasmid ( phrl - tk ) using lipofectamine plus reagents ( invitrogen , carlsbad , ca ) . after 4 hrs , 500 l of full medium was added into each well and then after 24 hrs , the cells were incubated with 100 nm 14,15-eet , 10m gw9662 and/or 20 m ciglitazone for 12 hrs , and then luciferase activities were measured . total rna from cell cultures was isolated with trizol reagent , and the amount of rna was measured spectrophotometrically . the obtained cdnas were then used as the templates for quantitative rt - pcr with the use of the brilliant sybr green qpcr master mix ( stratagene , la jolla , ca , usa ) . after treatments of 14,15-eet , 14,15-eet / auda and/or gw9662 , the cells were lysed using lysate buffer , and cell lysate supernatants were harvested by centrifugation at l0000 rpm for 10 min at 4c . proteins were separated in a sds- polyacrylamide gel ( 10% ) with 5% stacking gel in sds - tris - glycine running buffer . the membranes were incubated in ttbs buffer containing specific antibodies for 12 hrs , and then incubated with horseradish peroxidase - conjugated goat anti - rabbit igg ( 1:10000 , boehringer mannheim ) for 60 min . the immunodetection was accomplished by using an enhanced chemiluminescence detection system ( pierce chemical co. , usa ) and exposure to x - ray film . comparisons between groups were performed by a student 's paired two - tailed t test . one - way analysis of variance was used to examine differences in response to treatments and between groups . human lingual squamous cell carcinoma ( tca-8113 ) and umbilical vein endothelial ( huvec , ecv304 cells ) cell lines were obtained from shanghai institute of cell biology ( introduced from american type culture collection ) . when the cells were grown to 60% confluence , they were incubated with fbs - free dmem at 37 c for 12 hrs to allow for synchronization . then , auda and/or gw9662 ( 10 m ) were added into medium in the presence or absence of 14,15-eet ( 100 nm ) . after 12 or 24 hrs treatment , the medium was removed and replace with new medium containing 5 mg / ml mtt , and incubated for 4 hrs . the cells ( 1.510 cells ) were cultured in the presence of 14,15-eet , 14,15-eet / auda and/or gw9662 for 12 hrs in 24-wells plates , and then collected and fixed with 70% ethanol . the cells were resuspended and incubated in phosphate citrate buffer ( 4 mm citric acid and 192 mm na2hpo4 ) for 30 min at room temperature . the cells were collected by centrifugation at 1000 g for 10 min , and were resuspended in pbs containing propidium iodide / rnase ( 10 g / ml ) . the ratio of sub - g1 dna content were detected and analyzed using cellquest program on a facstar - plus flow cytometer ( bd biosciences , san jose , ca , usa ) . luciferase assays were performed using a dual- glo luciferase assay system ( promega , madison , wi , usa ) with phrl - tk vector as an internal control for normalization of transfection efficiency . transfection was performed in 24-well culture plates , and luciferase activities were measured using a luminometer ( bio - lumat lb9507 , berthold , wildbad , germany ) . briefly , cells were seeded into 24-well plates at a density of 810 cells / well , and cultured for 24 hrs . after removal of medium , the cells were co - transfected with 0.4 g of ppre - tk- luc , 0.4 g of pcmx / ppar and 0.1 g of control plasmid ( phrl - tk ) using lipofectamine plus reagents ( invitrogen , carlsbad , ca ) . after 4 hrs , 500 l of full medium was added into each well and then after 24 hrs , the cells were incubated with 100 nm 14,15-eet , 10m gw9662 and/or 20 m ciglitazone for 12 hrs , and then luciferase activities were measured . total rna from cell cultures was isolated with trizol reagent , and the amount of rna was measured spectrophotometrically . the obtained cdnas were then used as the templates for quantitative rt - pcr with the use of the brilliant sybr green qpcr master mix ( stratagene , la jolla , ca , usa ) . after treatments of 14,15-eet , 14,15-eet / auda and/or gw9662 , the cells were lysed using lysate buffer , and cell lysate supernatants were harvested by centrifugation at l0000 rpm for 10 min at 4c . proteins were separated in a sds- polyacrylamide gel ( 10% ) with 5% stacking gel in sds - tris - glycine running buffer . the membranes were incubated in ttbs buffer containing specific antibodies for 12 hrs , and then incubated with horseradish peroxidase - conjugated goat anti - rabbit igg ( 1:10000 , boehringer mannheim ) for 60 min . the immunodetection was accomplished by using an enhanced chemiluminescence detection system ( pierce chemical co. , usa ) and exposure to x - ray film . comparisons between groups were performed by a student 's paired two - tailed t test . one - way analysis of variance was used to examine differences in response to treatments and between groups . ppar has been proved to be a key transcription factor of adipocyte differentiation lipid and glucose homeostasis and an important target in type 2 diabetes and metabolic syndrome . moreover , they also appear to be expressed in monocytes / macrophages , dendritic cells , eosinophils , t cells , b cells and endothelial cells , etc ( 12 ) . to confirm whether ppar and seh were expressed in human carcinoma and ecv304 cells , western blot analysis was performed . results suggested that ppar and seh proteins were expressed in two tested cell lines , and have different degrees of intensity when treated with different concentrations of 14,15-eet . the degree of intensity of ppar in tca-8113 cell line was significantly higher than that of the ecv304 cells . results also suggested that the expression of ppar reached a maximum level after 24 hrs treatment with 14,15-eet within the range of 25 - 200 nm ( fig . effects of 14,15-eet treatment on the expression of ppar and seh in tca-8113 ( a ) and huvec ( b ) cells . ppar protein levels in carcinoma and cells were measured by treatment with different concentrations ( 0 - 200 nm ) of 14,15-eet after 24 hrs . total protein was extracted and analyzed by western blot as described in materials and methods . . lanes from 1 to 5 were 0 , 25 , 50 , 100 and 200 nm , respectively . -actin is shown as an internal control . as a ligand - activated transcription factor , the role of ppar in cancer remains a subject of debate ( 4 , 6 , 13 ) . based on luciferase activity assay system , the activation of ppar in carcinoma cells by 100 nm of 14,15-eet was investigated . as shown in figure 2 , luciferase activities showed 4.28 , 3.15 and 4.47- fold increase when the cells were incubated and transfected by using ppre - tk - luc and addition of 14,15-eet , 14,15-eet+gw9662 and ciglitazone , respectively . in addition , gw9662 , as a ppar antagonist , may inhibit the luciferase activities compared to that which was obtained by addition of 14,15-eet . under co - transfected ppre - tk - luc and pcmx3/ppar condition , activities resulting by the addition of 14,15-eet and ciglitazone compared to control increased significantly 7.75 and 5.13-fold , respectively . the third and sixth groups were co - transfected using 0.4 g ppre - tk - luc , 0.4 g pcmx3/ppar and 0.1 g control plasmid ( phrl - tk ) . other groups were transfected by using 0.4 g ppre - tk - luc and 0.1 g control plasmid ( phrl - tk ) . the fifth and sixth groups were administrated with ciglitazone ( 20 m ) as positive control . the third and fourth groups were then incubated with 14,15-eet ( 100 nm ) in the absence or presence of gw9662 . previous studies have indicated that eet may induce cell growth and apoptosis in various cancer cells ( 5 ) . however , the effects of 14,15-eet on the cell proliferation in human carcinoma cell is still unclear . in the present study , a human carcinoma cell was used as an in vitro model to evaluate whether exogenous eet - induced proliferative effects via ppar activation . as shown in figure 3 , results suggested that the addition of 14,15-eet ( 100 nm ) in carcinoma cells cultured for 12 and 24 hrs may stimulate cell proliferation compared to the control . the addition of gw9662 and auda in the cells had no effects compared to the control , but cell proliferation was inhibited by the addition of 14,15- eet and gw9662 . recent data have shown that ppar activation by thiazolidinediones such as troglitazone induced inhibition of a wide variety of cancer cells , suggesting that activation of ppar down - regulates cell growth ( 3 , 4 ) . results of this study suggested that 14,5-eet could up - regulate the expressions of ppar in the human carcinoma cells , and also showed that it may stimulate cell proliferation ( figs 2 and 3 ) . effects of exogenous 14,15-eet and 14,15-eet ( 100 nm)/auda carcinoma cell proliferation for 12 and 24 hrs . significant difference ( p < 0.05 ) is denoted as asterisk ( * ) between control and treatments . a reduction in cell proliferation , an increase in the rate of apoptosis , or both may explain the reduction of cell viability caused by ppar agonists . in a variety of cancer cells , ppar inhibits proliferation and cell cycle arrest at g0 /g1 restriction point ( 14 ) . to study the proliferative effect of 14,15-eet , the fate of 14,15-eet - treated carcinoma cells within the cell cycle was investigated using flowcytometry analysis . as shown in figure 4 and table 1 , exogenous 14,15-eet significantly increased the percentage ( 47.08% ) of cells during s - g2-m phase in tca-8113 cells compared to the control and that of vehicle . however , the proportion of s - g2-m phase cell populations compared to the control was reduced significantly by treatment with gw9662 . these results suggest that 14 , 15-eeet may have the ability to promote the proliferation of carcinoma cells and contribute to the anti - apoptosis effects in human carcinoma cells . effects of 14,15-eet on cell cycle progression in human carcinoma cell lines by treatment with 14,15-eet . tca-8113 cells were pretreated with auda ( 1 m ) for 2 h and then incubated with 14,15-eet ( 100 nm ) in the presence or absence of gw9662 ( 10 m ) . previous studies have shown that eet may bind to putative binding protein or receptor , which may trans - activate egfr and akt in regulation of the cell motility , and have revealed that expression of by epoxygenase is very strong and selective in human cancer tissues . the levels of phosphorylated egfr , pi3k / akt , and erk1/2 in tumor cells transfected with cytochrome epoxygenases were significantly upregulated . studies have also shown the status of signal molecules , such as egfr , pi3k , akt , and erk1/2 , in tumor cells after transfection with cyp genes . these results suggest that pi3k / akt and erk1/2 pathways are involved in the promotion of tumor cells by cyp epoxygenases overexpression ( 15 , 16 ) . results of this study suggest that the total amount of egfr , erk , and pi3 kinase / akt proteins were quite refractory to 14 , 15-eet and other additives , and their expression levels were correlated with these stimulators in human carcinoma cell ( figs 5 and 6 ) . the phosphorylated erk and pi3 kinase / akt pathways are induced significantly by auda and 14,15-eet treatments . however , the levels of pi3 kinase - akt pathway was reduced significantly by treatment with gw9662 compared to the control , suggesting that gw9662 , an antagonist of ppar , may block pi3 kinases - akt pathways slightly . the phosphorylated egfr was also elevated significantly by treatment with 14,15-eet , but no significant changes were observed by treatment with gw9662 . the changes in signal transduction pathways and transcriptional mechanisms induced by eet have been investigated , and recent studies are attempting to isolate an eet membrane receptor that mediates cell proliferation and anti - apoptosis in some tumor cell lines ( 6 , 17 ) . findings of this study suggest that exogenous eets significantly promote carcinoma cancer cell proliferation and inhibit apoptosis significantly via the activation of the egfr , erk and pi3 kinase / akt signaling pathways . effects of different treatments on the relative expression densitometry of egfr ( a ) , erk1/2 ( b ) , pi3k ( c ) and akt ( d ) pathways in human carcinoma cells . effects of 14,15-eet on the expression levels of pi3k - akt pathways in human carcinoma cells . tca-8113 cells were pretreated with auda ( 1 m ) for 2 hrs and then stimulated with 14,15-eet ( 100 nm ) in the presence or absence of gw9662 . after 8 hrs , the collected cell lysates were immunoblotted with anti - p - egfr antibody , and p - erk antibody . lanes from 1 to 8 were control , vehicle , gw9662 , auda , 14,15-eet , 14,15-eet+gw9662 , 14,15-eet+auda , 14,15-eet+ auda+gw9662 , respectively . ppar has been proved to be a key transcription factor of adipocyte differentiation lipid and glucose homeostasis and an important target in type 2 diabetes and metabolic syndrome . moreover , they also appear to be expressed in monocytes / macrophages , dendritic cells , eosinophils , t cells , b cells and endothelial cells , etc ( 12 ) . to confirm whether ppar and seh were expressed in human carcinoma and ecv304 cells , western blot analysis was performed . results suggested that ppar and seh proteins were expressed in two tested cell lines , and have different degrees of intensity when treated with different concentrations of 14,15-eet . the degree of intensity of ppar in tca-8113 cell line was significantly higher than that of the ecv304 cells . results also suggested that the expression of ppar reached a maximum level after 24 hrs treatment with 14,15-eet within the range of 25 - 200 nm ( fig . effects of 14,15-eet treatment on the expression of ppar and seh in tca-8113 ( a ) and huvec ( b ) cells . ppar protein levels in carcinoma and cells were measured by treatment with different concentrations ( 0 - 200 nm ) of 14,15-eet after 24 hrs . total protein was extracted and analyzed by western blot as described in materials and methods . . lanes from 1 to 5 were 0 , 25 , 50 , 100 and 200 nm , respectively . -actin is shown as an internal control . as a ligand - activated transcription factor , the role of ppar in cancer remains a subject of debate ( 4 , 6 , 13 ) . based on luciferase activity assay system , the activation of ppar in carcinoma cells by 100 nm of 14,15-eet was investigated . as shown in figure 2 , luciferase activities showed 4.28 , 3.15 and 4.47- fold increase when the cells were incubated and transfected by using ppre - tk - luc and addition of 14,15-eet , 14,15-eet+gw9662 and ciglitazone , respectively . in addition , gw9662 , as a ppar antagonist , may inhibit the luciferase activities compared to that which was obtained by addition of 14,15-eet . under co - transfected ppre - tk - luc and pcmx3/ppar condition , activities resulting by the addition of 14,15-eet and ciglitazone compared to control increased significantly 7.75 and 5.13-fold , respectively . the third and sixth groups were co - transfected using 0.4 g ppre - tk - luc , 0.4 g pcmx3/ppar and 0.1 g control plasmid ( phrl - tk ) . other groups were transfected by using 0.4 g ppre - tk - luc and 0.1 g control plasmid ( phrl - tk ) . the fifth and sixth groups were administrated with ciglitazone ( 20 m ) as positive control . the third and fourth groups were then incubated with 14,15-eet ( 100 nm ) in the absence or presence of gw9662 . previous studies have indicated that eet may induce cell growth and apoptosis in various cancer cells ( 5 ) . however , the effects of 14,15-eet on the cell proliferation in human carcinoma cell is still unclear . in the present study , a human carcinoma cell was used as an in vitro model to evaluate whether exogenous eet - induced proliferative effects via ppar activation . as shown in figure 3 , results suggested that the addition of 14,15-eet ( 100 nm ) in carcinoma cells cultured for 12 and 24 hrs may stimulate cell proliferation compared to the control . the addition of gw9662 and auda in the cells had no effects compared to the control , but cell proliferation was inhibited by the addition of 14,15- eet and gw9662 . recent data have shown that ppar activation by thiazolidinediones such as troglitazone induced inhibition of a wide variety of cancer cells , suggesting that activation of ppar down - regulates cell growth ( 3 , 4 ) . results of this study suggested that 14,5-eet could up - regulate the expressions of ppar in the human carcinoma cells , and also showed that it may stimulate cell proliferation ( figs 2 and 3 ) . effects of exogenous 14,15-eet and 14,15-eet ( 100 nm)/auda carcinoma cell proliferation for 12 and 24 hrs . significant difference ( p < 0.05 ) is denoted as asterisk ( * ) between control and treatments . a reduction in cell proliferation , an increase in the rate of apoptosis , or both may explain the reduction of cell viability caused by ppar agonists . in a variety of cancer cells , ppar inhibits proliferation and cell cycle arrest at g0 /g1 restriction point ( 14 ) . to study the proliferative effect of 14,15-eet , the fate of 14,15-eet - treated carcinoma cells within the cell cycle was investigated using flowcytometry analysis . as shown in figure 4 and table 1 , exogenous 14,15-eet significantly increased the percentage ( 47.08% ) of cells during s - g2-m phase in tca-8113 cells compared to the control and that of vehicle . however , the proportion of s - g2-m phase cell populations compared to the control was reduced significantly by treatment with gw9662 . these results suggest that 14 , 15-eeet may have the ability to promote the proliferation of carcinoma cells and contribute to the anti - apoptosis effects in human carcinoma cells . effects of 14,15-eet on cell cycle progression in human carcinoma cell lines by treatment with 14,15-eet . tca-8113 cells were pretreated with auda ( 1 m ) for 2 h and then incubated with 14,15-eet ( 100 nm ) in the presence or absence of gw9662 ( 10 m ) . previous studies have shown that eet may bind to putative binding protein or receptor , which may trans - activate egfr and akt in regulation of the cell motility , and have revealed that expression of by epoxygenase is very strong and selective in human cancer tissues . the levels of phosphorylated egfr , pi3k / akt , and erk1/2 in tumor cells transfected with cytochrome epoxygenases were significantly upregulated . studies have also shown the status of signal molecules , such as egfr , pi3k , akt , and erk1/2 , in tumor cells after transfection with cyp genes . these results suggest that pi3k / akt and erk1/2 pathways are involved in the promotion of tumor cells by cyp epoxygenases overexpression ( 15 , 16 ) . results of this study suggest that the total amount of egfr , erk , and pi3 kinase / akt proteins were quite refractory to 14 , 15-eet and other additives , and their expression levels were correlated with these stimulators in human carcinoma cell ( figs 5 and 6 ) . the phosphorylated erk and pi3 kinase / akt pathways are induced significantly by auda and 14,15-eet treatments . however , the levels of pi3 kinase - akt pathway was reduced significantly by treatment with gw9662 compared to the control , suggesting that gw9662 , an antagonist of ppar , may block pi3 kinases - akt pathways slightly . the phosphorylated egfr was also elevated significantly by treatment with 14,15-eet , but no significant changes were observed by treatment with gw9662 . the changes in signal transduction pathways and transcriptional mechanisms induced by eet have been investigated , and recent studies are attempting to isolate an eet membrane receptor that mediates cell proliferation and anti - apoptosis in some tumor cell lines ( 6 , 17 ) . findings of this study suggest that exogenous eets significantly promote carcinoma cancer cell proliferation and inhibit apoptosis significantly via the activation of the egfr , erk and pi3 kinase / akt signaling pathways . effects of different treatments on the relative expression densitometry of egfr ( a ) , erk1/2 ( b ) , pi3k ( c ) and akt ( d ) pathways in human carcinoma cells . effects of 14,15-eet on the expression levels of pi3k - akt pathways in human carcinoma cells . tca-8113 cells were pretreated with auda ( 1 m ) for 2 hrs and then stimulated with 14,15-eet ( 100 nm ) in the presence or absence of gw9662 . after 8 hrs , the collected cell lysates were immunoblotted with anti - p - egfr antibody , and p - erk antibody . lanes from 1 to 8 were control , vehicle , gw9662 , auda , 14,15-eet , 14,15-eet+gw9662 , 14,15-eet+auda , 14,15-eet+ auda+gw9662 , respectively . ppar has been proved to be a key transcription factor of adipocyte differentiation lipid and glucose homeostasis and an important target in type 2 diabetes and metabolic syndrome . moreover , they also appear to be expressed in monocytes / macrophages , dendritic cells , eosinophils , t cells , b cells and endothelial cells , etc ( 12 ) . to confirm whether ppar and seh were expressed in human carcinoma and ecv304 cells , western blot analysis was performed . results suggested that ppar and seh proteins were expressed in two tested cell lines , and have different degrees of intensity when treated with different concentrations of 14,15-eet . the degree of intensity of ppar in tca-8113 cell line was significantly higher than that of the ecv304 cells . results also suggested that the expression of ppar reached a maximum level after 24 hrs treatment with 14,15-eet within the range of 25 - 200 nm ( fig . effects of 14,15-eet treatment on the expression of ppar and seh in tca-8113 ( a ) and huvec ( b ) cells . ppar protein levels in carcinoma and cells were measured by treatment with different concentrations ( 0 - 200 nm ) of 14,15-eet after 24 hrs . total protein was extracted and analyzed by western blot as described in materials and methods . . lanes from 1 to 5 were 0 , 25 , 50 , 100 and 200 nm , respectively . as a ligand - activated transcription factor , the role of ppar in cancer remains a subject of debate ( 4 , 6 , 13 ) . based on luciferase activity assay system , as shown in figure 2 , luciferase activities showed 4.28 , 3.15 and 4.47- fold increase when the cells were incubated and transfected by using ppre - tk - luc and addition of 14,15-eet , 14,15-eet+gw9662 and ciglitazone , respectively . in addition , gw9662 , as a ppar antagonist , may inhibit the luciferase activities compared to that which was obtained by addition of 14,15-eet . under co - transfected ppre - tk - luc and pcmx3/ppar condition , activities resulting by the addition of 14,15-eet and ciglitazone compared to control increased significantly 7.75 and 5.13-fold , respectively . the third and sixth groups were co - transfected using 0.4 g ppre - tk - luc , 0.4 g pcmx3/ppar and 0.1 g control plasmid ( phrl - tk ) . other groups were transfected by using 0.4 g ppre - tk - luc and 0.1 g control plasmid ( phrl - tk ) . the fifth and sixth groups were administrated with ciglitazone ( 20 m ) as positive control . the third and fourth groups were then incubated with 14,15-eet ( 100 nm ) in the absence or presence of gw9662 . previous studies have indicated that eet may induce cell growth and apoptosis in various cancer cells ( 5 ) . however , the effects of 14,15-eet on the cell proliferation in human carcinoma cell is still unclear . in the present study , a human carcinoma cell was used as an in vitro model to evaluate whether exogenous eet - induced proliferative effects via ppar activation . as shown in figure 3 , results suggested that the addition of 14,15-eet ( 100 nm ) in carcinoma cells cultured for 12 and 24 hrs may stimulate cell proliferation compared to the control . the addition of gw9662 and auda in the cells had no effects compared to the control , but cell proliferation was inhibited by the addition of 14,15- eet and gw9662 . recent data have shown that ppar activation by thiazolidinediones such as troglitazone induced inhibition of a wide variety of cancer cells , suggesting that activation of ppar down - regulates cell growth ( 3 , 4 ) . results of this study suggested that 14,5-eet could up - regulate the expressions of ppar in the human carcinoma cells , and also showed that it may stimulate cell proliferation ( figs 2 and 3 ) . effects of exogenous 14,15-eet and 14,15-eet ( 100 nm)/auda carcinoma cell proliferation for 12 and 24 hrs . a reduction in cell proliferation , an increase in the rate of apoptosis , or both may explain the reduction of cell viability caused by ppar agonists . in a variety of cancer cells , ppar inhibits proliferation and cell cycle arrest at g0 /g1 restriction point ( 14 ) . to study the proliferative effect of 14,15-eet , the fate of 14,15-eet - treated carcinoma cells within the cell cycle was investigated using flowcytometry analysis . as shown in figure 4 and table 1 , exogenous 14,15-eet significantly increased the percentage ( 47.08% ) of cells during s - g2-m phase in tca-8113 cells compared to the control and that of vehicle . however , the proportion of s - g2-m phase cell populations compared to the control was reduced significantly by treatment with gw9662 . these results suggest that 14 , 15-eeet may have the ability to promote the proliferation of carcinoma cells and contribute to the anti - apoptosis effects in human carcinoma cells . effects of 14,15-eet on cell cycle progression in human carcinoma cell lines by treatment with 14,15-eet . tca-8113 cells were pretreated with auda ( 1 m ) for 2 h and then incubated with 14,15-eet ( 100 nm ) in the presence or absence of gw9662 ( 10 m ) . previous studies have shown that eet may bind to putative binding protein or receptor , which may trans - activate egfr and akt in regulation of the cell motility , and have revealed that expression of by epoxygenase is very strong and selective in human cancer tissues . the levels of phosphorylated egfr , pi3k / akt , and erk1/2 in tumor cells transfected with cytochrome epoxygenases were significantly upregulated . studies have also shown the status of signal molecules , such as egfr , pi3k , akt , and erk1/2 , in tumor cells after transfection with cyp genes . these results suggest that pi3k / akt and erk1/2 pathways are involved in the promotion of tumor cells by cyp epoxygenases overexpression ( 15 , 16 ) . results of this study suggest that the total amount of egfr , erk , and pi3 kinase / akt proteins were quite refractory to 14 , 15-eet and other additives , and their expression levels were correlated with these stimulators in human carcinoma cell ( figs 5 and 6 ) . the phosphorylated erk and pi3 kinase / akt pathways are induced significantly by auda and 14,15-eet treatments . however , the levels of pi3 kinase - akt pathway was reduced significantly by treatment with gw9662 compared to the control , suggesting that gw9662 , an antagonist of ppar , may block pi3 kinases - akt pathways slightly . the phosphorylated egfr was also elevated significantly by treatment with 14,15-eet , but no significant changes were observed by treatment with gw9662 . the changes in signal transduction pathways and transcriptional mechanisms induced by eet have been investigated , and recent studies are attempting to isolate an eet membrane receptor that mediates cell proliferation and anti - apoptosis in some tumor cell lines ( 6 , 17 ) . findings of this study suggest that exogenous eets significantly promote carcinoma cancer cell proliferation and inhibit apoptosis significantly via the activation of the egfr , erk and pi3 kinase / akt signaling pathways . effects of different treatments on the relative expression densitometry of egfr ( a ) , erk1/2 ( b ) , pi3k ( c ) and akt ( d ) pathways in human carcinoma cells . effects of 14,15-eet on the expression levels of pi3k - akt pathways in human carcinoma cells . tca-8113 cells were pretreated with auda ( 1 m ) for 2 hrs and then stimulated with 14,15-eet ( 100 nm ) in the presence or absence of gw9662 . after 8 hrs , the collected cell lysates were immunoblotted with anti - p - egfr antibody , and p - erk antibody . lanes from 1 to 8 were control , vehicle , gw9662 , auda , 14,15-eet , 14,15-eet+gw9662 , 14,15-eet+auda , 14,15-eet+ auda+gw9662 , respectively . results of this study suggest that 14,15-eet may promote the proliferation of tumor cells through activation of ppar , up - regulation of pi3 kinase / akt system in human carcinoma cells . these findings provide a novel clue regarding the role of 14,15- eet as a potential cancer therapeutic in tumor cells . importantly , the detailed mechanisms of 14,15-eet on carcinoma cells will be of high importance for future study .

background and the purpose of the study epoxyeicosatrienoic acids ( eets ) , which are cytochrome p450 epoxygenase metabolites of arachidonic acid , have anti - inflammatory effects , modulate smooth muscle proliferation , and inhibit smooth muscle migration . this study was designed to determine whether exogenous eets have any effect on the cell proliferation and apoptosis of carcinoma cell as well as the possible signaling pathways of eets in this regulation.methodsthe effects of eets on the proliferation and anti - apoptosis of human carcinoma cells were measured by mtt assay and flowcytometric analysis , and the regulation of ppar , epithelial growth factor receptor ( egfr ) , extracellular signal - regulated kinase ( erk ) , phosphatidylinositol 3 ( pi3)-kinase / akt pathways was investigated by reverse transcriptase polymerase chain reaction ( rt - pcr ) and western blot analysis.resultsresults of this study suggested that 14 , 15-eet may activate the expression of ppar in tca-8113 cells . 14,15-eet may stimulate cell proliferation , and increase the percentage of cells during s - g2-m phase in tca-8113 cells significantly . the levels of egfr , erk , and pi3 kinase / akt proteins were significantly induced by treatment of 14 , 15-eet and 14,15-eet / auda , but no significant changes were observed by addition of gw9662.conclusionthese findings suggest that exogenous 14,15-eet has potent inhibitory effect on proliferation , and could induce apoptosis in tca-8113 cell , and these changes are related to the expression of ppar , the activation of egfr , erk , and pi3 kinase / akt proteins .

INTRODUCTION MATERIAL AND METHODS Materials Assays of cell proliferation Assay by flowcytometry Assays of transfection and luciferase Quantification of mRNA levels by real-time PCR Assay of western blot Statistical analyses RESULTS AND DISCUSSION None Effects of 14,15-EET on expression of PPAR and sEH in human tumor and ECV304 cells Activation of PPAR by 14,15-EET in tumor cells Effects of 14,15-EET on carcinoma cell proliferation and cycle progression 14,15-EET stimulates cell proliferation via regulation of EGFR, PI3 kinase and AKT CONCLUSION

evidences from cell culture models show that blocking ppar activity reduces eets/ soluble epoxide hydrolase ( seh ) and inhibitor - mediated anti - inflammatory effect , indicating ppar is an effector of eets ( 2 ) . in lung cancers , the expression of ppar is found to be significantly lower in tumour than in adjacent non - tumour tissues , and there is a correlation between a lower survival rate and decreased ppar expression ( 3 , 4 ) . epoxyeicosatrienoic acids ( eets ) , are synthesized from arachidonic acid via cytochrome p450 enzymemediated pathway to four regioisomers , 5,6-eet , 8,9-eet , 11,12-eet , and 14,15-eet ( 5 ) . eets are also reported to have diverse physiological and pathophysiological functions , such as vasodilation by activating membrane maxi - ca - activated k ( bkca ) channels , anti - inflammatory effects , ion channel activation , promotion of the proliferation of vascular cells and angiogenesis , modulation of the proliferation , and migration of vessel smooth muscle cells ( 6 , 7 ) . recently , it has been found that eets in particular 11,12- and 14,15-eet , activate several intracellular signaling molecules including tyrosine kinases and phosphatases , p38 map kinase , extracellular regulated protein kinases 1/2 ( erk1/2 ) and map kinase phosphatases ( 8 , 9 ) . these findings led us to further investigate the effects and mechanisms of eets on proliferation , apoptosis and metastasis processes in tumour cells . in the present study , it was intended to verify the proliferative and anti - apoptotic effects of exogenous 14,15-eet , gw9662 and auda on carcinoma cancer cells . furthermore , the expression of ppar- , as well as regulation of egfr , erk1/2 , pi3k and akt proteins in carcinoma cancer cells was investigated . antibodies against epidermal growth factor receptor ( egfr ) , phosphorous egfr ( p - egfr ) , phosphatidylinositol 3-kinase ( pi3k ) , akt , seh and p - akt were purchased from cell signaling technology ( beverly , ma , usa ) . 14 , 15-epoxyeicosatrienoic acid ( 14,15- eet ) and adamantyl - ureido - dodecanoic acid ( auda ) were obtained from cayman chemical co. ( ann arbor , usa ) , and ciglitazone was from calbiochem ( san diego , ca , usa ) . after 4 hrs , 500 l of full medium was added into each well and then after 24 hrs , the cells were incubated with 100 nm 14,15-eet , 10m gw9662 and/or 20 m ciglitazone for 12 hrs , and then luciferase activities were measured . the obtained cdnas were then used as the templates for quantitative rt - pcr with the use of the brilliant sybr green qpcr master mix ( stratagene , la jolla , ca , usa ) . after treatments of 14,15-eet , 14,15-eet / auda and/or gw9662 , the cells were lysed using lysate buffer , and cell lysate supernatants were harvested by centrifugation at l0000 rpm for 10 min at 4c . antibodies against epidermal growth factor receptor ( egfr ) , phosphorous egfr ( p - egfr ) , phosphatidylinositol 3-kinase ( pi3k ) , akt , seh and p - akt were purchased from cell signaling technology ( beverly , ma , usa ) . 14 , 15-epoxyeicosatrienoic acid ( 14,15- eet ) and adamantyl - ureido - dodecanoic acid ( auda ) were obtained from cayman chemical co. ( ann arbor , usa ) , and ciglitazone was from calbiochem ( san diego , ca , usa ) . the obtained cdnas were then used as the templates for quantitative rt - pcr with the use of the brilliant sybr green qpcr master mix ( stratagene , la jolla , ca , usa ) . after treatments of 14,15-eet , 14,15-eet / auda and/or gw9662 , the cells were lysed using lysate buffer , and cell lysate supernatants were harvested by centrifugation at l0000 rpm for 10 min at 4c . after 4 hrs , 500 l of full medium was added into each well and then after 24 hrs , the cells were incubated with 100 nm 14,15-eet , 10m gw9662 and/or 20 m ciglitazone for 12 hrs , and then luciferase activities were measured . the obtained cdnas were then used as the templates for quantitative rt - pcr with the use of the brilliant sybr green qpcr master mix ( stratagene , la jolla , ca , usa ) . after treatments of 14,15-eet , 14,15-eet / auda and/or gw9662 , the cells were lysed using lysate buffer , and cell lysate supernatants were harvested by centrifugation at l0000 rpm for 10 min at 4c . the degree of intensity of ppar in tca-8113 cell line was significantly higher than that of the ecv304 cells . results also suggested that the expression of ppar reached a maximum level after 24 hrs treatment with 14,15-eet within the range of 25 - 200 nm ( fig . effects of 14,15-eet treatment on the expression of ppar and seh in tca-8113 ( a ) and huvec ( b ) cells . ppar protein levels in carcinoma and cells were measured by treatment with different concentrations ( 0 - 200 nm ) of 14,15-eet after 24 hrs . based on luciferase activity assay system , the activation of ppar in carcinoma cells by 100 nm of 14,15-eet was investigated . however , the effects of 14,15-eet on the cell proliferation in human carcinoma cell is still unclear . as shown in figure 3 , results suggested that the addition of 14,15-eet ( 100 nm ) in carcinoma cells cultured for 12 and 24 hrs may stimulate cell proliferation compared to the control . the addition of gw9662 and auda in the cells had no effects compared to the control , but cell proliferation was inhibited by the addition of 14,15- eet and gw9662 . results of this study suggested that 14,5-eet could up - regulate the expressions of ppar in the human carcinoma cells , and also showed that it may stimulate cell proliferation ( figs 2 and 3 ) . effects of exogenous 14,15-eet and 14,15-eet ( 100 nm)/auda carcinoma cell proliferation for 12 and 24 hrs . to study the proliferative effect of 14,15-eet , the fate of 14,15-eet - treated carcinoma cells within the cell cycle was investigated using flowcytometry analysis . as shown in figure 4 and table 1 , exogenous 14,15-eet significantly increased the percentage ( 47.08% ) of cells during s - g2-m phase in tca-8113 cells compared to the control and that of vehicle . however , the proportion of s - g2-m phase cell populations compared to the control was reduced significantly by treatment with gw9662 . these results suggest that 14 , 15-eeet may have the ability to promote the proliferation of carcinoma cells and contribute to the anti - apoptosis effects in human carcinoma cells . effects of 14,15-eet on cell cycle progression in human carcinoma cell lines by treatment with 14,15-eet . previous studies have shown that eet may bind to putative binding protein or receptor , which may trans - activate egfr and akt in regulation of the cell motility , and have revealed that expression of by epoxygenase is very strong and selective in human cancer tissues . the levels of phosphorylated egfr , pi3k / akt , and erk1/2 in tumor cells transfected with cytochrome epoxygenases were significantly upregulated . results of this study suggest that the total amount of egfr , erk , and pi3 kinase / akt proteins were quite refractory to 14 , 15-eet and other additives , and their expression levels were correlated with these stimulators in human carcinoma cell ( figs 5 and 6 ) . the phosphorylated erk and pi3 kinase / akt pathways are induced significantly by auda and 14,15-eet treatments . however , the levels of pi3 kinase - akt pathway was reduced significantly by treatment with gw9662 compared to the control , suggesting that gw9662 , an antagonist of ppar , may block pi3 kinases - akt pathways slightly . the phosphorylated egfr was also elevated significantly by treatment with 14,15-eet , but no significant changes were observed by treatment with gw9662 . the changes in signal transduction pathways and transcriptional mechanisms induced by eet have been investigated , and recent studies are attempting to isolate an eet membrane receptor that mediates cell proliferation and anti - apoptosis in some tumor cell lines ( 6 , 17 ) . findings of this study suggest that exogenous eets significantly promote carcinoma cancer cell proliferation and inhibit apoptosis significantly via the activation of the egfr , erk and pi3 kinase / akt signaling pathways . effects of different treatments on the relative expression densitometry of egfr ( a ) , erk1/2 ( b ) , pi3k ( c ) and akt ( d ) pathways in human carcinoma cells . effects of 14,15-eet on the expression levels of pi3k - akt pathways in human carcinoma cells . the degree of intensity of ppar in tca-8113 cell line was significantly higher than that of the ecv304 cells . effects of 14,15-eet treatment on the expression of ppar and seh in tca-8113 ( a ) and huvec ( b ) cells . ppar protein levels in carcinoma and cells were measured by treatment with different concentrations ( 0 - 200 nm ) of 14,15-eet after 24 hrs . based on luciferase activity assay system , the activation of ppar in carcinoma cells by 100 nm of 14,15-eet was investigated . however , the effects of 14,15-eet on the cell proliferation in human carcinoma cell is still unclear . as shown in figure 3 , results suggested that the addition of 14,15-eet ( 100 nm ) in carcinoma cells cultured for 12 and 24 hrs may stimulate cell proliferation compared to the control . the addition of gw9662 and auda in the cells had no effects compared to the control , but cell proliferation was inhibited by the addition of 14,15- eet and gw9662 . results of this study suggested that 14,5-eet could up - regulate the expressions of ppar in the human carcinoma cells , and also showed that it may stimulate cell proliferation ( figs 2 and 3 ) . effects of exogenous 14,15-eet and 14,15-eet ( 100 nm)/auda carcinoma cell proliferation for 12 and 24 hrs . to study the proliferative effect of 14,15-eet , the fate of 14,15-eet - treated carcinoma cells within the cell cycle was investigated using flowcytometry analysis . as shown in figure 4 and table 1 , exogenous 14,15-eet significantly increased the percentage ( 47.08% ) of cells during s - g2-m phase in tca-8113 cells compared to the control and that of vehicle . however , the proportion of s - g2-m phase cell populations compared to the control was reduced significantly by treatment with gw9662 . these results suggest that 14 , 15-eeet may have the ability to promote the proliferation of carcinoma cells and contribute to the anti - apoptosis effects in human carcinoma cells . previous studies have shown that eet may bind to putative binding protein or receptor , which may trans - activate egfr and akt in regulation of the cell motility , and have revealed that expression of by epoxygenase is very strong and selective in human cancer tissues . the levels of phosphorylated egfr , pi3k / akt , and erk1/2 in tumor cells transfected with cytochrome epoxygenases were significantly upregulated . results of this study suggest that the total amount of egfr , erk , and pi3 kinase / akt proteins were quite refractory to 14 , 15-eet and other additives , and their expression levels were correlated with these stimulators in human carcinoma cell ( figs 5 and 6 ) . the phosphorylated erk and pi3 kinase / akt pathways are induced significantly by auda and 14,15-eet treatments . however , the levels of pi3 kinase - akt pathway was reduced significantly by treatment with gw9662 compared to the control , suggesting that gw9662 , an antagonist of ppar , may block pi3 kinases - akt pathways slightly . the phosphorylated egfr was also elevated significantly by treatment with 14,15-eet , but no significant changes were observed by treatment with gw9662 . the changes in signal transduction pathways and transcriptional mechanisms induced by eet have been investigated , and recent studies are attempting to isolate an eet membrane receptor that mediates cell proliferation and anti - apoptosis in some tumor cell lines ( 6 , 17 ) . findings of this study suggest that exogenous eets significantly promote carcinoma cancer cell proliferation and inhibit apoptosis significantly via the activation of the egfr , erk and pi3 kinase / akt signaling pathways . effects of different treatments on the relative expression densitometry of egfr ( a ) , erk1/2 ( b ) , pi3k ( c ) and akt ( d ) pathways in human carcinoma cells . effects of 14,15-eet on the expression levels of pi3k - akt pathways in human carcinoma cells . the degree of intensity of ppar in tca-8113 cell line was significantly higher than that of the ecv304 cells . results also suggested that the expression of ppar reached a maximum level after 24 hrs treatment with 14,15-eet within the range of 25 - 200 nm ( fig . effects of 14,15-eet treatment on the expression of ppar and seh in tca-8113 ( a ) and huvec ( b ) cells . ppar protein levels in carcinoma and cells were measured by treatment with different concentrations ( 0 - 200 nm ) of 14,15-eet after 24 hrs . however , the effects of 14,15-eet on the cell proliferation in human carcinoma cell is still unclear . as shown in figure 3 , results suggested that the addition of 14,15-eet ( 100 nm ) in carcinoma cells cultured for 12 and 24 hrs may stimulate cell proliferation compared to the control . the addition of gw9662 and auda in the cells had no effects compared to the control , but cell proliferation was inhibited by the addition of 14,15- eet and gw9662 . results of this study suggested that 14,5-eet could up - regulate the expressions of ppar in the human carcinoma cells , and also showed that it may stimulate cell proliferation ( figs 2 and 3 ) . effects of exogenous 14,15-eet and 14,15-eet ( 100 nm)/auda carcinoma cell proliferation for 12 and 24 hrs . to study the proliferative effect of 14,15-eet , the fate of 14,15-eet - treated carcinoma cells within the cell cycle was investigated using flowcytometry analysis . as shown in figure 4 and table 1 , exogenous 14,15-eet significantly increased the percentage ( 47.08% ) of cells during s - g2-m phase in tca-8113 cells compared to the control and that of vehicle . however , the proportion of s - g2-m phase cell populations compared to the control was reduced significantly by treatment with gw9662 . these results suggest that 14 , 15-eeet may have the ability to promote the proliferation of carcinoma cells and contribute to the anti - apoptosis effects in human carcinoma cells . effects of 14,15-eet on cell cycle progression in human carcinoma cell lines by treatment with 14,15-eet . previous studies have shown that eet may bind to putative binding protein or receptor , which may trans - activate egfr and akt in regulation of the cell motility , and have revealed that expression of by epoxygenase is very strong and selective in human cancer tissues . the levels of phosphorylated egfr , pi3k / akt , and erk1/2 in tumor cells transfected with cytochrome epoxygenases were significantly upregulated . results of this study suggest that the total amount of egfr , erk , and pi3 kinase / akt proteins were quite refractory to 14 , 15-eet and other additives , and their expression levels were correlated with these stimulators in human carcinoma cell ( figs 5 and 6 ) . the phosphorylated erk and pi3 kinase / akt pathways are induced significantly by auda and 14,15-eet treatments . however , the levels of pi3 kinase - akt pathway was reduced significantly by treatment with gw9662 compared to the control , suggesting that gw9662 , an antagonist of ppar , may block pi3 kinases - akt pathways slightly . the phosphorylated egfr was also elevated significantly by treatment with 14,15-eet , but no significant changes were observed by treatment with gw9662 . the changes in signal transduction pathways and transcriptional mechanisms induced by eet have been investigated , and recent studies are attempting to isolate an eet membrane receptor that mediates cell proliferation and anti - apoptosis in some tumor cell lines ( 6 , 17 ) . findings of this study suggest that exogenous eets significantly promote carcinoma cancer cell proliferation and inhibit apoptosis significantly via the activation of the egfr , erk and pi3 kinase / akt signaling pathways . effects of different treatments on the relative expression densitometry of egfr ( a ) , erk1/2 ( b ) , pi3k ( c ) and akt ( d ) pathways in human carcinoma cells . effects of 14,15-eet on the expression levels of pi3k - akt pathways in human carcinoma cells . results of this study suggest that 14,15-eet may promote the proliferation of tumor cells through activation of ppar , up - regulation of pi3 kinase / akt system in human carcinoma cells .

a total of 220 patients with takayasu arteritis underwent ct examinations in our hospital during the 12 years , from january 1994 to december 2005 . to satisfy the inclusion criteria , the patients had to have active takayasu arteritis and to have had the follow - up ct examination done . the criteria for the active disease at the time of the initial ct included systemic features , an elevated erythrocyte sedimentation rate ( esr , normal range in females : 0 20 mm / hr , in males : 0 9 mm / hr ) and abnormal mural changes according to the initial ct evaluation . eighteen patients were included in this retrospective study to evaluate the interval changes of the ct findings in takayasu arteritis . their ages ranged from 11 to 53 years , with the mean age of 31.2 years at the time of the initial ct examination . from two to four ct examinations the clinical findings of the 18 patients are summarized in table 1 , including the number of the ct examinations , the modified ueda type of extent of disease , the levels of esr and c - reactive protein , along with the medical and surgical or interventional treatments . the esr was elevated in all except two patients ( cases 1 and 8) , who showed an elevated esr on a repeated examination performed within one month . the course of disease was modified ueda type i in four patients , type ii in two patients , type iii in nine patients and type iv in three patients . all the patients , except one patient with active pulmonary tuberculosis , were treated with prednisolone alone , or with methotrexate or cyclophosphamide . by the follow - up evaluation , the disease was defined as inactive if the esr was normal at least for six months without steroid medication . otherwise , the disease was defined as active . according to the above criteria , the disease was active in all 18 patients at the time of the initial ct evaluation . the disease was considered as inactive in five patients and active in 13 patients at the time of the last follow - up ct . because of the long period for collecting the cases , more than 10 years , various ct scanners were used for the patients scanning ; these scanners varied from a single slice spiral ct to a 16-channel multidetector ct ( mdct ) scanner including single slice spiral ct ( somatom plus - s , somatom plus-4 , siemens , erlangen , germany ) , the 4-channel mdct ( mx8000 , marconi medical systems , cleveland , oh ) , the 8-channel mdct ( lightspeed ultra , ge medical systems , milwaukee , wi ) and the 16-channel mdct ( sensation 16 , siemens , erlangen , germany ) . an 18- , or 20-gauge needle was placed into the antecubital vein , and 150 ml of nonionic contrast material , iopromide ( ultravist 370 ; schering , berlin , germany ) was injected at a rate of 2.5 3.0 ml / sec with using a power injector ( envision ct ; medrad , indianola , pa ) . we used the bolus - tracking method at the ascending aorta level and set 100 hu as the triggering threshold for the arterial phase . the venous phase scan was taken at 5 to 10 minutes after initiating the injection of the contrast material . the scanning parameters varied according to the type of the scanner , but the following basic concept for scanning of the aorta was followed : the slice thickness was kept as thin as possible for the scanning phase of the precontrast phase , the arterial phase and the venous phase . for the arterial phase , the slice thickness was 3 mm on the single spiral ct and the 4-channel mdct with 2 mm reconstruction intervals , and it was 1.5 mm on the 8-channel and the 16-channel mdct with 1 mm reconstruction intervals . for the precontrast and the delayed venous phase scans , the pitch value was 2 for the single spiral ct and 1 to 1.5 for mdct . mdct could cover the whole aorta during the time of a single breath - hold , and each scan was done with a single breath - hold . in case of the single slice spiral ct , the whole aorta for the arterial phase was covered with two breath - hold scans , one for the thoracic aorta and the other for the abdominal aorta . the high density of the aortic wall and the mural calcification on the precontrast ct scan and the low - attenuated ring on the arterial and the venous phase ct scan were examined by two collaborating radiologists . the mural findings of the follow - up ct were compared with those of the initial ct . in cases of multiple follow - up evaluations , the two radiologists measured the mural thickness on the transverse image of the arterial phase and the ct attenuation of the aortic wall in the precontrast , arterial and venous phases . the mean ct attenuation value in hounsfield units was calculated based on three or more measurements with a region of interest of 2 - 7 mm . the measurement was performed at the location with a maximum mural thickness , including the ascending aorta , the arch , the left common carotid and abdominal aorta . the arterial and delayed enhancements of the aortic wall were compared with the attenuation of the back muscle of the same image , and this was expressed as a ratio . the ratio of the attenuation of the enhanced aortic wall over the attenuation of the back muscle in the initial ct was compared with that of the follow - up ct . the luminal findings , in addition to the mural changes , were also considered on the initial and follow - up ct examinations . for the statistical tests between the two groups , mann - whitney u test was used for continuous variables , and the fisher exact test was used for categorical variables . the wilcoxon signed rank test was applied for statistically testing the follow - up evaluation . a two - tailed p value of 0.05 or less was considered to indicate statistical significance , and spss 10.0 statistical computer software ( spss , chicago , il ) was used . because of the long period for collecting the cases , more than 10 years , various ct scanners were used for the patients scanning ; these scanners varied from a single slice spiral ct to a 16-channel multidetector ct ( mdct ) scanner including single slice spiral ct ( somatom plus - s , somatom plus-4 , siemens , erlangen , germany ) , the 4-channel mdct ( mx8000 , marconi medical systems , cleveland , oh ) , the 8-channel mdct ( lightspeed ultra , ge medical systems , milwaukee , wi ) and the 16-channel mdct ( sensation 16 , siemens , erlangen , germany ) . an 18- , or 20-gauge needle was placed into the antecubital vein , and 150 ml of nonionic contrast material , iopromide ( ultravist 370 ; schering , berlin , germany ) was injected at a rate of 2.5 3.0 ml / sec with using a power injector ( envision ct ; medrad , indianola , pa ) . we used the bolus - tracking method at the ascending aorta level and set 100 hu as the triggering threshold for the arterial phase . the venous phase scan was taken at 5 to 10 minutes after initiating the injection of the contrast material . the scanning parameters varied according to the type of the scanner , but the following basic concept for scanning of the aorta was followed : the slice thickness was kept as thin as possible for the scanning phase of the precontrast phase , the arterial phase and the venous phase . for the arterial phase , the slice thickness was 3 mm on the single spiral ct and the 4-channel mdct with 2 mm reconstruction intervals , and it was 1.5 mm on the 8-channel and the 16-channel mdct with 1 mm reconstruction intervals . for the precontrast and the delayed venous phase scans , the pitch value was 2 for the single spiral ct and 1 to 1.5 for mdct . mdct could cover the whole aorta during the time of a single breath - hold , and each scan was done with a single breath - hold . in case of the single slice spiral ct , the whole aorta for the arterial phase was covered with two breath - hold scans , one for the thoracic aorta and the other for the abdominal aorta . the high density of the aortic wall and the mural calcification on the precontrast ct scan and the low - attenuated ring on the arterial and the venous phase ct scan were examined by two collaborating radiologists . the mural findings of the follow - up ct were compared with those of the initial ct . in cases of multiple follow - up evaluations , the two radiologists measured the mural thickness on the transverse image of the arterial phase and the ct attenuation of the aortic wall in the precontrast , arterial and venous phases . the mean ct attenuation value in hounsfield units was calculated based on three or more measurements with a region of interest of 2 - 7 mm . the measurement was performed at the location with a maximum mural thickness , including the ascending aorta , the arch , the left common carotid and abdominal aorta . the arterial and delayed enhancements of the aortic wall were compared with the attenuation of the back muscle of the same image , and this was expressed as a ratio . the ratio of the attenuation of the enhanced aortic wall over the attenuation of the back muscle in the initial ct was compared with that of the follow - up ct . the luminal findings , in addition to the mural changes , were also considered on the initial and follow - up ct examinations . for the statistical tests between the two groups , mann - whitney u test was used for continuous variables , and the fisher exact test was used for categorical variables . the wilcoxon signed rank test was applied for statistically testing the follow - up evaluation . a two - tailed p value of 0.05 or less was considered to indicate statistical significance , and spss 10.0 statistical computer software ( spss , chicago , il ) was used . the mural changes found on the initial and follow - up ct evaluations of the 18 patients are summarized in table 2 . on the precontrast ct , the high density of the aortic wall was noted in 15 patients ( 83% ) . curvilinear or dot - like calcifications were found in six patients ( 33% ) . on the arterial phase of ct , the wall was slightly enhanced in various portions of the aorta and its branches , and the mean ratio of attenuation of the wall over that of the back muscle was 1.4 . a circumferential low - attenuation ring was demonstrated for the patients with substantial arterial enhancement . in the venous phase , the mural enhancement was more pronounced in all the patients with an attenuation ratio from 1.2 to 2.6 ( the mean value : 1.9 ) . the low - attenuation ring was well demonstrated between the contrast - filled lumen and the enhanced outer wall in 15 patients ( 83% ) in the venous phase ( fig . no significant stenosis was shown , even for the four patients with significant mural changes . variable degrees of the luminal changes , including stenosis and dilatation , were noted in the aorta and its branches in 14 patients . on the follow - up ct evaluation , with a mean follow - up period of 55.6 months , the precontrast image showed the high density ring in two more patients ( table 2 ) . the mean attenuation ( hu ) of the aortic wall on the precontrast phase increased significantly from 48.9 to 56.5 ( p = 0.04 ) ( table 3 ) . the mural calcifications noted in 10 patients were more extensive . the mean mural thickness measured on the arterial phase was 2.4 mm , and it was significantly decreased from 4.1 mm as measured by the initial ct evaluation ( p = 0.001 ) . the mean ct attenuation ratio of the wall ( aortic wall / muscle ) in the venous phase decreased significantly from 1.9 to 1.3 ( p = 0.001 ) . the low - attenuation ring in the venous phase was identified in seven patients ( 39% ) with active persistent disease . the mural thickness decreased , and the mural enhancement also decreased significantly in both the group of patients with inactive disease and in the group of patients with active persistent disease ( figs . 2 , 3 ) . in the group of the inactive disease patients ( n = 5 ) , the low - attenuation ring , initially shown in four patients ( 80% ) , disappeared in all the patients by the last follow - up ct ( fig . 2 ) . however , each of the individual ct findings had no statistically significant correlation with the esr - based disease activity on comparison between the both groups . luminal stenosis was improved along with the mural changes by the follow - up ct after medical treatment in two patients ( cases 7 and 16 ) . in one patient ( case 1 ) , the lumen of the abdominal aorta and the proximal left renal artery had recovered , while focal stenosis developed at the middle of the left renal artery . the mural changes found on the initial and follow - up ct evaluations of the 18 patients are summarized in table 2 . on the precontrast ct , the high density of the aortic wall was noted in 15 patients ( 83% ) . curvilinear or dot - like calcifications were found in six patients ( 33% ) . on the arterial phase of ct , the wall was slightly enhanced in various portions of the aorta and its branches , and the mean ratio of attenuation of the wall over that of the back muscle was 1.4 . a circumferential low - attenuation ring was demonstrated for the patients with substantial arterial enhancement . in the venous phase , the mural enhancement was more pronounced in all the patients with an attenuation ratio from 1.2 to 2.6 ( the mean value : 1.9 ) . the low - attenuation ring was well demonstrated between the contrast - filled lumen and the enhanced outer wall in 15 patients ( 83% ) in the venous phase ( fig . no significant stenosis was shown , even for the four patients with significant mural changes . variable degrees of the luminal changes , including stenosis and dilatation , were noted in the aorta and its branches in 14 patients . on the follow - up ct evaluation , with a mean follow - up period of 55.6 months , the precontrast image showed the high density ring in two more patients ( table 2 ) . the mean attenuation ( hu ) of the aortic wall on the precontrast phase increased significantly from 48.9 to 56.5 ( p = 0.04 ) ( table 3 ) . the mural calcifications noted in 10 patients were more extensive . the mean mural thickness measured on the arterial phase was 2.4 mm , and it was significantly decreased from 4.1 mm as measured by the initial ct evaluation ( p = 0.001 ) . the mean ct attenuation ratio of the wall ( aortic wall / muscle ) in the venous phase decreased significantly from 1.9 to 1.3 ( p = 0.001 ) . the low - attenuation ring in the venous phase was identified in seven patients ( 39% ) with active persistent disease . the mural thickness decreased , and the mural enhancement also decreased significantly in both the group of patients with inactive disease and in the group of patients with active persistent disease ( figs . 2 , 3 ) . in the group of the inactive disease patients ( n = 5 ) , the low - attenuation ring , initially shown in four patients ( 80% ) , disappeared in all the patients by the last follow - up ct ( fig . 2 ) . however , each of the individual ct findings had no statistically significant correlation with the esr - based disease activity on comparison between the both groups . luminal stenosis was improved along with the mural changes by the follow - up ct after medical treatment in two patients ( cases 7 and 16 ) . in one patient ( case 1 ) , the lumen of the abdominal aorta and the proximal left renal artery had recovered , while focal stenosis developed at the middle of the left renal artery . various imaging modalities may reveal the mural changes in patients with takayasu arteritis during the early phase of the disease with or without such luminal changes , as dilatation and stenosis ( 6 , 8 - 10 ) . ct may reveal mural thickening with the arterial and venous enhancement ( 5 , 6 , 8 , 9 ) . sonography shows a homogenous concentric mural thickening of the aorta and its branches , such as the carotid or subclavian arteries ( 9 , 11 ) . the mr imaging reveals the mural thickening of the aorta , the pulmonary artery and its branches , and especially on the gadolinium enhanced t1 weighted images . the mural thickening and the enhancement , as evaluated by each modality , may decrease after appropriate treatment ( 9 - 13 ) . in this retrospective study , we evaluated the mural changes on the follow - up ct examination for the patients with active disease at the time of the initial ct examination . there have been a few reports on the detailed analysis of the mural changes on the follow - up ct evaluation in a group of patients . paul et al . reported on their results of electron beam ct follow - up , in which they noted a decrease of the mural thickening and an increase of the mural calcifications . the outer wall enhancement , initially noted in 81% cases , was found in 37% case on the last ct scans ( 8) . for our results , the mural thickness decreased significantly from 4.1 mm to 2.4 mm . other mural changes seen on the follow - up ct evaluation included increased attenuation of the aortic wall and more extensive mural calcifications on the precontrast ct . the mural enhancement in the venous phase was decreased significantly on the follow - up ct evaluation . it is very difficult to estimate the activity of this disease with using a single parameter of the clinical or imaging diagnosis . there are 4 criteria for the disease activity : systemic features , an elevated esr , features of vascular ischemia and the typical angiographic features ( 7 ) . systemic features such as neck or shoulder pain may fail to represent the real activity of the disease . the angiography may be normal even with an extensive wall thickening if the mural changes did not cause luminal changes , such as narrowing , irregularities or aneurysm ( 6 , 9 ) . so , any cross - sectional imaging modality , including sonography , ct and mr , may be added to make the criteria of activity complete in respect of the angiographic feature . the disease should be regarded as active if there is any new focus or an aggravation of the mural changes according to a cross - sectional imaging modality . on the contrary , the mural changes may resolve completely with appropriate treatment , resulting in an inactivated form of disease . reported that the contrast - enhanced mr imaging showed more enhancement of the thickened aortic wall as compared with the myocardium , suggesting active disease . for determining the progress of the disease activity , they reported an agreement rate for contrast - enhanced mr imaging as 88.5% with the clinical findings : 92.3% with the esr and 84.6% with the c - reactive protein ( 13 ) . in this study , the disease was defined as inactive by the follow - up evaluation if the esr was normal at least for six months even without steroid medication . after appropriate treatment , the mural thickness decreased significantly in all five cases with inactive disease , as was established by the follow - up ct evaluation . for the microscopic findings of takayasu arteritis , the inflammatory reactions affect mainly the adventitia and the media . the media shows vascularization , and the capillaries originating at the transition zone of the adventitia proliferate to the entire medial layer . following this neovascularization , inflammatory tissue invades the media with infiltration of lymphocytes , plasma cells and giant cells ( 14 ) . the active inflammation of the outer wall contributes mainly to the thickening of the aortic wall in the early phase of takayasu arteritis . the mural enhancement in the arterial or venous phase is most likely associated with the neovascularization and the productive inflammation of the media and the adventitia . the intima is thickened with an increase of the basophilic ground substance during the acute phase ( 14 ) . it can be seen as a radiolucent ring with a good contrast between the enhanced outer wall and the contrast filled arterial lumen . an improvement of the inflammatory process of the aortic wall may be manifested as a decrease of the thickness , a decrease of the mural enhancement and disappearance of the inner low - attenuation ring on the follow - up ct evaluation . ct may reflect the mural changes on the follow - up evaluation of active takayasu arteritis . however , there was no statistical significance in the correlation between the ct findings and activity of the disease on the basis of the esr in this study . though the esr is a sensitive parameter of active inflammation , it is nonspecific and has certain limitations in representing the activity of this disease . the esr has been reported to be persistently elevated during clinical remission in 56% of the cases ( 7 ) . the ct protocol in this study was variable because different ct scanners were used for the patients who were selected retrospectively over a long period . on the ct angiography for patients with takayasu arteritis , the carotid bifurcation should be included into the examination because involvement of the common carotid artery is common in takayasu arteritis . the thinner the slice thickness is , the easier it is to detect stenosis of the aortic branches such as the renal artery . in addition to the arterial phase data , the venous phase imaging also reveals important mural changes , including the delayed enhancement and the inner low - attenuation ring . however , the thin section acquisition and the additional venous phase imaging may be associated with exposure of patients to a considerable dose of radiation . decreasing the tube current and increasing the slice thickness up to 5 mm in the venous phase it was a retrospective study of ct follow - up in patients with takayasu arteritis with the different intervals between the initial and the ct follow - up examinations being from 10 months to 10 years . there could be bias in selection of the patients with active takayasu arteritis since the clinical criteria of activity were not perfect . the number of cases was not adequate to provide a sufficient statistical analysis with many ct findings . the ct imaging protocols and the imaging systems were not identical , which was due to the evolution of the ct technology during the study period . the regimen and the dosage of the medical treatment were different because the patients included into the study were treated by different clinicians . in summary , the mural changes on the follow - up ct evaluation of patients with active takayasu arteritis include a decrease of the mural thickness , a decrease of the mural enhancement and disappearance of the low - attenuation ring in the venous phase , and an increase of the mural attenuation and calcifications in the precontrast phase . further investigations are necessary to establish the correlation between these mural changes noted over a period of time with the luminal changes and the long - term prognosis of patients with active takayasu arteritis .

objectivewe wanted to evaluate the mural changes by ct on the follow - up examination of patients with active takayasu arteritis.materials and methodsthe study included 18 patients , ( 4 males and 14 females ) , with active takayasu arteritis . a total of 44 ct examinations were done during the follow - up period ( mean : 55.6 months ) . at the time of the last follow - up ct , the disease , on the basis of the erythrocyte sedimentation rate ( esr ) , was found to be inactive in five patients and the disease was active and persistent in 13 patients . the thickness and ct attenuation of the aortic wall on the precontrast , arterial and venous phases were measured on the initial and the follow - up ct examinations . the ratio of the mural attenuation over that of the back muscle on the initial ct was compared with the ratio found on the follow - up ct.resultsthe initial ct findings included high density and calcifications of the aortic wall in the precontrast images and a thickened wall with enhancements in the arterial and the venous phases . a low - attenuation ring was demonstrated in the venous phase in 15 patients ( 83% ) . on the follow - up evaluation , the mean mural thickness decreased significantly from 4.1 mm to 2.4 mm . the mean mural attenuation ratio in the venous phase decreased significantly from 1.9 to 1.3 ( p = 0.001 ) . the low attenuation ring was identified in seven patients ( 39% ) who had only with active , persistent takayasu arteritis.conclusionthe mural changes demonstrated by the follow - up ct evaluations for the patients with active takayasu arteritis included a decrease of the mural thickness and enhancement , disappearance of the low - attenuation ring on the venous phase , and an increase of the mural attenuation and calcification on the precontrast phase .

MATERIALS AND METHODS CT Techniques The Data Analysis RESULTS The Initial CT Findings The Follow-up CT findings (Tables 2, 3 and 4) DISCUSSION

a total of 220 patients with takayasu arteritis underwent ct examinations in our hospital during the 12 years , from january 1994 to december 2005 . to satisfy the inclusion criteria , the patients had to have active takayasu arteritis and to have had the follow - up ct examination done . the criteria for the active disease at the time of the initial ct included systemic features , an elevated erythrocyte sedimentation rate ( esr , normal range in females : 0 20 mm / hr , in males : 0 9 mm / hr ) and abnormal mural changes according to the initial ct evaluation . eighteen patients were included in this retrospective study to evaluate the interval changes of the ct findings in takayasu arteritis . their ages ranged from 11 to 53 years , with the mean age of 31.2 years at the time of the initial ct examination . from two to four ct examinations the clinical findings of the 18 patients are summarized in table 1 , including the number of the ct examinations , the modified ueda type of extent of disease , the levels of esr and c - reactive protein , along with the medical and surgical or interventional treatments . the course of disease was modified ueda type i in four patients , type ii in two patients , type iii in nine patients and type iv in three patients . all the patients , except one patient with active pulmonary tuberculosis , were treated with prednisolone alone , or with methotrexate or cyclophosphamide . by the follow - up evaluation , the disease was defined as inactive if the esr was normal at least for six months without steroid medication . otherwise , the disease was defined as active . according to the above criteria , the disease was active in all 18 patients at the time of the initial ct evaluation . the disease was considered as inactive in five patients and active in 13 patients at the time of the last follow - up ct . because of the long period for collecting the cases , more than 10 years , various ct scanners were used for the patients scanning ; these scanners varied from a single slice spiral ct to a 16-channel multidetector ct ( mdct ) scanner including single slice spiral ct ( somatom plus - s , somatom plus-4 , siemens , erlangen , germany ) , the 4-channel mdct ( mx8000 , marconi medical systems , cleveland , oh ) , the 8-channel mdct ( lightspeed ultra , ge medical systems , milwaukee , wi ) and the 16-channel mdct ( sensation 16 , siemens , erlangen , germany ) . we used the bolus - tracking method at the ascending aorta level and set 100 hu as the triggering threshold for the arterial phase . the venous phase scan was taken at 5 to 10 minutes after initiating the injection of the contrast material . the scanning parameters varied according to the type of the scanner , but the following basic concept for scanning of the aorta was followed : the slice thickness was kept as thin as possible for the scanning phase of the precontrast phase , the arterial phase and the venous phase . for the arterial phase , the slice thickness was 3 mm on the single spiral ct and the 4-channel mdct with 2 mm reconstruction intervals , and it was 1.5 mm on the 8-channel and the 16-channel mdct with 1 mm reconstruction intervals . for the precontrast and the delayed venous phase scans , the pitch value was 2 for the single spiral ct and 1 to 1.5 for mdct . mdct could cover the whole aorta during the time of a single breath - hold , and each scan was done with a single breath - hold . in case of the single slice spiral ct , the whole aorta for the arterial phase was covered with two breath - hold scans , one for the thoracic aorta and the other for the abdominal aorta . the high density of the aortic wall and the mural calcification on the precontrast ct scan and the low - attenuated ring on the arterial and the venous phase ct scan were examined by two collaborating radiologists . the mural findings of the follow - up ct were compared with those of the initial ct . in cases of multiple follow - up evaluations , the two radiologists measured the mural thickness on the transverse image of the arterial phase and the ct attenuation of the aortic wall in the precontrast , arterial and venous phases . the arterial and delayed enhancements of the aortic wall were compared with the attenuation of the back muscle of the same image , and this was expressed as a ratio . the ratio of the attenuation of the enhanced aortic wall over the attenuation of the back muscle in the initial ct was compared with that of the follow - up ct . the luminal findings , in addition to the mural changes , were also considered on the initial and follow - up ct examinations . for the statistical tests between the two groups , mann - whitney u test was used for continuous variables , and the fisher exact test was used for categorical variables . the wilcoxon signed rank test was applied for statistically testing the follow - up evaluation . because of the long period for collecting the cases , more than 10 years , various ct scanners were used for the patients scanning ; these scanners varied from a single slice spiral ct to a 16-channel multidetector ct ( mdct ) scanner including single slice spiral ct ( somatom plus - s , somatom plus-4 , siemens , erlangen , germany ) , the 4-channel mdct ( mx8000 , marconi medical systems , cleveland , oh ) , the 8-channel mdct ( lightspeed ultra , ge medical systems , milwaukee , wi ) and the 16-channel mdct ( sensation 16 , siemens , erlangen , germany ) . we used the bolus - tracking method at the ascending aorta level and set 100 hu as the triggering threshold for the arterial phase . the scanning parameters varied according to the type of the scanner , but the following basic concept for scanning of the aorta was followed : the slice thickness was kept as thin as possible for the scanning phase of the precontrast phase , the arterial phase and the venous phase . for the arterial phase , the slice thickness was 3 mm on the single spiral ct and the 4-channel mdct with 2 mm reconstruction intervals , and it was 1.5 mm on the 8-channel and the 16-channel mdct with 1 mm reconstruction intervals . for the precontrast and the delayed venous phase scans , the pitch value was 2 for the single spiral ct and 1 to 1.5 for mdct . mdct could cover the whole aorta during the time of a single breath - hold , and each scan was done with a single breath - hold . in case of the single slice spiral ct , the whole aorta for the arterial phase was covered with two breath - hold scans , one for the thoracic aorta and the other for the abdominal aorta . the high density of the aortic wall and the mural calcification on the precontrast ct scan and the low - attenuated ring on the arterial and the venous phase ct scan were examined by two collaborating radiologists . the mural findings of the follow - up ct were compared with those of the initial ct . in cases of multiple follow - up evaluations , the two radiologists measured the mural thickness on the transverse image of the arterial phase and the ct attenuation of the aortic wall in the precontrast , arterial and venous phases . the measurement was performed at the location with a maximum mural thickness , including the ascending aorta , the arch , the left common carotid and abdominal aorta . the arterial and delayed enhancements of the aortic wall were compared with the attenuation of the back muscle of the same image , and this was expressed as a ratio . the ratio of the attenuation of the enhanced aortic wall over the attenuation of the back muscle in the initial ct was compared with that of the follow - up ct . the luminal findings , in addition to the mural changes , were also considered on the initial and follow - up ct examinations . for the statistical tests between the two groups , mann - whitney u test was used for continuous variables , and the fisher exact test was used for categorical variables . the wilcoxon signed rank test was applied for statistically testing the follow - up evaluation . the mural changes found on the initial and follow - up ct evaluations of the 18 patients are summarized in table 2 . on the precontrast ct , the high density of the aortic wall was noted in 15 patients ( 83% ) . on the arterial phase of ct , the wall was slightly enhanced in various portions of the aorta and its branches , and the mean ratio of attenuation of the wall over that of the back muscle was 1.4 . a circumferential low - attenuation ring was demonstrated for the patients with substantial arterial enhancement . in the venous phase , the mural enhancement was more pronounced in all the patients with an attenuation ratio from 1.2 to 2.6 ( the mean value : 1.9 ) . the low - attenuation ring was well demonstrated between the contrast - filled lumen and the enhanced outer wall in 15 patients ( 83% ) in the venous phase ( fig . no significant stenosis was shown , even for the four patients with significant mural changes . on the follow - up ct evaluation , with a mean follow - up period of 55.6 months , the precontrast image showed the high density ring in two more patients ( table 2 ) . the mean attenuation ( hu ) of the aortic wall on the precontrast phase increased significantly from 48.9 to 56.5 ( p = 0.04 ) ( table 3 ) . the mean mural thickness measured on the arterial phase was 2.4 mm , and it was significantly decreased from 4.1 mm as measured by the initial ct evaluation ( p = 0.001 ) . the mean ct attenuation ratio of the wall ( aortic wall / muscle ) in the venous phase decreased significantly from 1.9 to 1.3 ( p = 0.001 ) . the low - attenuation ring in the venous phase was identified in seven patients ( 39% ) with active persistent disease . the mural thickness decreased , and the mural enhancement also decreased significantly in both the group of patients with inactive disease and in the group of patients with active persistent disease ( figs . in the group of the inactive disease patients ( n = 5 ) , the low - attenuation ring , initially shown in four patients ( 80% ) , disappeared in all the patients by the last follow - up ct ( fig . however , each of the individual ct findings had no statistically significant correlation with the esr - based disease activity on comparison between the both groups . luminal stenosis was improved along with the mural changes by the follow - up ct after medical treatment in two patients ( cases 7 and 16 ) . in one patient ( case 1 ) , the lumen of the abdominal aorta and the proximal left renal artery had recovered , while focal stenosis developed at the middle of the left renal artery . the mural changes found on the initial and follow - up ct evaluations of the 18 patients are summarized in table 2 . on the precontrast ct , the high density of the aortic wall was noted in 15 patients ( 83% ) . on the arterial phase of ct , the wall was slightly enhanced in various portions of the aorta and its branches , and the mean ratio of attenuation of the wall over that of the back muscle was 1.4 . a circumferential low - attenuation ring was demonstrated for the patients with substantial arterial enhancement . in the venous phase , the mural enhancement was more pronounced in all the patients with an attenuation ratio from 1.2 to 2.6 ( the mean value : 1.9 ) . the low - attenuation ring was well demonstrated between the contrast - filled lumen and the enhanced outer wall in 15 patients ( 83% ) in the venous phase ( fig . no significant stenosis was shown , even for the four patients with significant mural changes . on the follow - up ct evaluation , with a mean follow - up period of 55.6 months , the precontrast image showed the high density ring in two more patients ( table 2 ) . the mean attenuation ( hu ) of the aortic wall on the precontrast phase increased significantly from 48.9 to 56.5 ( p = 0.04 ) ( table 3 ) . the mean mural thickness measured on the arterial phase was 2.4 mm , and it was significantly decreased from 4.1 mm as measured by the initial ct evaluation ( p = 0.001 ) . the mean ct attenuation ratio of the wall ( aortic wall / muscle ) in the venous phase decreased significantly from 1.9 to 1.3 ( p = 0.001 ) . the low - attenuation ring in the venous phase was identified in seven patients ( 39% ) with active persistent disease . the mural thickness decreased , and the mural enhancement also decreased significantly in both the group of patients with inactive disease and in the group of patients with active persistent disease ( figs . in the group of the inactive disease patients ( n = 5 ) , the low - attenuation ring , initially shown in four patients ( 80% ) , disappeared in all the patients by the last follow - up ct ( fig . however , each of the individual ct findings had no statistically significant correlation with the esr - based disease activity on comparison between the both groups . luminal stenosis was improved along with the mural changes by the follow - up ct after medical treatment in two patients ( cases 7 and 16 ) . in one patient ( case 1 ) , the lumen of the abdominal aorta and the proximal left renal artery had recovered , while focal stenosis developed at the middle of the left renal artery . various imaging modalities may reveal the mural changes in patients with takayasu arteritis during the early phase of the disease with or without such luminal changes , as dilatation and stenosis ( 6 , 8 - 10 ) . ct may reveal mural thickening with the arterial and venous enhancement ( 5 , 6 , 8 , 9 ) . the mr imaging reveals the mural thickening of the aorta , the pulmonary artery and its branches , and especially on the gadolinium enhanced t1 weighted images . the mural thickening and the enhancement , as evaluated by each modality , may decrease after appropriate treatment ( 9 - 13 ) . in this retrospective study , we evaluated the mural changes on the follow - up ct examination for the patients with active disease at the time of the initial ct examination . there have been a few reports on the detailed analysis of the mural changes on the follow - up ct evaluation in a group of patients . reported on their results of electron beam ct follow - up , in which they noted a decrease of the mural thickening and an increase of the mural calcifications . the outer wall enhancement , initially noted in 81% cases , was found in 37% case on the last ct scans ( 8) . for our results , the mural thickness decreased significantly from 4.1 mm to 2.4 mm . other mural changes seen on the follow - up ct evaluation included increased attenuation of the aortic wall and more extensive mural calcifications on the precontrast ct . the mural enhancement in the venous phase was decreased significantly on the follow - up ct evaluation . the disease should be regarded as active if there is any new focus or an aggravation of the mural changes according to a cross - sectional imaging modality . on the contrary , the mural changes may resolve completely with appropriate treatment , resulting in an inactivated form of disease . reported that the contrast - enhanced mr imaging showed more enhancement of the thickened aortic wall as compared with the myocardium , suggesting active disease . for determining the progress of the disease activity , they reported an agreement rate for contrast - enhanced mr imaging as 88.5% with the clinical findings : 92.3% with the esr and 84.6% with the c - reactive protein ( 13 ) . in this study , the disease was defined as inactive by the follow - up evaluation if the esr was normal at least for six months even without steroid medication . after appropriate treatment , the mural thickness decreased significantly in all five cases with inactive disease , as was established by the follow - up ct evaluation . for the microscopic findings of takayasu arteritis , the inflammatory reactions affect mainly the adventitia and the media . the media shows vascularization , and the capillaries originating at the transition zone of the adventitia proliferate to the entire medial layer . the active inflammation of the outer wall contributes mainly to the thickening of the aortic wall in the early phase of takayasu arteritis . the mural enhancement in the arterial or venous phase is most likely associated with the neovascularization and the productive inflammation of the media and the adventitia . the intima is thickened with an increase of the basophilic ground substance during the acute phase ( 14 ) . an improvement of the inflammatory process of the aortic wall may be manifested as a decrease of the thickness , a decrease of the mural enhancement and disappearance of the inner low - attenuation ring on the follow - up ct evaluation . ct may reflect the mural changes on the follow - up evaluation of active takayasu arteritis . however , there was no statistical significance in the correlation between the ct findings and activity of the disease on the basis of the esr in this study . on the ct angiography for patients with takayasu arteritis , the carotid bifurcation should be included into the examination because involvement of the common carotid artery is common in takayasu arteritis . in addition to the arterial phase data , the venous phase imaging also reveals important mural changes , including the delayed enhancement and the inner low - attenuation ring . however , the thin section acquisition and the additional venous phase imaging may be associated with exposure of patients to a considerable dose of radiation . decreasing the tube current and increasing the slice thickness up to 5 mm in the venous phase it was a retrospective study of ct follow - up in patients with takayasu arteritis with the different intervals between the initial and the ct follow - up examinations being from 10 months to 10 years . there could be bias in selection of the patients with active takayasu arteritis since the clinical criteria of activity were not perfect . the regimen and the dosage of the medical treatment were different because the patients included into the study were treated by different clinicians . in summary , the mural changes on the follow - up ct evaluation of patients with active takayasu arteritis include a decrease of the mural thickness , a decrease of the mural enhancement and disappearance of the low - attenuation ring in the venous phase , and an increase of the mural attenuation and calcifications in the precontrast phase . further investigations are necessary to establish the correlation between these mural changes noted over a period of time with the luminal changes and the long - term prognosis of patients with active takayasu arteritis .

, , nevertheless , some literatures reported serious under - usage of omt in appropriate candidates after hospital discharge . , most of existing evidence about secondary prevention drugs was examined single drug effect but not combined effect and was from pre - reperfusion era . in contemporary era of increasing implementation of reperfusion therapies and aspirin or statin use , it is currently unclear whether if all cad patients could receive similar benefits from omt at discharge . in addition , it is not clear whether each of evidence - based drugs is indispensable in omt , or that the effect of individual drug would be modified by the combination of other secondary prevention drugs . therefore , the aim of this study was to investigate the usage rate and influential factors of omt at discharge , as well as its impact on survival in cad patients with different subgroups . this study included patients from cad database of west china hospital , sichuan university from july 2008 to october 2012 . the inclusion criteria was cad patients with an angiographically confirmed stenosis 50% in at least one major coronary artery , including those with stable cad and those who were stabilized after 15 days after an acute coronary syndrome ( acs ) . the exclusion criteria are as follows : ( 1 ) in - hospital mortality ; ( 2 ) history of gastrointestinal or cerebral hemorrhage ; ( 3 ) malignant tumor , connective tissue disease , creatinine 225 mol / l or acute bronchial asthma patients ; and ( 4 ) incomplete follow - up . all subjects signed an informed consent prior to the inclusion in the cad database , and this study was approved by the ethics committee from our institute 's ethics review board . omt was defined as combination of antiplatelet [ dual antiplatelet therapy ( dapt ) for pci or acs patients , and one for stable angina patients without pci ] , statins , beta blockers ( bbs ) , and angiotensin converting enzyme ( ace ) inhibitor or angiotensin receptor blocker ( arb ) , which was in line with the definition of omt in previous published studies . , , follow - up of patients was conducted primarily by telephone , and if necessary , hospital - visits . the primary end - point of the study was death from any cause ( all - cause mortality ) . patients were divided into omt group and non - omt group according to medication at discharge . continuous variables , if were normal distribution , were expressed as mean sd ; otherwise , they were expressed as median and interquartile range . initially , shapiro - wilk test was used to examine the normal distribution and homogeneity of variance of continuous data . if both of above were met , student 's t - test or analysis of variance ( anova ) was conducted to compare baseline characteristics between two groups or multiple groups , respectively . multiple comparisons were performed with s - n - k method . in the condition of non - normal distribution or unequal variances , wilcoxon rank sum test were performed . pearson or fisher test were performed to compared the variables with categorical variables between groups . kaplan - meier method was used to constructed survival curve , and the differences between curves were examined using log - rank test . multivariate logistic regression was conducted to detect the possible influential factors of omt at discharge . multivariate cox regression was performed to examine the predictive factors of long - term outcome . both of traditional cardiovascular risk factors , such as age , sex , smoking history , pre - hypertension and pre - diabetes , and variables with p < 0.2 in the baseline comparison were included into multivariate analysis . besides , subgroup analyses were conducted between following groups : ( 1 ) acs and stable cad ; ( 2 ) men and women ; ( 3 ) revascularization or not ; ( 4 ) follow - up duration of < 24 months or 24 months ; and ( 5 ) medication with omt , 3 types of drugs and 2 types of drugs . two - sided p value < 0.05 indicated statistical significance . this study included patients from cad database of west china hospital , sichuan university from july 2008 to october 2012 . the inclusion criteria was cad patients with an angiographically confirmed stenosis 50% in at least one major coronary artery , including those with stable cad and those who were stabilized after 15 days after an acute coronary syndrome ( acs ) . the exclusion criteria are as follows : ( 1 ) in - hospital mortality ; ( 2 ) history of gastrointestinal or cerebral hemorrhage ; ( 3 ) malignant tumor , connective tissue disease , creatinine 225 mol / l or acute bronchial asthma patients ; and ( 4 ) incomplete follow - up . all subjects signed an informed consent prior to the inclusion in the cad database , and this study was approved by the ethics committee from our institute 's ethics review board . omt was defined as combination of antiplatelet [ dual antiplatelet therapy ( dapt ) for pci or acs patients , and one for stable angina patients without pci ] , statins , beta blockers ( bbs ) , and angiotensin converting enzyme ( ace ) inhibitor or angiotensin receptor blocker ( arb ) , which was in line with the definition of omt in previous published studies . , , follow - up of patients was conducted primarily by telephone , and if necessary , hospital - visits . the primary end - point of the study was death from any cause ( all - cause mortality ) . patients were divided into omt group and non - omt group according to medication at discharge . continuous variables , if were normal distribution , were expressed as mean sd ; otherwise , they were expressed as median and interquartile range . initially , shapiro - wilk test was used to examine the normal distribution and homogeneity of variance of continuous data . if both of above were met , student 's t - test or analysis of variance ( anova ) was conducted to compare baseline characteristics between two groups or multiple groups , respectively . multiple comparisons were performed with s - n - k method . in the condition of non - normal distribution or unequal variances , wilcoxon rank sum test were performed . pearson or fisher test were performed to compared the variables with categorical variables between groups . kaplan - meier method was used to constructed survival curve , and the differences between curves were examined using log - rank test . multivariate logistic regression was conducted to detect the possible influential factors of omt at discharge . multivariate cox regression was performed to examine the predictive factors of long - term outcome . both of traditional cardiovascular risk factors , such as age , sex , smoking history , pre - hypertension and pre - diabetes , and variables with p < 0.2 in the baseline comparison besides , subgroup analyses were conducted between following groups : ( 1 ) acs and stable cad ; ( 2 ) men and women ; ( 3 ) revascularization or not ; ( 4 ) follow - up duration of < 24 months or 24 months ; and ( 5 ) medication with omt , 3 types of drugs and 2 types of drugs . two - sided p value < 0.05 indicated statistical significance . from july 2008 to october 2012 , a total of 3714 patients were included into our center 's cad database , of which , 347 patients were lost during follow - up . after exclusion according to aforementioned study criteria , 3176 patients were finally included in current study . patients with omt at discharge were more likely to have hypertension , diabetes mellitus , or prior myocardial infarction compared with non - omt group ; furthermore , they had higher heart rate , blood pressure at admission , and shorter hospital stay duration . although usage rate of omt was low in both groups before admission , it was higher in omt groups than non - omt groups ( 11.3% vs. 4.0% , p < 0.001 ) . patients receiving omt were more likely to be discharged from department of cardiology than those not receiving omt ( 94.4% vs. 87.3% ) ( p < 0.001 ) . non - omt group has a higher percentage of left ventricular ejection fraction ( lvef ) in the normal range ( 55% ) , while omt group has a higher proportion of patients without lvef examination during hospitalization . there was no significant difference in age , gender , smoking , ethnicity , marriage status , laboratory values , canadian cardiovascular society ( ccs ) classes of angina and revascularization therapies . the types of medications used before and after admission are shown in figure 2 and figure 3 . acs : acute coronary syndrome ; bmi : body mass index ; cabg : coronary artery bypass grafting ; ccs : canadian cardiovascular society ; dbp : diastolic blood pressure ; hdl - c : high density lipoprotein cholesterol ; ldl - c : low density lipoprotein cholesterol ; lvef : left ventricular ejection fraction ; omt : optimal medical therapy ; pci : percutaneous coronary intervention ; plt : platelets ; sbp : systolic blood pressure ; tc : total cholesterol ; tg : triglyceride ; wbc : white blood cell . beta blockers and angiotensin converting enzyme inhibitor / angiotensin receptor blockers were significantly lesser compared with antiplatelet drug or statins . acei : angiotensin converting enzyme inhibitor ; arb : angiotensin receptor blocker ; omt : optimal medical therapy . multiple logistics regression analyses indicated that pre - hypertension , pre - diabetes , faster heart rate , higher sbp , discharge form department of cardiology ( or : 1.82 , 95% ci : 1.292.59 , p = 0.001 ) , and omt before admission ( or : 3.21 , 95% ci : 2.324.44 , p < 0.001 ) were associated with omt at discharge , while older age and longer length of hospital stay were associated with a lower likelihood to receive omt ( table 2 ) . adjusted for five traditional cardiovascular risk factors ( age , sex , pre - hypertension , pre - diabetes , current smoker ) and variables with p < 0.2 at baseline between omt and non - omt groups . omt : optimal medical therapy ; sbp : systolic blood pressure . after a median follow - up of 27.1 months , all - cause mortality occurred in 217/3176 ( 6.8% ) patients [ omt : 69 ( 5.5% ) , non - omt : 148 ( 7.7% ) , log - rank test : p = 0.01 ] ( figure 4 ) . multiple cox regression analyses indicates omt at discharge was associated with a 35% reduced risk of all - cause mortality [ ( hr : 0.65 , 0.450.95 ) , p = 0.025 ] . other factors associated with all - cause mortality included age , ethnic minorities , lvef , creatinine , white blood cell count , and revascularization therapy ( table 3 ) . further analysis adjusting for lvef ( categorical variable ) and prior myocardial infarction ( mi ) did not change the results ( data were not shown ) . a further analysis indicates omt at discharge on the outcomes of survival differs across subgroups , especially in consideration of follow - up duration ( figure 5 ) . at follow - up 24 months , the benefit of omt at discharge was most apparent , while there was no statistical significance below 24 months of follow - up . both in acs patients and in men , omt at discharge were associated with reduced risk of all - cause mortality ( hr : 0.65 , 95% ci : 0.460.90 and 0.63 , 95% ci : 0.450.88 , respectively ) . both patients with or without revascularization therapies were able to receive benefit with omt at discharge , and the benefits were more evident at 24 months follow - up . adjusted for five traditional risk factors ( age , sex , pre - hypertension , pre - diabetes , current smoker ) and variables with p < 0.2 in the baseline comparison between patients who were survival and who were dead . cabg : coronary artery bypass grafting ; lvef : left ventricular ejection fraction ; omt : indicates optimal medical therapy ; pci : percutaneous coronary intervention ; wbc : white blood cell . acs : acute coronary syndrome ; chd : coronary heart disease ; omt : optimal medical therapy . efficacy of types of secondary prevention drugs at discharge differs between acs and stable angina patients . in acs patients , a trend towards reduced risk of all - cause mortality was observed with 3-drug therapy in comparison with 2 drug therapy , while omt maximized the clinical benefits of evidence - based drugs ( table 4 ) . for stable angina patients however , the survival benefits of omt and 3-drug therapy were similar compared to those with 2-drug therapy . model 1 : adjusted for age , sex , pre - hypertension , pre - diabetes and current smoker ; model 2 : adjusted for model 1 + body mass index , acs , nation and revascularization ; model 3 : adjusted for model 2 + marital status , previous myocardial infarction , previous percutaneous coronary intervention , omt before admission , serum creatinine , glucose , triglyceride , low density lipoprotein , white blood cell , platelet and systolic blood pressure . acs : acute coronary syndrome ; chd : coronary heart disease ; omt : optimal medical therapy . the principle findings of this study suggests : ( 1 ) there is a serious under - usage of omt in cad patients , only 39.8% received omt at discharge ; ( 2 ) omt at discharge was associated with decreased risk of mortality , and subgroup analyses indicates this positive impact is more apparent at 24 months follow - up ; ( 3 ) patients with or without revascularization therapies were both able to receive benefit from omt at discharge ; and for those with revascularization therapies , the positive impact was more apparent at longer - term follow - up ; and ( 4 ) acs patients were able to receive maximal survival benefit from omt at discharge compared to 2-drug therapy . for stable cad patients however , several countries reported that omt was achieved in < 50% of acs patients , , , and in current study , only 40.2% of acs accepted omt at discharge . for patients with stable angina , the united states national cardiovascular disease registry revealed that 65% of them were on 3-drug therapy ( antiplatelet , statins and bb ) and only 38.9% received omt , which is similar with present study ( 62.5% ) . the lack of omt at discharge may be due to non - compliance of patients and inadequate patient education or counseling . perhaps the value of omt at discharge is underestimated due to the limited evidence of studies without robust conclusions on the prognostic implications of omt with subgroups . the conceivable illusion that patients who had revascularization therapy do n't necessitate omt at discharge ensues . , antiplatelet and statin drugs are currently recommended for all cad patients without contraindications ; , thus , were commonly used ( 98.3% and 93.0% , respectively ) . bbs and ace inhibitors or angiotensin receptor blockers ( arbs ) however , are more strongly recommended for cad patients with hypertension , diabetes mellitus and/or heart failure . it is worthy to note that higher percentage of patients in the omt group had baseline comorbidities . this suggests it may be appropriate to stringent future applications of omt at discharge in non - contraindicated hypertensive and/or diabetic cad patients . previous studies indicated that revascularized patients are less likely to fill prescriptions , thus , a lower secondary preventive medical care . , the results of the courage trial showed that in patients who received optimal drug therapy . pci therapy did not further decrease the risk of adverse cardiovascular events syntax trial showed that omt in the long run reduced 36% relative risk of pci or coronary artery bypass grafting ( cabg ) therapy and 27% risk of the composite end point of death , stroke and myocardial infarction . a south korean registered research showed that omt in - hospital was associated with a 21% reduction of major adverse cardiovascular events in patients with acute coronary syndrome . furthermore , a canadian registry study showed that compared with those taking either the 0 or 1 secondary prevention drug , omt was associated with a 50% reduced risk of all - cause mortality reduction , in patients with acs . in the subgroup analyses , omt at discharge was associated with better survival in both patients with or without revascularization therapies . for revascularized patients , this survival benefit is revealed only after 24 months follow - up . possible explanation for this trend is that , the benefit of omt at short - term follow - up is diluted due to revascularization therapies . in patients without revascularization therapies , omt at discharge was not associated with an obvious survival trend at short - term follow - up . nevertheless , non - revascularized cad patients might be frailer and accompanied with multiple adverse comorbidities ; if a patient 's general condition was promising , omt at discharge could still reduce the risk of all - cause mortality at long - term follow - up . these results suggest that it is important for patients to be adamant with omt at discharge , asthe survival benefit of omt would maximize at long - term follow - up . discharge medication was an important factor for the long - term outcomes of cad patients as present database revealed that patients with omt at discharge were 2.4 times more likely to continue omt during follow - up compared with non - omt group . guideline also recommended physicians to initiate secondary preventive medical care prior to hospital discharge to improve patient compliances . , , there was a sex - related difference on the outcomes of omt at discharge . male cad patients were more likely to receive substantial survival benefits , with a 37% reduction in all - cause mortality , while female patients with omt on the other hand were not associated with an obvious survival trend . possible explanation is the fact that female cad patients are often more elderly , non - compliant , and complicated with multiple baseline adverse comorbidities , which might undermine the benefits of omt . , moreover , relative small percentage of female in our study might have no enough power to detect the significance . our results suggest omt at discharge was associated with a 38% reduced risk of all - cause mortality in acs patients . further subgroup analyses showed that acs patients with omt at discharge had significantly better survival compared to those with 2-drug combination therapy , while 3-drug combination therapy did not . it is likely that the rennin angiotensin aldosterone system and sympathetic nervous system is more activated in acs patients ; , therefore , the positive impact of omt was more apparent , and should be implemented in all acs patients , if tolerated . for stable angina patients , omt was not superior than the combination of the three types of the evidence - based drugs . this result is in line with the peace study , which revealed that the presence of ace inhibitors along with antiplatelet , statins and bbs in stable cad patients was not associated with additional benefits . similarly , another study reported that bbs along with antiplatelet , statins and ace inhibitors in stable cad patients did not improve survival . nevertheless , due to the relatively small number of patients in our subgroup analyses , this result merits further study . in addition , we failed to identify superiority between bbs and ace inhibitors on the basis of antiplatelet and statin therapy in stable cad patients . we also found several factors , including the discharged department and pre - admission omt , were associated with omt at discharge . it is likely that cardiologist have a more related and extensive knowledge on the management of secondary prevention . besides , previous study showed that patients were more compliant with a drug they had ever used in the past . therefore , omt at discharge was more acceptable in these patients , with less unfavorable drug - associated complications . firstly , this was a single center observational study , therefore , the non - randomization and potential selection bias could have resulted in confounding . in addition , it is possible the rate of omt at discharge were underestimated because omt contraindication was not reported . therefore , this study could have overstated the actual proportion of non - omt group . furthermore , the present study primarily focused on the hard endpoint , all - cause mortality , while other important endpoints , such as mi , hf and stroke , which has a significant impact on the quality of life were not reported ; nevertheless , the present study might be underpowered to test the association between omt and individual endpoints . last but not least important , comprehensive treatment is as vital as secondary prevention for the long - term outcome of patients with cad . in the present study , we focused on the effect of omt on the prevention of further cardiovascular events in patients with cad only while did not assess the impact of comprehensive treatment or rehabilitation on the outcome , which might result in bias and exaggerated the effect of omt . lack of omt at discharge is still a serious problem amongst cad patients as omt was associated significant reduced all - cause mortality . patients with omt should adhere to the evidence - based medication as the clinical benefit might be more apparent in the long - run . omt of four - drug combination maximize the benefits in patients with acs , while omt of three - drug combination was sufficient to maximize the benefits in patients with stable cad . firstly , this was a single center observational study , therefore , the non - randomization and potential selection bias could have resulted in confounding . in addition , it is possible the rate of omt at discharge were underestimated because omt contraindication was not reported . therefore , this study could have overstated the actual proportion of non - omt group . furthermore , the present study primarily focused on the hard endpoint , all - cause mortality , while other important endpoints , such as mi , hf and stroke , which has a significant impact on the quality of life were not reported ; nevertheless , the present study might be underpowered to test the association between omt and individual endpoints . last but not least important , comprehensive treatment is as vital as secondary prevention for the long - term outcome of patients with cad . in the present study , we focused on the effect of omt on the prevention of further cardiovascular events in patients with cad only while did not assess the impact of comprehensive treatment or rehabilitation on the outcome , which might result in bias and exaggerated the effect of omt . lack of omt at discharge is still a serious problem amongst cad patients as omt was associated significant reduced all - cause mortality . patients with omt should adhere to the evidence - based medication as the clinical benefit might be more apparent in the long - run . omt of four - drug combination maximize the benefits in patients with acs , while omt of three - drug combination was sufficient to maximize the benefits in patients with stable cad .

objectiveto analyze the current usage of optimal medical therapy ( omt ) , influencing factors , and the predictive value of omt for all - cause mortality in coronary artery disease ( cad ) patients with different subgroups.methodsa total of 3176 cad patients confirmed by coronary angiography were included . omt was defined as the combination of anti - platelet drugs , statins , beta blockers , and angiotensin converting enzyme inhibitors or angiotensin receptor blockers . factors for omt and its prognostic value were analyzed in cad patients across different subgroups.resultsout of 3176 patients , only 39.8% ( n = 1265 ) were on omt at discharge . factors associated with omt at discharge were pre - admission omt and discharge department . all - cause mortality occurred in 6.8% ( n = 217 ) of patients . multivariate analyses indicated that omt was significantly associated with reduced all - cause mortality ( hr : 0.65 , 95% ci : 0.450.95 ; p = 0.025 ) . sub - group analyses indicate that male acute coronary syndrome ( acs ) patients were more likely to receive survival benefits with omt at discharge . the positive impact of omt at discharge was more apparent after 24 months , regardless of revascularization therapy . four - drug combination of omt was superior to 3-drug combination therapy in acs patients but not in stable patients.conclusionsomt was associated with significant improvement in survival in patients with cad . the positive impact of omt was distinct in the cad patients with different characteristics .

Introduction Methods Patients inclusion Consent and ethics Data collection Statistical analyses Results Discussion Study limitations Conclusions

, , nevertheless , some literatures reported serious under - usage of omt in appropriate candidates after hospital discharge . , most of existing evidence about secondary prevention drugs was examined single drug effect but not combined effect and was from pre - reperfusion era . in contemporary era of increasing implementation of reperfusion therapies and aspirin or statin use , it is currently unclear whether if all cad patients could receive similar benefits from omt at discharge . in addition , it is not clear whether each of evidence - based drugs is indispensable in omt , or that the effect of individual drug would be modified by the combination of other secondary prevention drugs . therefore , the aim of this study was to investigate the usage rate and influential factors of omt at discharge , as well as its impact on survival in cad patients with different subgroups . the inclusion criteria was cad patients with an angiographically confirmed stenosis 50% in at least one major coronary artery , including those with stable cad and those who were stabilized after 15 days after an acute coronary syndrome ( acs ) . all subjects signed an informed consent prior to the inclusion in the cad database , and this study was approved by the ethics committee from our institute 's ethics review board . omt was defined as combination of antiplatelet [ dual antiplatelet therapy ( dapt ) for pci or acs patients , and one for stable angina patients without pci ] , statins , beta blockers ( bbs ) , and angiotensin converting enzyme ( ace ) inhibitor or angiotensin receptor blocker ( arb ) , which was in line with the definition of omt in previous published studies . , , follow - up of patients was conducted primarily by telephone , and if necessary , hospital - visits . the primary end - point of the study was death from any cause ( all - cause mortality ) . patients were divided into omt group and non - omt group according to medication at discharge . in the condition of non - normal distribution or unequal variances , wilcoxon rank sum test were performed . kaplan - meier method was used to constructed survival curve , and the differences between curves were examined using log - rank test . multivariate logistic regression was conducted to detect the possible influential factors of omt at discharge . multivariate cox regression was performed to examine the predictive factors of long - term outcome . both of traditional cardiovascular risk factors , such as age , sex , smoking history , pre - hypertension and pre - diabetes , and variables with p < 0.2 in the baseline comparison were included into multivariate analysis . besides , subgroup analyses were conducted between following groups : ( 1 ) acs and stable cad ; ( 2 ) men and women ; ( 3 ) revascularization or not ; ( 4 ) follow - up duration of < 24 months or 24 months ; and ( 5 ) medication with omt , 3 types of drugs and 2 types of drugs . the inclusion criteria was cad patients with an angiographically confirmed stenosis 50% in at least one major coronary artery , including those with stable cad and those who were stabilized after 15 days after an acute coronary syndrome ( acs ) . all subjects signed an informed consent prior to the inclusion in the cad database , and this study was approved by the ethics committee from our institute 's ethics review board . omt was defined as combination of antiplatelet [ dual antiplatelet therapy ( dapt ) for pci or acs patients , and one for stable angina patients without pci ] , statins , beta blockers ( bbs ) , and angiotensin converting enzyme ( ace ) inhibitor or angiotensin receptor blocker ( arb ) , which was in line with the definition of omt in previous published studies . , , follow - up of patients was conducted primarily by telephone , and if necessary , hospital - visits . the primary end - point of the study was death from any cause ( all - cause mortality ) . patients were divided into omt group and non - omt group according to medication at discharge . in the condition of non - normal distribution or unequal variances , wilcoxon rank sum test were performed . kaplan - meier method was used to constructed survival curve , and the differences between curves were examined using log - rank test . multivariate logistic regression was conducted to detect the possible influential factors of omt at discharge . multivariate cox regression was performed to examine the predictive factors of long - term outcome . both of traditional cardiovascular risk factors , such as age , sex , smoking history , pre - hypertension and pre - diabetes , and variables with p < 0.2 in the baseline comparison besides , subgroup analyses were conducted between following groups : ( 1 ) acs and stable cad ; ( 2 ) men and women ; ( 3 ) revascularization or not ; ( 4 ) follow - up duration of < 24 months or 24 months ; and ( 5 ) medication with omt , 3 types of drugs and 2 types of drugs . from july 2008 to october 2012 , a total of 3714 patients were included into our center 's cad database , of which , 347 patients were lost during follow - up . after exclusion according to aforementioned study criteria , 3176 patients were finally included in current study . patients with omt at discharge were more likely to have hypertension , diabetes mellitus , or prior myocardial infarction compared with non - omt group ; furthermore , they had higher heart rate , blood pressure at admission , and shorter hospital stay duration . although usage rate of omt was low in both groups before admission , it was higher in omt groups than non - omt groups ( 11.3% vs. 4.0% , p < 0.001 ) . patients receiving omt were more likely to be discharged from department of cardiology than those not receiving omt ( 94.4% vs. 87.3% ) ( p < 0.001 ) . non - omt group has a higher percentage of left ventricular ejection fraction ( lvef ) in the normal range ( 55% ) , while omt group has a higher proportion of patients without lvef examination during hospitalization . acs : acute coronary syndrome ; bmi : body mass index ; cabg : coronary artery bypass grafting ; ccs : canadian cardiovascular society ; dbp : diastolic blood pressure ; hdl - c : high density lipoprotein cholesterol ; ldl - c : low density lipoprotein cholesterol ; lvef : left ventricular ejection fraction ; omt : optimal medical therapy ; pci : percutaneous coronary intervention ; plt : platelets ; sbp : systolic blood pressure ; tc : total cholesterol ; tg : triglyceride ; wbc : white blood cell . beta blockers and angiotensin converting enzyme inhibitor / angiotensin receptor blockers were significantly lesser compared with antiplatelet drug or statins . acei : angiotensin converting enzyme inhibitor ; arb : angiotensin receptor blocker ; omt : optimal medical therapy . multiple logistics regression analyses indicated that pre - hypertension , pre - diabetes , faster heart rate , higher sbp , discharge form department of cardiology ( or : 1.82 , 95% ci : 1.292.59 , p = 0.001 ) , and omt before admission ( or : 3.21 , 95% ci : 2.324.44 , p < 0.001 ) were associated with omt at discharge , while older age and longer length of hospital stay were associated with a lower likelihood to receive omt ( table 2 ) . adjusted for five traditional cardiovascular risk factors ( age , sex , pre - hypertension , pre - diabetes , current smoker ) and variables with p < 0.2 at baseline between omt and non - omt groups . omt : optimal medical therapy ; sbp : systolic blood pressure . after a median follow - up of 27.1 months , all - cause mortality occurred in 217/3176 ( 6.8% ) patients [ omt : 69 ( 5.5% ) , non - omt : 148 ( 7.7% ) , log - rank test : p = 0.01 ] ( figure 4 ) . multiple cox regression analyses indicates omt at discharge was associated with a 35% reduced risk of all - cause mortality [ ( hr : 0.65 , 0.450.95 ) , p = 0.025 ] . other factors associated with all - cause mortality included age , ethnic minorities , lvef , creatinine , white blood cell count , and revascularization therapy ( table 3 ) . a further analysis indicates omt at discharge on the outcomes of survival differs across subgroups , especially in consideration of follow - up duration ( figure 5 ) . at follow - up 24 months , the benefit of omt at discharge was most apparent , while there was no statistical significance below 24 months of follow - up . both in acs patients and in men , omt at discharge were associated with reduced risk of all - cause mortality ( hr : 0.65 , 95% ci : 0.460.90 and 0.63 , 95% ci : 0.450.88 , respectively ) . both patients with or without revascularization therapies were able to receive benefit with omt at discharge , and the benefits were more evident at 24 months follow - up . adjusted for five traditional risk factors ( age , sex , pre - hypertension , pre - diabetes , current smoker ) and variables with p < 0.2 in the baseline comparison between patients who were survival and who were dead . cabg : coronary artery bypass grafting ; lvef : left ventricular ejection fraction ; omt : indicates optimal medical therapy ; pci : percutaneous coronary intervention ; wbc : white blood cell . acs : acute coronary syndrome ; chd : coronary heart disease ; omt : optimal medical therapy . efficacy of types of secondary prevention drugs at discharge differs between acs and stable angina patients . in acs patients , a trend towards reduced risk of all - cause mortality was observed with 3-drug therapy in comparison with 2 drug therapy , while omt maximized the clinical benefits of evidence - based drugs ( table 4 ) . for stable angina patients however , the survival benefits of omt and 3-drug therapy were similar compared to those with 2-drug therapy . model 1 : adjusted for age , sex , pre - hypertension , pre - diabetes and current smoker ; model 2 : adjusted for model 1 + body mass index , acs , nation and revascularization ; model 3 : adjusted for model 2 + marital status , previous myocardial infarction , previous percutaneous coronary intervention , omt before admission , serum creatinine , glucose , triglyceride , low density lipoprotein , white blood cell , platelet and systolic blood pressure . acs : acute coronary syndrome ; chd : coronary heart disease ; omt : optimal medical therapy . the principle findings of this study suggests : ( 1 ) there is a serious under - usage of omt in cad patients , only 39.8% received omt at discharge ; ( 2 ) omt at discharge was associated with decreased risk of mortality , and subgroup analyses indicates this positive impact is more apparent at 24 months follow - up ; ( 3 ) patients with or without revascularization therapies were both able to receive benefit from omt at discharge ; and for those with revascularization therapies , the positive impact was more apparent at longer - term follow - up ; and ( 4 ) acs patients were able to receive maximal survival benefit from omt at discharge compared to 2-drug therapy . for stable cad patients however , several countries reported that omt was achieved in < 50% of acs patients , , , and in current study , only 40.2% of acs accepted omt at discharge . for patients with stable angina , the united states national cardiovascular disease registry revealed that 65% of them were on 3-drug therapy ( antiplatelet , statins and bb ) and only 38.9% received omt , which is similar with present study ( 62.5% ) . the lack of omt at discharge may be due to non - compliance of patients and inadequate patient education or counseling . perhaps the value of omt at discharge is underestimated due to the limited evidence of studies without robust conclusions on the prognostic implications of omt with subgroups . the conceivable illusion that patients who had revascularization therapy do n't necessitate omt at discharge ensues . , antiplatelet and statin drugs are currently recommended for all cad patients without contraindications ; , thus , were commonly used ( 98.3% and 93.0% , respectively ) . bbs and ace inhibitors or angiotensin receptor blockers ( arbs ) however , are more strongly recommended for cad patients with hypertension , diabetes mellitus and/or heart failure . it is worthy to note that higher percentage of patients in the omt group had baseline comorbidities . this suggests it may be appropriate to stringent future applications of omt at discharge in non - contraindicated hypertensive and/or diabetic cad patients . previous studies indicated that revascularized patients are less likely to fill prescriptions , thus , a lower secondary preventive medical care . , the results of the courage trial showed that in patients who received optimal drug therapy . pci therapy did not further decrease the risk of adverse cardiovascular events syntax trial showed that omt in the long run reduced 36% relative risk of pci or coronary artery bypass grafting ( cabg ) therapy and 27% risk of the composite end point of death , stroke and myocardial infarction . a south korean registered research showed that omt in - hospital was associated with a 21% reduction of major adverse cardiovascular events in patients with acute coronary syndrome . furthermore , a canadian registry study showed that compared with those taking either the 0 or 1 secondary prevention drug , omt was associated with a 50% reduced risk of all - cause mortality reduction , in patients with acs . in the subgroup analyses , omt at discharge was associated with better survival in both patients with or without revascularization therapies . for revascularized patients , this survival benefit is revealed only after 24 months follow - up . possible explanation for this trend is that , the benefit of omt at short - term follow - up is diluted due to revascularization therapies . in patients without revascularization therapies , omt at discharge was not associated with an obvious survival trend at short - term follow - up . nevertheless , non - revascularized cad patients might be frailer and accompanied with multiple adverse comorbidities ; if a patient 's general condition was promising , omt at discharge could still reduce the risk of all - cause mortality at long - term follow - up . these results suggest that it is important for patients to be adamant with omt at discharge , asthe survival benefit of omt would maximize at long - term follow - up . discharge medication was an important factor for the long - term outcomes of cad patients as present database revealed that patients with omt at discharge were 2.4 times more likely to continue omt during follow - up compared with non - omt group . , , there was a sex - related difference on the outcomes of omt at discharge . male cad patients were more likely to receive substantial survival benefits , with a 37% reduction in all - cause mortality , while female patients with omt on the other hand were not associated with an obvious survival trend . possible explanation is the fact that female cad patients are often more elderly , non - compliant , and complicated with multiple baseline adverse comorbidities , which might undermine the benefits of omt . our results suggest omt at discharge was associated with a 38% reduced risk of all - cause mortality in acs patients . further subgroup analyses showed that acs patients with omt at discharge had significantly better survival compared to those with 2-drug combination therapy , while 3-drug combination therapy did not . it is likely that the rennin angiotensin aldosterone system and sympathetic nervous system is more activated in acs patients ; , therefore , the positive impact of omt was more apparent , and should be implemented in all acs patients , if tolerated . for stable angina patients , omt was not superior than the combination of the three types of the evidence - based drugs . this result is in line with the peace study , which revealed that the presence of ace inhibitors along with antiplatelet , statins and bbs in stable cad patients was not associated with additional benefits . similarly , another study reported that bbs along with antiplatelet , statins and ace inhibitors in stable cad patients did not improve survival . nevertheless , due to the relatively small number of patients in our subgroup analyses , this result merits further study . in addition , we failed to identify superiority between bbs and ace inhibitors on the basis of antiplatelet and statin therapy in stable cad patients . we also found several factors , including the discharged department and pre - admission omt , were associated with omt at discharge . besides , previous study showed that patients were more compliant with a drug they had ever used in the past . therefore , omt at discharge was more acceptable in these patients , with less unfavorable drug - associated complications . in addition , it is possible the rate of omt at discharge were underestimated because omt contraindication was not reported . furthermore , the present study primarily focused on the hard endpoint , all - cause mortality , while other important endpoints , such as mi , hf and stroke , which has a significant impact on the quality of life were not reported ; nevertheless , the present study might be underpowered to test the association between omt and individual endpoints . last but not least important , comprehensive treatment is as vital as secondary prevention for the long - term outcome of patients with cad . in the present study , we focused on the effect of omt on the prevention of further cardiovascular events in patients with cad only while did not assess the impact of comprehensive treatment or rehabilitation on the outcome , which might result in bias and exaggerated the effect of omt . lack of omt at discharge is still a serious problem amongst cad patients as omt was associated significant reduced all - cause mortality . patients with omt should adhere to the evidence - based medication as the clinical benefit might be more apparent in the long - run . omt of four - drug combination maximize the benefits in patients with acs , while omt of three - drug combination was sufficient to maximize the benefits in patients with stable cad . in addition , it is possible the rate of omt at discharge were underestimated because omt contraindication was not reported . furthermore , the present study primarily focused on the hard endpoint , all - cause mortality , while other important endpoints , such as mi , hf and stroke , which has a significant impact on the quality of life were not reported ; nevertheless , the present study might be underpowered to test the association between omt and individual endpoints . last but not least important , comprehensive treatment is as vital as secondary prevention for the long - term outcome of patients with cad . in the present study , we focused on the effect of omt on the prevention of further cardiovascular events in patients with cad only while did not assess the impact of comprehensive treatment or rehabilitation on the outcome , which might result in bias and exaggerated the effect of omt . lack of omt at discharge is still a serious problem amongst cad patients as omt was associated significant reduced all - cause mortality . patients with omt should adhere to the evidence - based medication as the clinical benefit might be more apparent in the long - run . omt of four - drug combination maximize the benefits in patients with acs , while omt of three - drug combination was sufficient to maximize the benefits in patients with stable cad .

we simulated the intentional aerosolized release of rift valley fever virus ( rvfv ) in a semirural community ( population 300,000 ) in the southern part of the united states . we videotaped a series of simulated print and television ( local , network , and cable ) news reports over a fictional 9-day crisis period . we invited four groups ( medical first - responders , medical first - responder spouses or partners , journalists , and others ) within the selected community to view the videotape and answer questions about their reactions . the story of the simulated outbreak unfolded in a series of video reports from federal and local governments and the news media ( appendix 1 ) . health agency news bulletins were provided by the centers for diseases control and prevention ; news reports by television reporters or news anchors ; and community reports by local officials , including the mayor . the reports began with recognition in the community of an unusual infection affecting humans and certain farm animals and continued during the next 9 days with an epidemiologic investigation and the identification by federal authorities of intentional release of rvfv . reports included a detailed press conference by federal health authorities describing routes of transmission , prevention measures , signs and symptoms of infection , and medical management of the disease . the news conference , held in the state capital the day after the presence of rvfv infection in the united states was announced , was immediately followed by a panel discussion ( by nongovernment experts ) on rvfv . differences of opinion on clinical , epidemiologic , and biological issues among rvfv experts were reported . confusion arose in the community over disease management ( e.g. , the effectiveness of the antiviral drug ribavirin , the need for rvfv vaccine , and who should receive the vaccine ) and over the potential for infected persons to serve as reservoirs and carriers of the virus elsewhere during the few days when viral titers are especially high . governors of adjoining states questioned the adequacy of mosquito - control and animal quarantine measures , given the lack of a control model for the spread of rvfv infection in industrialized countries . although official quarantine measures were not taken , final video reports showed a gradual de facto isolation of the city . the questionnaire ( appendix 2 ) , which included multiple - choice , open - ended questions , and opportunities for additional comments , was distributed to all participants . questions addressed job abandonment , quarantine compliance , demand for drugs and vaccine , information requirements , and other issues of community interest . set 1 was given after a disease of unknown etiology affecting humans and some farm animals was recognized in the community . these questions focused on willingness to remain at work , the types and sources of information that influenced the decision to work or not work , and actions regarding families and loved ones . set 2 was given after the disease was identified as rvfv infection , federal health authorities briefed the public about this infection at a press conference , and a panel of nongovernment experts discussed the disease on television . these questions tested the participants understanding of rvfv routes of transmission and preventive measures and the participants satisfaction with information from government and nongovernment sources . sets 3 and 4 followed a period of growing confusion and anxiety caused by changing and sometimes conflicting authoritative statements and tested participants requests for medication , including ribavirin ( set 3 ) , and for rvfv vaccine ( set 4 ) . set 5 , given after the participants learned that the outbreak of rvfv was intentional , reassessed decisions and actions regarding job and family concerns and information needed to make these decisions . set 6 followed a period of increasing anxiety over a now - confirmed bioterrorism - related outbreak that could spread to humans and cattle in the state and in adjoining states , over the ability of the government to stop the spread of the infection , and over the de facto isolation of the community . these questions surveyed participants reactions to rumors of possible quarantine and to sources of information deemed reliable and influential in decision making now that the threat had become more complicated , personal , and disruptive . the number of candidates contacted to assemble groups of at least 30 participants followed guidelines from marketing study groups ( harwell productions , pers . one hundred thirty - eight medical first - responders ( responders ) were invited to participate in one study group ; 58 responded to our invitation , 45 registered , and 38 attended . reflecting the make - up of medical first - responders in the community , one third of the group s participants were fire department emergency medical services personnel , and two thirds were emergency department personnel ( nurses , physicians , technicians ) from the area s major medical centers . eighty - three spouses or partners of responders ( hereafter termed spouses ) were invited to form a second study group ; 47 responded , 44 registered , and 32 attended . fifty - seven members of the local print and tv news media ( hereafter , termed the media ) were invited to a third study group ; 50 responded , 42 registered , and 34 attended . three hundred fifty invitations were sent to rank - and - file residents of the community ( hereafter termed residents ) to form a final study group . postal service as undeliverable ; 73 responded , 46 registered , and 47 attended ( 1 registered participant failed to appear , but 2 invitees who had not registered arrived and participated ) . names of persons invited to participate in the first three groups were drawn blindly from rosters of eligible candidates prepared by their employers . rosters of eligible candidates for the residents category were prepared from census tract data and cross - indexed telephone books ( table ) . sex and age distributions within the four study groups were representative of the population segments from which their members were drawn , as were the educational levels of the responder , spouse , and media groups . the educational distribution of residents was not representative of the metropolitan area ; those who chose to participate in this group had more formal education than area residents in general . according to 2000 census data for this area , 24.2% of the population have no high school diploma or equivalency , 30.2% are high school graduates , 22.4% have some college education , 12.7% have either an associate or full college degree , and 6.1% have graduate or professional degrees . we asked all participants if they had family members and loved ones in the area to assess potential conflict in job loyalty versus safety of one s family and loved ones during a disaster . the media group was unique in the large number of its members who answered no to this question . the story of the simulated outbreak unfolded in a series of video reports from federal and local governments and the news media ( appendix 1 ) . health agency news bulletins were provided by the centers for diseases control and prevention ; news reports by television reporters or news anchors ; and community reports by local officials , including the mayor . the reports began with recognition in the community of an unusual infection affecting humans and certain farm animals and continued during the next 9 days with an epidemiologic investigation and the identification by federal authorities of intentional release of rvfv . reports included a detailed press conference by federal health authorities describing routes of transmission , prevention measures , signs and symptoms of infection , and medical management of the disease . the news conference , held in the state capital the day after the presence of rvfv infection in the united states was announced , was immediately followed by a panel discussion ( by nongovernment experts ) on rvfv . differences of opinion on clinical , epidemiologic , and biological issues among rvfv experts were reported . confusion arose in the community over disease management ( e.g. , the effectiveness of the antiviral drug ribavirin , the need for rvfv vaccine , and who should receive the vaccine ) and over the potential for infected persons to serve as reservoirs and carriers of the virus elsewhere during the few days when viral titers are especially high . governors of adjoining states questioned the adequacy of mosquito - control and animal quarantine measures , given the lack of a control model for the spread of rvfv infection in industrialized countries . although official quarantine measures were not taken , final video reports showed a gradual de facto isolation of the city . the questionnaire ( appendix 2 ) , which included multiple - choice , open - ended questions , and opportunities for additional comments , was distributed to all participants . questions addressed job abandonment , quarantine compliance , demand for drugs and vaccine , information requirements , and other issues of community interest . set 1 was given after a disease of unknown etiology affecting humans and some farm animals was recognized in the community . these questions focused on willingness to remain at work , the types and sources of information that influenced the decision to work or not work , and actions regarding families and loved ones . set 2 was given after the disease was identified as rvfv infection , federal health authorities briefed the public about this infection at a press conference , and a panel of nongovernment experts discussed the disease on television . these questions tested the participants understanding of rvfv routes of transmission and preventive measures and the participants satisfaction with information from government and nongovernment sources . sets 3 and 4 followed a period of growing confusion and anxiety caused by changing and sometimes conflicting authoritative statements and tested participants requests for medication , including ribavirin ( set 3 ) , and for rvfv vaccine ( set 4 ) . set 5 , given after the participants learned that the outbreak of rvfv was intentional , reassessed decisions and actions regarding job and family concerns and information needed to make these decisions . set 6 followed a period of increasing anxiety over a now - confirmed bioterrorism - related outbreak that could spread to humans and cattle in the state and in adjoining states , over the ability of the government to stop the spread of the infection , and over the de facto isolation of the community . these questions surveyed participants reactions to rumors of possible quarantine and to sources of information deemed reliable and influential in decision making now that the threat had become more complicated , personal , and disruptive . the number of candidates contacted to assemble groups of at least 30 participants followed guidelines from marketing study groups ( harwell productions , pers . one hundred thirty - eight medical first - responders ( responders ) were invited to participate in one study group ; 58 responded to our invitation , 45 registered , and 38 attended . reflecting the make - up of medical first - responders in the community , one third of the group s participants were fire department emergency medical services personnel , and two thirds were emergency department personnel ( nurses , physicians , technicians ) from the area s major medical centers . eighty - three spouses or partners of responders ( hereafter termed spouses ) were invited to form a second study group ; 47 responded , 44 registered , and 32 attended . fifty - seven members of the local print and tv news media ( hereafter , termed the media ) were invited to a third study group ; 50 responded , 42 registered , and 34 attended . three hundred fifty invitations were sent to rank - and - file residents of the community ( hereafter termed residents ) to form a final study group . postal service as undeliverable ; 73 responded , 46 registered , and 47 attended ( 1 registered participant failed to appear , but 2 invitees who had not registered arrived and participated ) . names of persons invited to participate in the first three groups were drawn blindly from rosters of eligible candidates prepared by their employers . rosters of eligible candidates for the residents category were prepared from census tract data and cross - indexed telephone books ( table ) . sex and age distributions within the four study groups were representative of the population segments from which their members were drawn , as were the educational levels of the responder , spouse , and media groups . the educational distribution of residents was not representative of the metropolitan area ; those who chose to participate in this group had more formal education than area residents in general . according to 2000 census data for this area , 24.2% of the population have no high school diploma or equivalency , 30.2% are high school graduates , 22.4% have some college education , 12.7% have either an associate or full college degree , and 6.1% have graduate or professional degrees . we asked all participants if they had family members and loved ones in the area to assess potential conflict in job loyalty versus safety of one s family and loved ones during a disaster . the media group was unique in the large number of its members who answered no to this question . when asked about the transmission of rvfv , 30% to 35% of all group participants knew that the virus is transmitted by mosquitoes and not from person to person . rvfv transmission information was given by federal authorities at the televised press conference , as well as by academic experts interviewed on television after the press conference . the largest group who thought the risk for person - to - person transmission was considerable was responders . most participants wanted additional information from local public health authorities , and 48% to 75% wanted information from both government and nongovernment sources . in all four groups , participants who expressed interest in nonmedically indicated antibiotic or ribavirin treatment were in the minority . the largest minority to demand such medications was in the group of spouses ; however , the demanded medication was primarily for their first - responder companions , not for themselves . in all four groups , approximately 50% of participants said that they would compete for rvfv vaccine for themselves and their families ; the largest demand came from the media . when asked if they would demand vaccine for themselves as a quid pro quo for remaining on the job during the crisis , 26% of responders said they would , and 21% said they were uncertain . sixty - three percent of spouses said they would want their mates to demand vaccine . reacting to rumors of quarantine ( not to an official quarantine announcement ) , 59% of media and 75% of residents said they would comply and not try to leave ; 6% of residents and 13% of spouses said they would try to leave regardless of consequences ; 4% of residents and 15% of media said they would obey but try to leave if necessary . in all groups , most participants stated that their willingness to comply would increase if they were assured that quarantine was absolutely necessary and that it would work . in all groups , a majority faulted federal authorities for holding the joint press conference in the state capital , rather than at the scene of the outbreak 250 miles away . early in the simulated outbreak , before the disease was identified and its implications were known , pluralities in all groups wanted health information from local public health authorities . responders wanted the information delivered within the chain of command at work ; ranking second as the desired source of this information for all groups , except responders , was the private physician . after the disease was identified and terrorism was determined the cause , no single source of information at any level was chosen as desirable by any majority . small pluralities in each group chose as the most reliable source of information the head of the federal team working at the outbreak site , the president , a physician from a federal agency , or when asked whom the participants considered most influential in decisions they would make about work , family , and themselves , once again no single authority figure emerged as the majority s selection in any group . selections with small pluralities were family and loved ones , the president , and the head of the federal team working at the outbreak site . half the participants in all groups chose other . within other , 34% of the participants chose another federal medical or nonmedical official , 4% chose state officials , 5% chose national ( not local ) media , 11% said they did not know at that time in the outbreak who would be most influential in their personal decision making , and 47% chose local leaders , including government and nongovernment officials . the media indicated they would turn to a variety of local sources for their assignments . most of these sources were professors in the local medical school , but other sources included the reporters personal physicians and veterinarians . most participants in all four study groups indicated they would remain on the job throughout the crisis . initially , when the outbreak of an as - yet - unknown disease in the area was recognized , 97% of responders , 94% of media , and 77% of residents said they would remain at work ; 11% of residents opted not to work , and the remainder said they were students , unemployed , or retired . ninety - one percent of spouses said they would encourage their mates to continue working . after the disease was identified and the outbreak was recognized as an act of bioterrorism , 95% , 71% , and 65% of responders , media , and residents , respectively , said they would continue working , and 78% of spouses said they would want their mates to remain on the job . in all groups , most participants said they would continue to work , provided that they received information about medical issues ( particularly transmission and prevention ) , their work sites were adequately protected , and the community were unlikely to be exposed to another act of bioterrorism . seventy - seven percent of media said that if their work put them at risk , they would expect their employers to provide protective measures ( from insecticides to vaccine ) and necessary medication and treatment . just before conclusion of the video , responders and spouses were asked how important it was to reach agreement with their partners on whether to stay at work , seek medicines , and send family members out of town . twenty - six percent of responders said that concurrence would be essential , whereas 53% of spouses thought such agreement was essential . the study was based on a simulated outbreak ; therefore , the participants reactions were to a simulated , not an actual , crisis . for budgetary reasons , we could not recruit and compensate sufficient numbers of community residents to provide a statistical sample of the populations from which they were drawn ; therefore , as with some forms of market research , participants answers to questions reflect their opinions and not those of their peers . sample bias may have resulted from the absence of persons invited to participate who declined to do so ; this may be especially important among residents , whose undereducated members were underrepresented . the choice of rvfv as the disseminated agent may have muted the responses of the participants because it has a relatively low death rate ( 1% to 10% ) and does not have the high fear factor of some other diseases such as smallpox . finally , the community we chose has its own customs , traditions , and ways of coping with a crisis that may not be shared by communities elsewhere . for example , disagreements between responders and spouses over reporting for duty during the crisis and demands for vaccine as quid pro quo for staying on the job could influence staffing levels and responder job performance in an actual bioterrorism - related outbreak . risk communication messages may need to be crafted , tailored to the needs and concerns of first - responders and families , and delivered separately . journalists are key participants in risk communication ( 1216 ) , yet in this study , the media exhibited more fear than any group other than spouses , made high demands for vaccine , had the poorest understanding of medical issues associated with rvfv , and were most likely to stay away from work after terrorism was recognized . had this been an actual bioterrorism - related outbreak , the media might not have served effectively as conduits of information to the public because they had not been adequately educated to eliminate confusion and dispel fear about their personal safety . participants in this study were not unique in their wariness of sole - source information , however authoritative or expert ( 1720 ) . members of the actual community in which the simulated bioterrorism - related outbreak occurred wanted information from varied sources , even if the sources they mentioned differed in quality and reliability . their reactions suggest that bioterrorism training should include information management for risk communicators and public affairs officers who have the responsibility of providing timely and accurate information to dispel the fog of rumors and misinformation present during the aftermath of an intentional disease outbreak . journalists and other media specialists should participate actively in scenarios and other similar exercises to gain insight into the complexity of information management in a bioterrorism - related crisis . as the simulated outbreak became more complicated and personally threatening , participants indicated a preference for information from local government and nongovernment sources or from federal officials at the outbreak site . recognized , respected community leaders ( e.g. , private physicians , government and nongovernment officials ) are likely to provide guidance in a bioterrorism - related crisis , just as they do in other crises . training in the strategy and tactics of risk communication ( 2124 ) should be expanded to include them . in an actual bioterrorism - related outbreak , these local leaders , supported by federal health authorities , should take the lead in communicating with local residents . the study was based on a simulated outbreak ; therefore , the participants reactions were to a simulated , not an actual , crisis . for budgetary reasons , we could not recruit and compensate sufficient numbers of community residents to provide a statistical sample of the populations from which they were drawn ; therefore , as with some forms of market research , participants answers to questions reflect their opinions and not those of their peers . sample bias may have resulted from the absence of persons invited to participate who declined to do so ; this may be especially important among residents , whose undereducated members were underrepresented . the choice of rvfv as the disseminated agent may have muted the responses of the participants because it has a relatively low death rate ( 1% to 10% ) and does not have the high fear factor of some other diseases such as smallpox . finally , the community we chose has its own customs , traditions , and ways of coping with a crisis that may not be shared by communities elsewhere . for example , disagreements between responders and spouses over reporting for duty during the crisis and demands for vaccine as quid pro quo for staying on the job could influence staffing levels and responder job performance in an actual bioterrorism - related outbreak . risk communication messages may need to be crafted , tailored to the needs and concerns of first - responders and families , and delivered separately . journalists are key participants in risk communication ( 1216 ) , yet in this study , the media exhibited more fear than any group other than spouses , made high demands for vaccine , had the poorest understanding of medical issues associated with rvfv , and were most likely to stay away from work after terrorism was recognized . had this been an actual bioterrorism - related outbreak , the media might not have served effectively as conduits of information to the public because they had not been adequately educated to eliminate confusion and dispel fear about their personal safety . participants in this study were not unique in their wariness of sole - source information , however authoritative or expert ( 1720 ) . members of the actual community in which the simulated bioterrorism - related outbreak occurred wanted information from varied sources , even if the sources they mentioned differed in quality and reliability . their reactions suggest that bioterrorism training should include information management for risk communicators and public affairs officers who have the responsibility of providing timely and accurate information to dispel the fog of rumors and misinformation present during the aftermath of an intentional disease outbreak . journalists and other media specialists should participate actively in scenarios and other similar exercises to gain insight into the complexity of information management in a bioterrorism - related crisis . as the simulated outbreak became more complicated and personally threatening , participants indicated a preference for information from local government and nongovernment sources or from federal officials at the outbreak site . recognized , respected community leaders ( e.g. , private physicians , government and nongovernment officials ) are likely to provide guidance in a bioterrorism - related crisis , just as they do in other crises . training in the strategy and tactics of risk communication ( 2124 ) should be expanded to include them . in an actual bioterrorism - related outbreak , these local leaders , supported by federal health authorities , should take the lead in communicating with local residents . these events did not occur . note : the following questionnaire is from an outbreak simulation .

to assess community needs for public information during a bioterrorism - related crisis , we simulated an intentional rift valley fever outbreak in a community in the southern part of the united states . we videotaped a series of simulated print and television news reports over a fictional 9-day crisis period and invited various groups ( e.g. , first - responders and their spouses or partners , journalists ) within the selected community to view the videotape and respond to questions about their reactions . all responses were given anonymously . first - responders and their spouses or partners varied in their reactions about how the crisis affected family harmony and job performance . local journalists exhibited considerable personal fear and confusion . all groups demanded , and put more trust in , information from local sources . these findings may have implications for risk communication during bioterrorism - related outbreaks .

Methods The Video The Questionnaire Participants Results Discussion and Conclusions Supplementary Material

we simulated the intentional aerosolized release of rift valley fever virus ( rvfv ) in a semirural community ( population 300,000 ) in the southern part of the united states . we videotaped a series of simulated print and television ( local , network , and cable ) news reports over a fictional 9-day crisis period . we invited four groups ( medical first - responders , medical first - responder spouses or partners , journalists , and others ) within the selected community to view the videotape and answer questions about their reactions . the story of the simulated outbreak unfolded in a series of video reports from federal and local governments and the news media ( appendix 1 ) . health agency news bulletins were provided by the centers for diseases control and prevention ; news reports by television reporters or news anchors ; and community reports by local officials , including the mayor . reports included a detailed press conference by federal health authorities describing routes of transmission , prevention measures , signs and symptoms of infection , and medical management of the disease . the news conference , held in the state capital the day after the presence of rvfv infection in the united states was announced , was immediately followed by a panel discussion ( by nongovernment experts ) on rvfv . differences of opinion on clinical , epidemiologic , and biological issues among rvfv experts were reported . confusion arose in the community over disease management ( e.g. , the effectiveness of the antiviral drug ribavirin , the need for rvfv vaccine , and who should receive the vaccine ) and over the potential for infected persons to serve as reservoirs and carriers of the virus elsewhere during the few days when viral titers are especially high . the questionnaire ( appendix 2 ) , which included multiple - choice , open - ended questions , and opportunities for additional comments , was distributed to all participants . questions addressed job abandonment , quarantine compliance , demand for drugs and vaccine , information requirements , and other issues of community interest . set 1 was given after a disease of unknown etiology affecting humans and some farm animals was recognized in the community . these questions focused on willingness to remain at work , the types and sources of information that influenced the decision to work or not work , and actions regarding families and loved ones . these questions tested the participants understanding of rvfv routes of transmission and preventive measures and the participants satisfaction with information from government and nongovernment sources . sets 3 and 4 followed a period of growing confusion and anxiety caused by changing and sometimes conflicting authoritative statements and tested participants requests for medication , including ribavirin ( set 3 ) , and for rvfv vaccine ( set 4 ) . set 6 followed a period of increasing anxiety over a now - confirmed bioterrorism - related outbreak that could spread to humans and cattle in the state and in adjoining states , over the ability of the government to stop the spread of the infection , and over the de facto isolation of the community . these questions surveyed participants reactions to rumors of possible quarantine and to sources of information deemed reliable and influential in decision making now that the threat had become more complicated , personal , and disruptive . the number of candidates contacted to assemble groups of at least 30 participants followed guidelines from marketing study groups ( harwell productions , pers . one hundred thirty - eight medical first - responders ( responders ) were invited to participate in one study group ; 58 responded to our invitation , 45 registered , and 38 attended . reflecting the make - up of medical first - responders in the community , one third of the group s participants were fire department emergency medical services personnel , and two thirds were emergency department personnel ( nurses , physicians , technicians ) from the area s major medical centers . eighty - three spouses or partners of responders ( hereafter termed spouses ) were invited to form a second study group ; 47 responded , 44 registered , and 32 attended . fifty - seven members of the local print and tv news media ( hereafter , termed the media ) were invited to a third study group ; 50 responded , 42 registered , and 34 attended . three hundred fifty invitations were sent to rank - and - file residents of the community ( hereafter termed residents ) to form a final study group . postal service as undeliverable ; 73 responded , 46 registered , and 47 attended ( 1 registered participant failed to appear , but 2 invitees who had not registered arrived and participated ) . names of persons invited to participate in the first three groups were drawn blindly from rosters of eligible candidates prepared by their employers . sex and age distributions within the four study groups were representative of the population segments from which their members were drawn , as were the educational levels of the responder , spouse , and media groups . the educational distribution of residents was not representative of the metropolitan area ; those who chose to participate in this group had more formal education than area residents in general . according to 2000 census data for this area , 24.2% of the population have no high school diploma or equivalency , 30.2% are high school graduates , 22.4% have some college education , 12.7% have either an associate or full college degree , and 6.1% have graduate or professional degrees . we asked all participants if they had family members and loved ones in the area to assess potential conflict in job loyalty versus safety of one s family and loved ones during a disaster . the story of the simulated outbreak unfolded in a series of video reports from federal and local governments and the news media ( appendix 1 ) . the reports began with recognition in the community of an unusual infection affecting humans and certain farm animals and continued during the next 9 days with an epidemiologic investigation and the identification by federal authorities of intentional release of rvfv . reports included a detailed press conference by federal health authorities describing routes of transmission , prevention measures , signs and symptoms of infection , and medical management of the disease . the news conference , held in the state capital the day after the presence of rvfv infection in the united states was announced , was immediately followed by a panel discussion ( by nongovernment experts ) on rvfv . differences of opinion on clinical , epidemiologic , and biological issues among rvfv experts were reported . confusion arose in the community over disease management ( e.g. , the effectiveness of the antiviral drug ribavirin , the need for rvfv vaccine , and who should receive the vaccine ) and over the potential for infected persons to serve as reservoirs and carriers of the virus elsewhere during the few days when viral titers are especially high . although official quarantine measures were not taken , final video reports showed a gradual de facto isolation of the city . the questionnaire ( appendix 2 ) , which included multiple - choice , open - ended questions , and opportunities for additional comments , was distributed to all participants . questions addressed job abandonment , quarantine compliance , demand for drugs and vaccine , information requirements , and other issues of community interest . set 1 was given after a disease of unknown etiology affecting humans and some farm animals was recognized in the community . these questions focused on willingness to remain at work , the types and sources of information that influenced the decision to work or not work , and actions regarding families and loved ones . set 2 was given after the disease was identified as rvfv infection , federal health authorities briefed the public about this infection at a press conference , and a panel of nongovernment experts discussed the disease on television . sets 3 and 4 followed a period of growing confusion and anxiety caused by changing and sometimes conflicting authoritative statements and tested participants requests for medication , including ribavirin ( set 3 ) , and for rvfv vaccine ( set 4 ) . set 6 followed a period of increasing anxiety over a now - confirmed bioterrorism - related outbreak that could spread to humans and cattle in the state and in adjoining states , over the ability of the government to stop the spread of the infection , and over the de facto isolation of the community . these questions surveyed participants reactions to rumors of possible quarantine and to sources of information deemed reliable and influential in decision making now that the threat had become more complicated , personal , and disruptive . one hundred thirty - eight medical first - responders ( responders ) were invited to participate in one study group ; 58 responded to our invitation , 45 registered , and 38 attended . reflecting the make - up of medical first - responders in the community , one third of the group s participants were fire department emergency medical services personnel , and two thirds were emergency department personnel ( nurses , physicians , technicians ) from the area s major medical centers . eighty - three spouses or partners of responders ( hereafter termed spouses ) were invited to form a second study group ; 47 responded , 44 registered , and 32 attended . fifty - seven members of the local print and tv news media ( hereafter , termed the media ) were invited to a third study group ; 50 responded , 42 registered , and 34 attended . three hundred fifty invitations were sent to rank - and - file residents of the community ( hereafter termed residents ) to form a final study group . postal service as undeliverable ; 73 responded , 46 registered , and 47 attended ( 1 registered participant failed to appear , but 2 invitees who had not registered arrived and participated ) . names of persons invited to participate in the first three groups were drawn blindly from rosters of eligible candidates prepared by their employers . sex and age distributions within the four study groups were representative of the population segments from which their members were drawn , as were the educational levels of the responder , spouse , and media groups . the educational distribution of residents was not representative of the metropolitan area ; those who chose to participate in this group had more formal education than area residents in general . according to 2000 census data for this area , 24.2% of the population have no high school diploma or equivalency , 30.2% are high school graduates , 22.4% have some college education , 12.7% have either an associate or full college degree , and 6.1% have graduate or professional degrees . we asked all participants if they had family members and loved ones in the area to assess potential conflict in job loyalty versus safety of one s family and loved ones during a disaster . the media group was unique in the large number of its members who answered no to this question . most participants wanted additional information from local public health authorities , and 48% to 75% wanted information from both government and nongovernment sources . in all four groups , participants who expressed interest in nonmedically indicated antibiotic or ribavirin treatment were in the minority . the largest minority to demand such medications was in the group of spouses ; however , the demanded medication was primarily for their first - responder companions , not for themselves . in all four groups , approximately 50% of participants said that they would compete for rvfv vaccine for themselves and their families ; the largest demand came from the media . when asked if they would demand vaccine for themselves as a quid pro quo for remaining on the job during the crisis , 26% of responders said they would , and 21% said they were uncertain . in all groups , most participants stated that their willingness to comply would increase if they were assured that quarantine was absolutely necessary and that it would work . in all groups , a majority faulted federal authorities for holding the joint press conference in the state capital , rather than at the scene of the outbreak 250 miles away . early in the simulated outbreak , before the disease was identified and its implications were known , pluralities in all groups wanted health information from local public health authorities . responders wanted the information delivered within the chain of command at work ; ranking second as the desired source of this information for all groups , except responders , was the private physician . small pluralities in each group chose as the most reliable source of information the head of the federal team working at the outbreak site , the president , a physician from a federal agency , or when asked whom the participants considered most influential in decisions they would make about work , family , and themselves , once again no single authority figure emerged as the majority s selection in any group . selections with small pluralities were family and loved ones , the president , and the head of the federal team working at the outbreak site . half the participants in all groups chose other . within other , 34% of the participants chose another federal medical or nonmedical official , 4% chose state officials , 5% chose national ( not local ) media , 11% said they did not know at that time in the outbreak who would be most influential in their personal decision making , and 47% chose local leaders , including government and nongovernment officials . the media indicated they would turn to a variety of local sources for their assignments . most of these sources were professors in the local medical school , but other sources included the reporters personal physicians and veterinarians . most participants in all four study groups indicated they would remain on the job throughout the crisis . initially , when the outbreak of an as - yet - unknown disease in the area was recognized , 97% of responders , 94% of media , and 77% of residents said they would remain at work ; 11% of residents opted not to work , and the remainder said they were students , unemployed , or retired . after the disease was identified and the outbreak was recognized as an act of bioterrorism , 95% , 71% , and 65% of responders , media , and residents , respectively , said they would continue working , and 78% of spouses said they would want their mates to remain on the job . in all groups , most participants said they would continue to work , provided that they received information about medical issues ( particularly transmission and prevention ) , their work sites were adequately protected , and the community were unlikely to be exposed to another act of bioterrorism . just before conclusion of the video , responders and spouses were asked how important it was to reach agreement with their partners on whether to stay at work , seek medicines , and send family members out of town . for budgetary reasons , we could not recruit and compensate sufficient numbers of community residents to provide a statistical sample of the populations from which they were drawn ; therefore , as with some forms of market research , participants answers to questions reflect their opinions and not those of their peers . sample bias may have resulted from the absence of persons invited to participate who declined to do so ; this may be especially important among residents , whose undereducated members were underrepresented . the choice of rvfv as the disseminated agent may have muted the responses of the participants because it has a relatively low death rate ( 1% to 10% ) and does not have the high fear factor of some other diseases such as smallpox . finally , the community we chose has its own customs , traditions , and ways of coping with a crisis that may not be shared by communities elsewhere . for example , disagreements between responders and spouses over reporting for duty during the crisis and demands for vaccine as quid pro quo for staying on the job could influence staffing levels and responder job performance in an actual bioterrorism - related outbreak . risk communication messages may need to be crafted , tailored to the needs and concerns of first - responders and families , and delivered separately . journalists are key participants in risk communication ( 1216 ) , yet in this study , the media exhibited more fear than any group other than spouses , made high demands for vaccine , had the poorest understanding of medical issues associated with rvfv , and were most likely to stay away from work after terrorism was recognized . had this been an actual bioterrorism - related outbreak , the media might not have served effectively as conduits of information to the public because they had not been adequately educated to eliminate confusion and dispel fear about their personal safety . participants in this study were not unique in their wariness of sole - source information , however authoritative or expert ( 1720 ) . members of the actual community in which the simulated bioterrorism - related outbreak occurred wanted information from varied sources , even if the sources they mentioned differed in quality and reliability . their reactions suggest that bioterrorism training should include information management for risk communicators and public affairs officers who have the responsibility of providing timely and accurate information to dispel the fog of rumors and misinformation present during the aftermath of an intentional disease outbreak . journalists and other media specialists should participate actively in scenarios and other similar exercises to gain insight into the complexity of information management in a bioterrorism - related crisis . as the simulated outbreak became more complicated and personally threatening , participants indicated a preference for information from local government and nongovernment sources or from federal officials at the outbreak site . recognized , respected community leaders ( e.g. , private physicians , government and nongovernment officials ) are likely to provide guidance in a bioterrorism - related crisis , just as they do in other crises . training in the strategy and tactics of risk communication ( 2124 ) should be expanded to include them . in an actual bioterrorism - related outbreak , these local leaders , supported by federal health authorities , should take the lead in communicating with local residents . for budgetary reasons , we could not recruit and compensate sufficient numbers of community residents to provide a statistical sample of the populations from which they were drawn ; therefore , as with some forms of market research , participants answers to questions reflect their opinions and not those of their peers . sample bias may have resulted from the absence of persons invited to participate who declined to do so ; this may be especially important among residents , whose undereducated members were underrepresented . the choice of rvfv as the disseminated agent may have muted the responses of the participants because it has a relatively low death rate ( 1% to 10% ) and does not have the high fear factor of some other diseases such as smallpox . for example , disagreements between responders and spouses over reporting for duty during the crisis and demands for vaccine as quid pro quo for staying on the job could influence staffing levels and responder job performance in an actual bioterrorism - related outbreak . risk communication messages may need to be crafted , tailored to the needs and concerns of first - responders and families , and delivered separately . journalists are key participants in risk communication ( 1216 ) , yet in this study , the media exhibited more fear than any group other than spouses , made high demands for vaccine , had the poorest understanding of medical issues associated with rvfv , and were most likely to stay away from work after terrorism was recognized . had this been an actual bioterrorism - related outbreak , the media might not have served effectively as conduits of information to the public because they had not been adequately educated to eliminate confusion and dispel fear about their personal safety . participants in this study were not unique in their wariness of sole - source information , however authoritative or expert ( 1720 ) . members of the actual community in which the simulated bioterrorism - related outbreak occurred wanted information from varied sources , even if the sources they mentioned differed in quality and reliability . their reactions suggest that bioterrorism training should include information management for risk communicators and public affairs officers who have the responsibility of providing timely and accurate information to dispel the fog of rumors and misinformation present during the aftermath of an intentional disease outbreak . journalists and other media specialists should participate actively in scenarios and other similar exercises to gain insight into the complexity of information management in a bioterrorism - related crisis . as the simulated outbreak became more complicated and personally threatening , participants indicated a preference for information from local government and nongovernment sources or from federal officials at the outbreak site . recognized , respected community leaders ( e.g. , private physicians , government and nongovernment officials ) are likely to provide guidance in a bioterrorism - related crisis , just as they do in other crises . training in the strategy and tactics of risk communication ( 2124 ) should be expanded to include them . in an actual bioterrorism - related outbreak , these local leaders , supported by federal health authorities , should take the lead in communicating with local residents .